Rbcs gene for regulating biomass of lettuce, regulating module and application thereof

Abstract

The application discloses an RbcS gene for regulating the biomass of lettuce, a regulation module and application thereof, relates to the technical field of molecular biology, and relates to a nucleic acid molecule for regulating the biomass of lettuce, wherein the nucleic acid molecule is a gene as shown in (a), (b) or (c) below: (a) an LsRbcS2 gene with a CDS sequence as shown in SEQ ID NO: 3, or an LsRbcS6 gene with a CDS sequence as shown in SEQ ID NO: 4; (b) a nucleic acid molecule for encoding an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; (c) a variant sequence with at least 95% sequence identity to the nucleotide sequence defined in (a) or (b), and derived from lettuce, and encoding a protein with the function of a small subunit of Rubisco. The screened LsRbcS2 and LsRbcS6 can be used as a target for molecular marker assisted selection, and are used for early screening of high-yield varieties; and the negative regulation relationship between LsKN1 and LsRbcS6 also provides a new idea for gene editing improvement, and the combination of the LsRbcS6 promoter and the LsKN1 can be optimized to precisely regulate photosynthetic efficiency.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the RbcS gene, regulatory module, and application of the gene that regulates lettuce biomass. Background Technology

[0002] Lettuce (Lactuca sativa L.), belonging to the genus Lactuca in the family Asteraceae, is an annual or biennial herbaceous plant and one of the most important leafy vegetables widely cultivated and consumed globally, especially in temperate and subtropical regions. Originating in the Mediterranean region, lettuce has undergone long-term domestication and varietal improvement, resulting in various types including head lettuce, loose-leaf lettuce, and Roman lettuce, adapted to different climatic conditions and cultivation methods. According to statistics from the Food and Agriculture Organization of the United Nations (http: / / www.fao.org / faostat / en / ), global lettuce planting area and production have been on the rise in recent years. Data shows that the total global lettuce planting area is 1.2264 million hectares, of which China accounts for 606,400 hectares, or 49.45% of the global total; global lettuce production reached 27.6602 million tons, with China producing 14.3231 million tons, accounting for 51.78% of global lettuce production. The continued growth in global lettuce planting area and production reflects its important position in the vegetable industry.

[0003] Lettuce is widely favored by consumers for its crisp texture, rich nutrition, and versatility in preparation. High levels of vitamin C, vitamin K, folic acid, and minerals such as potassium, calcium, and magnesium have been detected in lettuce leaves. It is also rich in dietary fiber, making it a highly nutritious vegetable and an important source of nutrients in the daily diet. Furthermore, lettuce contains various bioactive compounds, including flavonoids, phenolic acids, carotenoids, and sesquiterpene lactones, which possess antioxidant, anti-inflammatory, and anti-tumor properties.

[0004] In recent years, the market demand for lettuce has been increasing year by year, and public attention and scientific research value have also risen accordingly. The main edible part of most lettuce is the leaf. As a leafy vegetable, the economic yield of lettuce directly comes from the accumulation of leaf biomass, and leaf growth is highly dependent on the efficiency of photosynthesis. Photosynthetic efficiency is positively correlated with crop yield, and increasing the expression level of photosynthesis-related genes helps promote biomass accumulation. The RbcS gene, as the encoding gene of the Rubisco small subunit, directly affects the assembly and activity of the Rubisco holoenzyme, thereby regulating photosynthetic efficiency and biomass formation.

[0005] Lettuce often faces various stresses during its growth and development, such as soil salinization, water deficit, and abnormal temperatures. These stresses can lead to decreased photosynthetic efficiency, inhibited growth, and reduced yield. Plants respond to environmental stress by regulating the expression of photosynthesis-related genes. Changes in the expression of RbcS gene family members under stress conditions reflect their potential functions in adversity adaptation. Studies have shown that photosynthetic efficiency is positively correlated with crop yield, and increasing the expression level of photosynthesis-related genes helps promote biomass accumulation.

[0006] In existing technologies, the functions of RbcS genes in multiple species have been reported. For example, in rice, OsRbcS2 and OsRbcS4 are expressed at the highest levels in leaves, and their co-silenced mutants exhibit decreased Rubisco content and yield. In tobacco, plants with silenced RbcS show decreased Rubisco expression, leading to reduced photosynthetic rate, plant growth, and biomass accumulation. In Arabidopsis, RbcS1A and RbcS3B contribute to Rubisco accumulation, and double mutant plants show significantly inhibited photosynthetic rate and growth. These studies indicate that RbcS genes play an indispensable positive regulatory role in plant photosynthesis and biomass accumulation, and direct manipulation of the RbcS gene itself affects yield traits.

[0007] However, current technologies mainly focus on directly manipulating the RbcS gene itself or studying its expression changes under different stress conditions. The upstream transcriptional regulatory mechanisms of the RbcS gene, particularly the existence of specific negative regulators and how they affect biomass accumulation, remain unclear. In lettuce, no systematic identification of the RbcS gene family or studies on its upstream regulatory network have been reported. Therefore, identifying key upstream factors regulating RbcS expression in lettuce and elucidating their regulatory mechanisms are of great significance for high-yield molecular breeding of lettuce. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide the RbcS gene, regulatory module and its application for regulating lettuce biomass.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0010] A nucleic acid molecule that regulates lettuce biomass, said nucleic acid molecule being a gene as shown in (a), (b), or (c) below:

[0011] (a) The LsRbcS2 gene with a CDS sequence as shown in SEQ ID NO:3, or the LsRbcS6 gene with a CDS sequence as shown in SEQ ID NO:4;

[0012] (b) Nucleic acid molecules encoding amino acid sequences as shown in SEQ ID NO:1 or SEQ ID NO:2;

[0013] (c) A variant sequence that has at least 95% sequence identity with the nucleotide sequence defined in (a) or (b) and is derived from lettuce and encodes a protein with the function of the Rubisco small subunit.

[0014] A recombinant expression vector includes the aforementioned nucleic acid molecule and a promoter operatively linked upstream of the nucleic acid molecule.

[0015] A method for increasing the aboveground biomass of lettuce includes: introducing the above-mentioned recombinant expression vector into lettuce cells to obtain transgenic lettuce plants overexpressing the LsRbcS2 and / or LsRbcS6 genes, so as to increase the fresh weight, dry weight and leaf area of ​​lettuce leaves.

[0016] A method to increase the aboveground biomass of lettuce involves inhibiting the expression of the endogenous LsKN1 gene or the activity of its encoded transcription factor in lettuce plants, thereby upregulating the expression of the LsRbcS6 gene, which in turn increases the Rubisco enzyme activity and chlorophyll content in lettuce leaves, ultimately increasing the aboveground biomass.

[0017] The nucleotide sequence of the LsKN1 gene is shown in SEQ ID NO:5, and the amino acid sequence encoding the transcription factor is shown in SEQ ID NO:6.

[0018] Application of nucleic acid molecules or the above-mentioned recombinant expression vectors in the breeding of high-yield lettuce varieties.

[0019] A method for screening high-yielding lettuce germplasm, characterized by comprising the following steps:

[0020] (1) Total RNA was extracted from the lettuce leaves to be tested and cDNA was obtained by reverse transcription;

[0021] (2) The expression levels of LsRbcS2 and / or LsRbcS6 genes in cDNA were detected by qRT-PCR using specific primers.

[0022] (3) Using wild-type lettuce as a control, plants with significantly higher LsRbcS2 and / or LsRbcS6 gene expression levels than the control were selected as high-yield candidate germplasm.

[0023] Further optimization includes detecting the expression level of the LsKN1 gene and selecting plants with significantly lower LsKN1 expression levels than the control as high-yield candidate germplasm.

[0024] A functional module for regulating lettuce biomass, the functional module being composed of the LsKN1 gene and its encoded transcription factor, and the LsRbcS6 gene; the transcription factor encoded by the LsKN1 gene specifically binds to the promoter region of the LsRbcS6 gene, negatively regulating the expression of LsRbcS6.

[0025] The promoter sequence of the LsRbcS6 gene is shown in SEQ ID NO:7.

[0026] More preferably, the negative regulation relieves the negative regulation of LsRbcS6 by inhibiting the expression of the LsKN1 gene or inhibiting the activity of its encoded transcription factor, thereby upregulating the expression of LsRbcS6.

[0027] More preferably, the methods for inhibiting LsKN1 gene expression include gene silencing, gene knockout, gene editing, RNA interference, antisense RNA, or artificial miRNA.

[0028] The beneficial effects of adopting the above technical solution are as follows: In this invention, our research group conducted whole-genome identification and bioinformatics analysis of the lettuce RbcS gene family for the first time, successfully screening two major genes related to lettuce aboveground biomass, LsRbcS2 and LsRbcS6, and preliminarily studied their biological functions in promoting aboveground biomass and leaf size. We also discovered for the first time that LsKN1 can negatively regulate the expression of LsRbcS6, revealing a new function of LsKN1 beyond regulating leaf morphology and bolting traits. This research provides directly applicable gene resources for high-yield lettuce breeding. The screened LsRbcS2 and LsRbcS6 can serve as targets for marker-assisted selection for early screening of high-yield varieties. The negative regulatory relationship between LsKN1 and LsRbcS6 also provides new ideas for gene editing improvement, allowing for precise regulation of photosynthetic efficiency by optimizing the binding of the LsRbcS6 promoter to LsKN1. The research findings not only provide new insights into the molecular mechanisms of lettuce yield traits, but also reserve key gene resources for high-yield breeding practices, possessing potential application and economic value. Attached Figure Description

[0029] Figure 1 This is a phylogenetic tree constructed based on the RbcS protein sequences of six plants. Note: At Arabidopsis thaliana, Ls lettuce, Han sunflower, Nt tobacco, Zm maize, Sl tomato.

[0030] Figure 2 This is a diagram showing the conserved motifs, domains, and gene structure of LsRbcS. Note: Different colors represent different conserved motifs. PLN02289 is a conserved domain of the RbcS protein. UTR is the non-coding region, and CDS is the coding region.

[0031] Figure 3 This is a map showing the distribution of the lettuce RbcS gene on the chromosome.

[0032] Figure 4 This is a collinearity diagram of the RbcS gene in lettuce. The red line represents the collinearity relationship within the LsRbcS gene. Chr represents chromosomes, and the color scale represents the chromosome gene density (0~12, blue for low and red for high).

[0033] Figure 5 This is a collinearity diagram of RbcS in Arabidopsis thaliana, lettuce, and sunflower, with different colors representing each species and chromosome numbers labeled numerically.

[0034] Figure 6 The predicted three-dimensional structure of the lettuce RbcS protein.

[0035] Figure 7 Diagram of cis-acting elements in the promoter of the lettuce RbcS gene.

[0036] Figure 8 This diagram illustrates the expression of the lettuce RbcS gene under salt stress. 0h, 6h, 12h, 24h, and 48h represent 0 hours, 6 hours, 12 hours, 24 hours, and 48 hours after salt stress treatment, respectively. LsRbcS1~LsRbcS7 are lettuce RbcS family genes. Color changes represent logFC relative to the 0h control, with red indicating high expression and blue indicating low expression, ranging from -1.5 to 1.5.

[0037] Figure 9 This diagram illustrates the expression of the RbcS gene in lettuce under drought stress. 0h, 6h, 12h, 24h, and 48h represent 0 hours, 6 hours, 12 hours, and 24 hours after drought stress treatment, respectively. The color changes represent the logFC relative to the 0h control, with red indicating high expression and blue indicating low expression, and the values ​​range from -1.5 to 1.5.

[0038] Figure 10 This diagram illustrates the expression of the RbcS gene in lettuce after 48 hours of dark stress and 24 hours of light recovery. 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h represent 0 hours, 6 hours, 12 hours, 24 hours, and 48 hours after dark stress treatment, and 24 hours after light recovery, respectively. The color changes represent the logFC relative to the 0 h control, with red indicating high expression and blue indicating low expression, and the values ​​range from -1.5 to 1.5.

[0039] Figure 11This diagram illustrates the comparison of the expression of RbcS gene family members in two lettuce varieties. The black bars represent Yingxia lettuce, and the gray bars represent Lollo Rosso lettuce. **** indicates P < 0.0001. Error bars represent the standard error of biological replication (SE, n = 3).

[0040] Figure 12 The diagram illustrates the correlation between the expression levels of LsRbcS2 and LsRbcS6 and biomass traits. The ac plot shows the correlation between the relative expression level of LsRbcS2 and the aboveground fresh weight, aboveground dry weight, and leaf area of ​​the two lettuce varieties; the df plot shows the correlation between the relative expression level of LsRbcS6 and the aboveground fresh weight, aboveground dry weight, and leaf area of ​​the two lettuce varieties; r is the Pearson correlation coefficient, and P < 0.01 indicates a highly significant correlation; all data are the average of three biological replicates.

[0041] Figure 13 Subcellular localization map of the p1300-GFP-LsRbcS2 and p1300-GFP-LsRbcS6 fusion proteins.

[0042] Figure 14 The graphs show the changes in the aboveground dry and fresh weight and leaf area of ​​T2 generation transgenic Arabidopsis thaliana. One-way ANOVA was used, with P < 0.05 as the significance criterion.

[0043] Figure 15 This diagram illustrates the relative expression levels of LsRbcS6 and LsRbcS2 in different strains after VIGS silencing. pTRV2 represents lettuce injected with the empty pTRV2 vector, while pTRV2-LsRbcS2 and pTRV2-LsRbcS6 represent lettuce injected with the recombinant pTRV2-LsRbcS2 and pTRV2-LsRbcS6 vectors, respectively. One-way ANOVA was used, with P < 0.05 considered significant.

[0044] Figure 16 A comparative diagram showing the effects of LsRbcS6 and LsRbcS2 gene silencing on plant phenotypes. Scale bar = 10 cm.

[0045] Figure 17 The diagram illustrates the effects of LsRbcS2 and LsRbcS6 gene silencing on plant biomass and leaf area. One-way ANOVA was used, with P < 0.05 as the significance criterion.

[0046] Figure 18 This is a schematic diagram of the yeast self-activation experiment results for LsRbcS6.

[0047] Figure 19This is a schematic diagram of the results of a yeast one-hybrid experiment using LsRbcS6 and LsKN1.

[0048] Figure 20 This diagram illustrates the comparison of dry and fresh weight and leaf area of ​​mutants of the LsKN1 homolog Atstm in Arabidopsis thaliana and plants overexpressing LsKN1-OE. One-way ANOVA was used, with P < 0.05 as the significance criterion.

[0049] Figure 21 This is a schematic diagram showing the relative expression levels of LsKN1 in different strains after VIGS silencing. pTRV2 represents lettuce injected with the empty pTRV2 vector culture, and pTRV2-LsKN1 represents lettuce injected with the pTRV2-LsKN1 recombinant vector culture. One-way ANOVA was used, with P < 0.05 as the significance criterion.

[0050] Figure 22 The diagram shows the effect of LsKN1 gene silencing on plant phenotype. Scale bar = 10 cm.

[0051] Figure 23 This is a schematic diagram showing the effects of LsKN1 gene silencing on dry and fresh weight and leaf area. One-way ANOVA was used, with P < 0.05 as the significance criterion.

[0052] Figure 24 This is a schematic diagram showing the expression levels of LsRbcS6 in each line after VIGS silencing LsKN1. One-way ANOVA was used, with P < 0.05 as the significance criterion.

[0053] Figure 25 The diagram shows the Rubisco enzyme activity results in pTRV2 and pTRV2-LsKN1. One-way ANOVA was used, and P < 0.05 was used as the significance criterion.

[0054] Figure 26 A schematic diagram showing the chlorophyll content results in pTRV2 and pTRV2-LsKN1.

[0055] Figure 27 Phenotypic diagrams for WT and LsKN1-OE, scale bar = 10 cm.

[0056] Figure 28 The diagram shows the biomass and leaf area results of LsKN1-OE and WT. One-way ANOVA was used, and P < 0.05 was used as the significance criterion.

[0057] Figure 29 This is a schematic diagram showing the expression levels of LsRbcS6 in WT and OE-LsKN1.

[0058] Figure 30 The diagram shows the Rubisco enzyme activity results in WT and LsKN1-OE. One-way ANOVA was used, and P < 0.05 was used as the significance criterion.

[0059] Figure 31 A schematic diagram showing the chlorophyll content results in WT and LsKN1-OE. Detailed Implementation

[0060] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. In the following description of the embodiments, specific details such as particular system structures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that the present application can also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, and methods are omitted so as not to obscure the description of this application with unnecessary detail.

[0061] It should be understood that, when used in this specification and the appended claims, the term "comprising" indicates the presence of a described feature, integral, step, operation, element, and / or component, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or a collection thereof. It should also be understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations. As used in this specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."

[0062] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance. References to "one embodiment" or "some embodiments" in this application mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0063] Example 1: Identification and Expression Pattern Analysis of the LsRbcS Gene Family

[0064] 1.1 Experimental Materials

[0065] 1.1.1 Plant Samples and Culture

[0066] The plant materials used in this chapter are mainly cultivated lettuce. Seeds of *Lactuca sativa* L. 'YingXia' and *Lactuca sativa* L. 'Lollo Rosso' were purchased from Sichuan Zhongdu Gaoke Seed Industry Co., Ltd. and Guangzhou Haomei Horticulture Co., Ltd., respectively. Seeds of *Nicotiana benthamiana* L. used in the subcellular localization experiments were obtained from the laboratory.

[0067] Lettuce cultivation: After soaking, the lettuce seeds were placed in a light-incubation room (22℃, 16 h light / 8 h darkness, 50% humidity) to germinate. Once the seeds showed signs of germination, they were sown in sterilized substrate and cultured under the same conditions, with regular watering. Leaves were collected at 14 days (seedling stage), 28 days (heading stage), and 42 days (early stage of fleshy stem / head formation) after sowing as materials for subsequent experiments.

[0068] Four-week-old lettuce plants were subjected to stress treatments with polyethylene glycol (PEG), sodium chloride (NaCl), and gibberellin (GA3), respectively. The treatments were as follows: 30% (w / v) PEG-8000 solution and 150 mmol·L⁻¹ NaCl solution were placed in the lettuce seedling trays and subjected to stress for 48 hours. Lettuce leaves were collected at 0, 6, 12, 24, and 48 hours after stress treatment for subsequent experiments.

[0069] Light and dark treatment: Lettuce was cultured in darkness for 48 h, followed by 24 h of light exposure. Leaf samples were collected at 0, 6, 12, 24, and 48 h after the start of the dark treatment and 24 h after the return to light, and were used as experimental materials for subsequent experiments.

[0070] Sterilized tobacco seeds were sown on sterile culture medium and then placed in an artificial climate chamber (light / dark cycle 14h / 10h, constant temperature 25℃, air humidity 70%) for 4-5 weeks.

[0071] 1.1.2 Bioinformatics Analysis Tools

[0072] The software and online analysis websites used in this chapter are as follows:

[0073] Table 1.1 Software and Online Analytics Websites

[0074]

[0075] 1.1.3 Experimental Instruments and Reagents

[0076] The experimental instruments and reagents used in this chapter are as follows:

[0077] Table 1.2 Experimental Instruments and Reagents

[0078]

[0079] 1.1.4 Strains and Plasmids

[0080] The strains and plasmids used in the experiment are as follows.

[0081] Table 1.3 Information on strains and plasmids

[0082]

[0083] 1.2 Experimental Methods

[0084] 1.2.1 Identification of members of the lettuce RbcS family and analysis of their protein physicochemical properties

[0085] A local protein library was constructed based on the genomic data of cultivated lettuce (Lactuca sativa V7) downloaded from the Ensembl Plants database. Subsequently, using the Arabidopsis RbcS protein obtained from the TAIR database as the query sequence, a BLASTP search was performed to obtain initial candidates. Finally, the Rubisco small subunit HMM model (Rubisco_ssu_N, PF12338; Rubisco_sc_dom, PF00101) downloaded from the Pfam database was used to screen the whole lettuce protein sequences using HMMER (E value ≤ 1e-5) to ensure the reliability of the identification results. The candidate protein sequences obtained from the BLAST and HMMER screenings were merged and submitted to the SMART, NCBI CDD, and Pfam online databases for conserved domain verification. The non-redundant sequences obtained by the above methods were ultimately identified as members of the lettuce RbcS gene family and used as the subjects for subsequent research. The physicochemical properties of the identified LsRbcS protein were evaluated using the ExPASyProtParam online website, including parameters such as molecular weight (MW) and isoelectric point (pI). At the same time, the subcellular localization of the protein was predicted using the WoLF PSORT tool.

[0086] 1.2.2 Phylogenetic analysis of the lettuce RbcS gene

[0087] To elucidate the phylogenetic relationships of the lettuce RbcS gene, RbcS protein sequences from Arabidopsis thaliana, sunflower, tobacco, and maize were obtained from the Phytozome database as references. All protein sequences were aligned using MEGA11 software, and a phylogenetic tree was constructed using the neighbor-joining method, with 1000 bootstrap resampling runs for confidence testing. Subsequently, the generated phylogenetic tree was visualized and refined using the Evolview online tool.

[0088] 1.2.2.1 Analysis of gene structure and conserved protein motifs of the lettuce RbcS gene

[0089] Conserved motif analysis was performed on the identified protein sequences using the MEME Suite online tool, with parameters set as follows: a maximum of 10 motifs and a motif width ranging from 6 to 50 amino acids. All candidate protein sequences were submitted to the NCBI Conserved Domain Database for domain analysis and identification. Finally, based on genome annotation files, the gene structure, domains, and conserved motifs were visualized using the "Visualize Gene Structure" plugin in TBtools.

[0090] 1.2.2.2 Chromosomal localization and collinearity analysis of the lettuce RbcS gene

[0091] To study the chromosomal distribution of the LsRbcS gene in lettuce, the length information of each chromosome was obtained by searching the lettuce genome database. Based on the starting position of the LsRbcS gene and the full length data of its corresponding chromosome, the physical distribution map of each LsRbcS gene on the lettuce chromosome was drawn using Mapgene2chrom software.

[0092] To investigate the collinearity of the lettuce RbcS gene, whole-genome collinearity analysis was performed using the TBtools software package. The specific workflow was as follows: the GFF3 annotation file of the genome was parsed to obtain the precise coordinates of the genes on the chromosomes, and the length of each chromosome was determined by analyzing the FASTA file of the genome. Subsequently, the MCScanX algorithm, a plugin of TBtools, was used to detect collinear blocks within the lettuce genome and between it and the genomes of other selected species. Finally, the collinearity map was constructed using the advanced circos visualization tool. For inter-species collinearity analysis, reference genomes of species such as Arabidopsis thaliana and sunflower were used as references. Pairwise alignments were performed on the lettuce whole-genome sequence, and the MCScanX algorithm was also used to identify systemic homologous blocks. The collinearity results were then displayed using a multiple synteny plot.

[0093] 1.2.3 Three-dimensional structure prediction of lettuce RbcS gene

[0094] Using the SWISS-MODEL online platform, the LsRbcS protein sequence was submitted for homology modeling, and the template with the highest QMQE value was selected to construct its three-dimensional structural model.

[0095] 1.2.4 Cis-acting element analysis of the lettuce RbcS gene

[0096] Using TBtools software, the 2000 bp upstream of the translation initiation site of each gene was extracted from the genome. The obtained sequences were imported into the PlantCARE database for retrieval to predict the cis-regulatory elements contained within. The identification results were classified and statistically analyzed. Based on element function and distribution, cis-regulatory element maps were plotted using the ggplot2 graphical package in R to visualize the results.

[0097] 1.2.5 Total RNA extraction and cDNA synthesis from lettuce

[0098] The specific steps for RNA extraction are as follows: Weigh approximately 0.1 g of leaf tissue that has been flash-frozen in liquid nitrogen and stored at -80°C, place it in a pre-cooled grinding tube containing sterile grinding beads, and thoroughly homogenize it using a tissue homogenizer. RNA extraction is performed using the FastPure® UniversalPlant Total RNA Isolation Kit.

[0099] After extraction, RNA concentration and purity were measured by spectrophotometer (assessed by A260 / A280 ratio), and its integrity was verified by agarose gel electrophoresis.

[0100] RNA reverse transcription was performed using the HiScript® III All-in-one RT SuperMix Perfect for qPCR kit. The reaction mixture consisted of 1 μg total RNA, 1 μL Enzyme Mix, 4 μL 5× All-in-one SuperMix, and RNase-free water to a final volume of 20 μL. After thorough mixing and brief centrifugation, the PCR program was executed as follows: incubation at 50°C for 15 minutes, followed by inactivation at 85°C for 5 seconds. The cDNA product was stored at -20°C for later use.

[0101] 1.2.6 Analysis of lettuce transcriptome data

[0102] 1.2.6.1 Sequencing quality assessment, sequence alignment, and quantification of gene expression levels.

[0103] Transcriptome data for cultivated lettuce (Lola) and summer lettuce (Lettuce arugula) were downloaded from the NCBI website (National Center for Biotechnology Information). The SRR numbers of the samples were: SRR32702899, SRR32702900, SRR32702901, SRR32704475, SRR32704476, and SRR32704477. The initial data generated by the Illumina sequencing platform were stored in FASTQ format, with each read containing an identification tag, base sequence, and its corresponding quality score. Subsequently, the data were quality controlled using the Fastp tool (https: / / github.com / OpenGene / fastp).

[0104] This study used the lettuce genome V11 version data as a reference sequence. The filtered clean data was aligned to this reference genome using Hisat2 software. Subsequently, the obtained SAM format files were converted to BAM format using Samtools, and the alignment results were evaluated. StringTie software was then used to obtain the counts and FPKM values ​​for each gene. To eliminate the influence of differences in gene length, sequencing depth, and data volume between samples on expression level comparisons, the FPKM value was selected as a standardized quantitative indicator of gene expression levels.

[0105] 1.2.6.2 Principal Component Analysis of Samples

[0106] To visually represent the differences between samples, principal component analysis (PCA) was used to reduce the dimensionality of the whole gene expression data. The prcomp function from the stats package in R was used for PCA analysis, and scatter plots containing biological replicates of each sample were generated to assess intragroup consistency and intergroup variability.

[0107] 1.2.6.3 Differentially expressed gene analysis

[0108] Based on the gene counts data in 1.2.3.1, differential expression analysis was performed on data from different tissues and treatments of lettuce using the Deseq2 package in R. Lollo, with its smaller biomass, was used as the control group, and Yingxia was used as the treatment group. |log2(FoldChange)| >=1 and P-adj < 0.05 were used as the criteria for screening differentially expressed genes (DEGs).

[0109] 1.2.6.4 GO and KEGG enrichment analysis of differentially expressed genes

[0110] The selected DEGs were subjected to functional enrichment analysis using Gene Oncology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.kegg.jp. / ). The GO database systematically annotates gene functions, and its classification system comprises three components. The KEGG database integrates multi-level biological information from the molecular, cellular, species, and ecosystem levels, providing crucial support for interpreting systemic biological changes revealed by genome sequencing and other high-throughput data.

[0111] GO / KEGG enrichment analysis was performed using TBtools software, and the significant enrichment results were visualized using R language.

[0112] 1.2.7 Real-time quantitative PCR of lettuce RbcS gene

[0113] Qpcr primers for the LsRbcS gene were designed using SnapGene software. The primers were approximately 200 bp in length. To verify the specificity of the designed primers, the preliminary primer sequences were first submitted to the NCBI Primer-BLAST online platform for alignment analysis; relevant primer information is detailed in Table 1.6. Subsequently, following strict standard procedures, the specificity-verified sequences were sent to Sangon Biotech Co., Ltd. for primer synthesis. The lettuce Tubulin gene was selected as the internal reference gene.

[0114] Quantitative analysis was performed on a Bio-Rad Mini Option quantitative PCR instrument. Three biological replicates and three technical replicates were set up for each gene and internal control gene. Three biological replicate samples were prepared for each target gene and internal control gene, and three technical replicate assays were performed for each sample. The reaction system was prepared on ice.

[0115] Table 1.4 Reaction System Table

[0116]

[0117] To ensure the accuracy and reproducibility of the experimental results, the qRT-PCR reaction was performed on a LightCycler® 96 real-time quantitative PCR instrument (Roche Diagnostics, Germany).

[0118] Table 1.5 Specific thermal cycle parameters

[0119]

[0120] The 2⁻ΔΔCt method was used to analyze the quantitative data to assess the relative expression level of the target gene. Subsequently, the Tukey HSD multiple comparison test was used to assess the significance of differences between groups, with a statistical threshold set at p < 0.05. Finally, the results were presented as bar charts using GraphPad Prism software.

[0121] Table 1.6 LsRbcS gene qPCR primer sequences

[0122]

[0123] 1.3 Results and Analysis

[0124] 1.3.1 Physicochemical properties of lettuce RbcS family genes

[0125] Seven RbcS gene family members were identified in the lettuce genome. The coding sequences (CDS) of these family members range from 534 bp to 546 bp in length, encoding 177 to 181 amino acids. Their protein sizes are relatively conserved during evolution. The predicted relative molecular masses of the proteins range from 19766.88 to 20359.48 Da, with little variation. The calculated theoretical isoelectric point (pI) values ​​for each member range from 8.77 to 9.07, showing an overall alkaline characteristic. Subcellular localization predictions indicate that LsRbcS proteins are all located in chloroplasts (Table 1.7).

[0126] Table 1.7 Information on LsRbcS gene family members

[0127]

[0128] 1.3.2 Phylogenetic relationship of the RbcS gene in lettuce

[0129] Phylogenetic trees constructed from the RbcS amino acid sequences of Arabidopsis thaliana, lettuce, sunflower, tobacco, and maize show that members of the lettuce LsRbcS family (marked with purple dots) and some members of the sunflower HanRbcS family (marked with orange squares) are mixed in one subgroup, indicating a close evolutionary relationship. Meanwhile, Arabidopsis AtRbcS (marked with green inverted triangles), maize ZmRbcS (marked with blue checkmarks), and tobacco NtRbcS (marked with red pentagrams) each form a species-specific branch. In addition, another branch mixes RbcS members from sunflower, maize, tomato, and tobacco. Figure 1 ).

[0130] 1.3.2.1 Gene structure and protein motif analysis of lettuce RbcS

[0131] Analysis of the gene structure, domains, and conserved motifs of lettuce LsRbcS revealed the following results: Figure 2All seven LsRbcS members contain a typical RbcS domain (PLN02289), belonging to the Rubisco small subunit family, validating the accuracy of gene annotation at the protein functional structure level. Conserved motif analysis showed that all LsRbcS members contain the core motifs Motif1, Motif2, Motif3, Motif4, and Motif5, with a relatively consistent motif order; only LsRbcS2 additionally contains Motif6 and Motif8, suggesting that this member may exhibit unique functional differentiation. Domain analysis revealed that the RbcS domain (PLN02289) is located in the central region of all member protein sequences, indicating a relatively conserved position. Gene structure analysis revealed significant diversity in the LsRbcS family: except for LsRbcS2 and LsRbcS3, the other five members (LsRbcS1 / 4 / 5 / 6 / 7) all contain three CDS regions and two introns; while LsRbcS2 has three CDS regions, its introns are significantly longer, and LsRbcS3 contains only one CDS region with no obvious intron structure. Overall, conserved motifs and RbcS domains are strictly distributed within the CDS regions, and their arrangement is highly consistent across family members, reflecting the stability of the core functional structure of the LsRbcS family during evolution. Furthermore, variations in gene structure provide a basis for functional differentiation among family members.

[0132] 1.3.2.2 Chromosome distribution and collinearity analysis of lettuce RbcS family members

[0133] Genome-wide collinearity analysis showed that the seven LsRbcS genes were distributed on chromosomes 2 and 4. Figure 3 The LsRbcS family members are unevenly distributed on chromosomes. LsRbcS1, LsRbcS4, LsRbcS5, LsRbcS6, and LsRbcS7 are all located on chromosome 2, forming clusters in regions of approximately 154 Mb and 156 Mb. The other two members are located on chromosome 4. Our analysis of RbcS gene family replication events in lettuce revealed a pair of collinear homologous genes: RbcS2 on chromosome 4 and the RbcS gene cluster on chromosome 2. Figure 4 ).

[0134] By constructing an interspecific collinearity map, collinearity analysis showed that ( Figure 5 There are four pairs of homologous genes between lettuce and Arabidopsis thaliana, and six pairs between lettuce and sunflower. These homologous relationships mainly involve two collinear LsRbcS gene regions on chromosomes 2 and 4 of lettuce. The relatively few collinear relationships between lettuce and Arabidopsis thaliana suggest that the RbcS family of lettuce and sunflower, both belonging to the Asteraceae family, has a more closely related evolutionary history.

[0135] 1.3.3 Three-dimensional structure prediction of lettuce RbcS protein

[0136] The three-dimensional structure prediction results of lettuce RbcS protein showed that all seven members had similar folding patterns, and the secondary structure was mainly composed of random coils, α-helices, and β-sheets. Figure 6 The N-terminal domains of each member are highly conserved, assembled into a compact domain by α-helices and β-folds, containing a potential metal bonding site, and exhibiting a highly consistent spatial arrangement of secondary structural elements. In contrast, the C-terminal regions are primarily composed of random curls and loops, exhibiting differences in length and flexibility; for example, the C-terminus of LsRbcS3 forms additional local folds. This diversity in C-terminal structures may be the structural basis for the functional differentiation among family members.

[0137] 1.3.4 Analysis of cis-regulatory elements in the lettuce RbcS gene

[0138] Cis-acting elements, as specific recognition sites for transcriptional regulators, play a central role in gene transcriptional regulation. Analysis of cis-elements in the 1500 bp promoter region upstream of the start codon of the LsRbcS gene revealed that these elements can be categorized into three main types: elements involved in the light signal response (such as G-box, Box4, GATA-motif, TCT-motif, AE-box, etc.), elements mediating hormone responses (including ABRE, CGTCA-motif, ERE, GARE-motif, etc.), and elements related to the regulation of growth and development. Figure 7 In addition, several regulatory factors involved in the response to adversity stress were identified, such as MYB, MYC, and ARE.

[0139] 1.3.5 Expression dynamics of lettuce RbcS family members under different abiotic stresses

[0140] 1.3.5.1 Expression response of lettuce RbcS family members under salt and drought stress

[0141] Under salt and drought stress, the expression of all LsRbcS family members in *Lettuce sibiricum* showed a downregulation trend. A significant decrease occurred after 6 hours of treatment, and the expression continued to decline with increasing treatment time. Figure 8 , Figure 9 ).

[0142] 1.3.5.2 Expressive Responses of Lettuce RbcS Family Members Under Dark Coercion

[0143] Under darkness stress, most members of the LsRbcS family in *Lettuce arvense* showed a significant decrease in expression after 6 hours of treatment, and this decrease continued with prolonged treatment. Upon restoration of light, all family members showed some recovery in expression. Figure 10 ).

[0144] 1.3.6 Differences in the expression of RbcS family members in lettuce varieties with different biomass

[0145] 1.3.6.1 Analysis of differences in biomass and photosynthetic properties between the two varieties

[0146] Fresh weight, dry weight and leaf area of ​​two lettuce varieties (Yingxia Pointed Leaf Lettuce and Lola) were measured. Yingxia Pointed Leaf Lettuce was significantly higher than Lola in all three indicators, making it suitable as a representative material for subsequent analysis of the "high biomass" phenotype (Table 1.8).

[0147] Table 1.8 Comparison of biomass between two lettuce varieties

[0148]

[0149] Note: Different lowercase letters in the same line indicate significant differences between the two varieties (P<0.05); fresh weight and dry weight are in g / plant, and leaf area is in cm² / plant.

[0150] 1.3.6.2 Expression differences of lettuce RbcS family members among varieties

[0151] Transcriptome data analysis was performed on two cultivars, Yingxia Pointed Leaf Lettuce and Lola, which showed significant differences in dry weight, fresh weight, and leaf area. The results showed that the expression levels of LsRbcS2 and LsRbcS6 in both varieties were significantly higher than those in other members of the Lola family. Specifically, the expression levels of LsRbcS2 and LsRbcS6 in Yingxia were approximately 2.3 times and 3.8 times higher than those in Lola, respectively. Figure 11 ).

[0152] 1.3.6.3 Correlation analysis between expression levels and leaf biomass traits

[0153] Correlation analysis of the expression levels of LsRbcS2 and LsRbcS6 with the dry weight, fresh weight, and leaf area data of the two cultivars revealed highly significant positive correlations between the expression levels of both genes and the three biomass traits (P < 0.001). Specifically, the correlation coefficients between LsRbcS2 and leaf area, fresh weight, and dry weight were all 0.96, and LsRbcS6 also showed highly consistent strong correlations, at 0.96, 0.94, and 0.95, respectively. Figure 12 ).

[0154] This study is the first to discover that LsKN1 can bind to the key photosynthetic gene LsRbcS6 and negatively regulate its expression, thereby affecting biomass accumulation. Plants need to maintain a balance between morphogenesis and photosynthetic productivity during growth and development. On the one hand, leaf morphogenesis (such as curling and serration) requires energy and carbon skeleton consumption; on the other hand, the accumulation of photosynthetic products also depends on the photosynthetic system. Rubisco, as the rate-limiting enzyme in photosynthetic carbon assimilation, has its small subunit encoding gene RbcS, whose expression level directly determines the reserve of the Rubisco holoenzyme, thus affecting photosynthetic efficiency. The expression level of RbcS is positively correlated with the Rubisco holoenzyme content and CO2 fixation efficiency. Moderate upregulation of RbcS can increase the photosynthetic rate, but overexpression may lead to excessive allocation of nitrogen resources to the photosynthetic apparatus, which in turn inhibits leaf expansion and plant growth. LsKN1, as a transcription factor member of the KNOX family, has been shown to promote cytokinin synthesis and inhibit gibberellin synthesis, thereby regulating leaf curling, heading, and palmate notching. In the regulation of bolting in lettuce, LsKN1 directly binds to the gibberellin synthesis gene LsGA20ox1 and inhibits its transcription, while activating the expression of the gibberellin signaling inhibitor LsRGA1. This dual inhibition of the gibberellin pathway delays bolting and the plant's transition from vegetative to reproductive growth.

[0155] Example 2: Functional verification and interaction analysis of key genes in the lettuce RbcS gene family

[0156] 2.1 Experimental Materials

[0157] 2.1.1 Plant Samples and Culture

[0158] The plant materials used in this study included cultivated lettuce and Arabidopsis thaliana. *Lettuce simonii* was used in the VIGS experiment, and *Lettuce laurel* seeds were purchased from Sichuan Zhongdu Gaoke Seed Industry Co., Ltd. and Guangzhou Haomei Horticulture Co., Ltd. KN1-overexpressing lettuce seeds were kindly provided by Professor An Guanghui of Henan Agricultural University. The transgenic plants used for Arabidopsis thaliana genetic transformation were ATRbcS2B (AT5G38420) and STM (AT1G62360) gene knockout Arabidopsis thaliana, both purchased from Arashare.

[0159] Lettuce cultivation: Select plump lettuce seeds, soak them in clean water, and germinate them in a light-treated incubator with the following environmental parameters: 16 h light / 8 h darkness, constant temperature of 22℃, and humidity of 50%. Once the radicle begins to appear (showing white tips), transplant them into sterilized seedling substrate (provided by Zhengzhou Jinshiji Horticulture Materials Co., Ltd.). Cultivate in the same light-treated incubator as described above, watering regularly daily to maintain normal plant growth. VIGS experiment: When lettuce seedlings reach the five-leaf stage, select uniformly growing and robust plants as experimental materials. Randomly divide the plants into a blank control group, a negative control group, and an experimental group, with 30 plants in each group. After treatment, images of representative plants from each group are collected to record phenotypic changes.

[0160] Arabidopsis thaliana culture: After sterilization, Arabidopsis thaliana seeds were sown on sterile MS medium and cultured in a light-controlled incubator for 7 days. The seedlings were then transplanted into sterile potting soil for genetic transformation experiments.

[0161] 2.1.2 Test reagents and equipment

[0162] Experimental reagents: Total RNA was extracted from plant materials using the FastPure® Universal Plant Total RNA Isolation Kit from Nanjing Vazyme Biotech. Reverse transcription was performed using the HiScript® III All-in-one RT SuperMix Perfect for qPCR premix from Nanjing Vazyme Biotech. Real-time quantitative PCR (qRT-PCR) was performed using the CWBIO UltraSYBR Mixture (LowROX) reaction system from Beijing Kangwei Century Biotechnology. Conventional PCR amplification was performed using Vazyme's 2×Phanta Max Master Mix (DyePlus) high-fidelity enzyme premix, and the products were purified using Vazyme's FastPure Gel DNA Extraction Mini Kit. The purified DNA fragments were ligated to the T vector using Vazyme's 5 min TA / Blunt-Zero Cloning Kit, and plasmid extraction was performed using Vazyme's FastPure® Plasmid Mini Kit. Cloning was performed using the Vazyme ClonExpress® II One Step Cloning Kit for seamless ligation. Yeast single-hybrid interaction analysis was performed using the Y2HGold-GAL4 system yeast two-hybrid kit from Beijing Coolbio Technology Co., Ltd. LB liquid and solid culture medium powder required for bacterial culture were purchased from Solarbio. Restriction endonucleases used, including BamHI, XbaI, NcoI, SmaI, and EcoRI, were all purchased from Thermo Fisher Scientific. Other basic chemical reagents, such as 75% ethanol and deionized water (ddH2O), were of commonly used specifications.

[0163] Equipment: -80℃, -20℃ and 4℃ refrigerators; sample culture in an intelligent light-illuminated culture room; aseptic operations were performed in a laminar flow hood; solution preparation and processing involved a pure water system, ice maker, water bath, micropipette, centrifuge and high temperature autoclave; nucleic acid amplification and quantitative detection were performed using a PCR instrument and a Roche LightCycler® 96 real-time PCR system, respectively; electrophoresis analysis was performed using an electrophoresis instrument and a gel imaging system; nucleic acid concentration and purity were determined using a Thermo Fisher Scientific NanoDrop 2000 micro spectrophotometer.

[0164] 2.1.3 Strains and Plasmids

[0165] Information on the strains and plasmids used in the experiment is shown in the table.

[0166] Table 2.1 Information on strains and plasmids

[0167]

[0168] 2.1.4 Culture medium preparation

[0169] Both LB liquid and LB solid culture media used in this experiment were prepared according to the following methods:

[0170] LB solid culture medium preparation steps (1 L):

[0171] (1) Weigh 40 g of LB solid culture medium powder (containing agar), add it to 950 mL of deionized water, and stir to dissolve.

[0172] (2) Make up to a total volume of 1 L with deionized water.

[0173] (3) Adjust the pH to 7.0 with 5 mol / L NaOH solution.

[0174] (4) Sterilize at 121℃ with high pressure steam for 20 minutes.

[0175] (5) Cool the sterilized culture medium in a 55°C water bath.

[0176] (6) In a clean bench, add the required antibiotics to the cooled culture medium and mix thoroughly.

[0177] (7) Pour the culture medium into sterile petri dishes, about 10 mL per dish.

[0178] (8) Let it stand in the clean bench for 10-15 minutes until it solidifies.

[0179] (9) Seal the petri dish with sealing film, store it upside down in a refrigerator at 4°C, and use it within one month.

[0180] 2.2 Experimental Methods

[0181] 2.2.1 Construction of Recombinant Vector

[0182] 2.2.1.1 Full-length cloning of the target gene

[0183] Primer design and synthesis: Based on the coding sequence information (CDS) of LsRbcS2 and LsRbcS6, specific primers for amplifying their complete open reading frames were designed using snapgene software. Subsequently, the product specificity of the designed primers was evaluated using the NCBI online tool Primer-BLAST. The confirmed primers were synthesized by Zhengzhou Branch of Sangon Biotech Co., Ltd., and their nucleotide sequences are as follows:

[0184] Table 2.2 Primer sequences for LsRbcS6 and LsRbcS2 gene cloning

[0185]

[0186] Total RNA was extracted from leaves of the summer-blooming pointed-leaf lettuce variety and used as a template for reverse transcription to synthesize cDNA. The cDNA was then amplified using 2×Phanta Max Master Mix (Dye Plus) high-fidelity DNA polymerase premix (Nanjing Novizan).

[0187] Table 2.3 Specific Reaction System Table

[0188]

[0189] After amplification, the product was placed in a 1% agarose gel and electrophoresed at a constant voltage of 120 V for about 25 minutes. The bands were then observed and recorded using a gel imaging system.

[0190] 2.2.1.2 Purification of the target fragment

[0191] The amplification products were gel-harvested and purified using the FastPure Gel DNA Extraction Mini Kit (DC301, Nanjing Novizan) according to its instructions. The concentration and purity of the obtained products were then determined using a micro spectrophotometer and stored at -20°C for subsequent experiments.

[0192] 2.2.1.3 Target Fragment Fragment Ligation with T-Vector

[0193] The purified PCR product was ligated to the T vector using the 5 min TA / Blunt-Zero Cloning Kit (C601, Nanjing Novizan). The optimal amount of insert fragment was determined based on its length, calculated using the formula: Optimal amount (ng) = 0.05 × number of base pairs in the insert fragment. After preparing the ligation reaction system, the reaction tubes were placed on ice to maintain enzyme activity and ensure reaction efficiency.

[0194] 2.2.1.4 Transformation of Escherichia coli

[0195] Mix 1 µL of the ligation product with 50 µL of competent cells, incubate on ice for 30 min, then heat shock at 42 ˚C for 60 s, followed by an ice incubation for 2 min. Add 500 µL of antibiotic-free LB medium and revive at 37 ˚C, 200 rpm for 1 h. Simultaneously, preheat LB plates containing the appropriate antibiotic at 37 ˚C. Centrifuge the revival solution at 5000 rpm for 5 min, discard a portion of the supernatant (approximately 300 µL), and resuspend the cells. Spread 100 µL onto LB plates containing ampicillin and incubate upside down at 37 ˚C for 16 h. Pick a single colony and transfer it to 4 mL of LB liquid medium containing ampicillin, incubate at 37 ˚C, 200 rpm with shaking for 16 h, for subsequent molecular identification.

[0196] 2.2.1.5 Colony PCR

[0197] To screen for positive clones, 2 µL of the overnight culture from 2.2.1.4 was used as a PCR template and added to the pre-mixed PCR reaction system.

[0198] Table 2.4 Specific Reaction System

[0199]

[0200] After amplification, the products were analyzed by 1% agarose gel electrophoresis (conditions: 120 V, 20 minutes), and the bands were observed under a gel imaging system. PCR products containing bands of the expected size were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing verification.

[0201] 2.2.1.6 Plasmid Extraction

[0202] The remaining bacterial culture of the sequenced positive clone was inoculated into 10 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C with shaking at 200 rpm. Plasmids were extracted according to the FastPure® Plasmid Maxi Kit (DC201-01, Nanjing Novizan) instructions. The purified LsRbcS2 and LsRbcS6 recombinant plasmids were stored at -20°C for later use.

[0203] 2.2.2 Subcellular localization of LsRbcS2 and LsRbcS6

[0204] 2.2.2.1 Primer Design

[0205] Based on the CDS sequences of LsRbcS2 and LsRbcS6, BamHI and XbaI restriction sites were designed. Specific primers were designed based on the 15 bp before the BamHI restriction site and the 15 bp after the XbaI restriction site in the vector plasmid pCAMBIA1300-GFP. The primer sequences were synthesized by Zhengzhou Branch of Sangon Biotech Co., Ltd.

[0206] Table 2.5 Primer sequences for cloning the LsRbcS2 and LsRbcS6 genes

[0207]

[0208] 2.2.2.2 Extraction of p1300-GFP empty vector

[0209] The pCAMBIA1300-GFP *E. coli* strain, stored at -80°C, was amplified and activated. The bacterial culture was then inoculated into 5 mL of ampicillin LB broth and cultured overnight at 37°C with shaking at 200 rpm. Subsequently, the cultured bacterial cells were collected, and plasmid extraction was performed strictly according to the FastPure® Plasmid Maxi Kit method.

[0210] 2.2.2.3 Linearized Carrier

[0211] Thaw 1 µg of pCAMBIA1300-GFP plasmid on ice and prepare the double enzyme digestion reaction system on ice.

[0212] Table 2.6 Reaction System

[0213]

[0214] Gently blow the prepared system until it is evenly mixed, and incubate at 37°C for 15 minutes and at 65°C for 20 minutes.

[0215] 2.2.2.4 Seamless Cloning

[0216] Seamless cloning was performed using the ClonExpress® II One Step Cloning Kit. The target fragment was ligated to the double-digested linearized vector according to the manufacturer's instructions. Single colonies were picked and incubated overnight at 37°C with shaking at 200 rpm in LB broth containing 3 mL of kanamycin. Colony PCR amplification was performed using the bacterial culture as a template. PCR products from positive clones with correct band sizes were sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing verification.

[0217] The LsRbcS2, LsRbcS6, and LsKN1 gene fragments were inserted into the p1300 vector and recombined into p1300-GFP-LsRbcS2 and p1300-GFP-LsRbcS6, respectively. These fragments were then transformed into clonal competent cells DH5α, plated on LB medium containing kanamycin, and cultured at 37°C for 16 h.

[0218] Single colonies were picked from plates cultured for 16 h and placed in 4 mL of liquid LB medium supplemented with kanamycin. The culture was incubated at 37°C and 200 rpm for 16 h. Colony PCR was performed on the bacterial culture, and the products were detected by 1% agarose gel electrophoresis. The gel was run at 120 V for about 25 min, and the gel was analyzed using a gel imaging system.

[0219] PCR products were obtained from the Zhengzhou branch of Sangon Biotech Co., Ltd. After confirming the sequencing results were correct, p1300-GFP-LsRbcS2 and p1300-GFP-LsRbcS6 were extracted using method 2.2.1.6, and the plasmids were analyzed for concentration.

[0220] 2.2.2.5 Plasmid Extraction

[0221] Positive clones verified by sequencing were selected, and the remaining bacterial culture was inoculated into 10 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C and 200 rpm with shaking. Subsequently, high-purity plasmids were extracted from the culture according to the instructions of the FastPure® Plasmid MaxiKit (DC201-01, Nanjing Novizan). Finally, the purified target plasmid containing the target gene was stored at -20°C for later use.

[0222] 2.2.2.6 Agrobacterium-mediated transformation

[0223] The recombinant plasmid extracted in section 2.2.2.5 was introduced into Agrobacterium GV3101 competent cells via heat shock after its concentration was determined. The specific steps are as follows:

[0224] The competent cells, thawed on ice, were gently mixed with 100 ng–1 µg of plasmid DNA, incubated on ice for 5 min, and then flash-frozen in liquid nitrogen for 5 min. They were then heat-shocked in a 37 °C water bath for 5 min, followed by another 5 min on ice. 800 µL of antibiotic-free LB medium was added, and the cells were incubated at 28 °C and 200 rpm for 3 h. The resuscitation solution was centrifuged at 6000 rpm for 1 min, the supernatant was discarded, and the cells were resuspended and plated on LB agar plates containing rifampicin and kanamycin. The plates were incubated upside down at 28 °C for 3 days. Single colonies were picked and incubated in 3 mL of LB liquid medium containing the corresponding antibiotics, with shaking, at 28 °C and 200 rpm for 48 h. Colony PCR was performed using the bacterial culture as a template. PCR products from positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Strains with correct sequencing results were stored at -80 °C after adding glycerol.

[0225] 2.2.2.7 Agrobacterium treatment

[0226] The remaining bacterial culture from section 2.2.2.6 with correct sequencing results was placed in 20 mL of LB liquid medium containing kanamycin and rifampin and cultured at 28°C and 200 rpm until the logarithmic growth phase of Agrobacterium (OD600 = 0.5~0.6).

[0227] Collect bacterial cells by centrifugation at 5000 rpm for 10 min, and suspend the Agrobacterium cells in infection solution (containing 10 mM MgCl2, 10 mM MES, 150 µM acetylsylcholine, pH=5.6) until OD600=1.0. Let stand at room temperature for 2-3 h.

[0228] 2.2.2.8 Instantaneous transformation of tobacco

[0229] Correctly sequenced positive Agrobacterium bacteria were inoculated into 20 mL of LB liquid medium containing the appropriate antibiotic and cultured at 28°C with shaking until the logarithmic growth phase. The bacterial suspension was centrifuged at 5000 rpm for 10 min, and the bacterial resuspended in the infection solution (10 mmol·L⁻¹MgCl₂, 10 mmol·L⁻¹MES, 150 μmol·L⁻¹acetylsuccinone, pH 5.6). OD₂ was adjusted. 600 Up to version 1.0.

[0230] A small hole was gently punctured on the underside of a tobacco leaf using a sterile needle. Bacterial solution was then drawn up with a syringe (without the needle) and injected into the leaf abaxial surface, allowing the solution to penetrate the mesophyll tissue. The plants were then cultured in the dark for 12 hours after injection, and then transferred to normal culture conditions. Approximately 48 hours later, fluorescence signals were observed under a laser confocal microscope.

[0231] 2.2.3 Arabidopsis transformation

[0232] 2.2.3.1 Primer Design

[0233] Based on the CDS sequences of LsRbcS2, LsRbcS6, and LsKN1, XbaI and BamHI restriction sites were designed. Specific primers were designed based on the 15 bp before the XbaI restriction site and the 15 bp after the BamHI site in the vector plasmid p1300-35S-mCherry-Flag. Primer sequences were synthesized by Zhengzhou Branch of Sangon Biotech Co., Ltd.

[0234] Table 2.7 Primer sequences for cloning the LsRbcS2, LsRbcS6, and LsKN1 genes

[0235]

[0236] The PCR reaction system and procedure are the same as in 2.2.1.1. After the reaction, the product is purified using the method in 2.2.1.2.

[0237] 2.2.3.2 Linearized Carrier

[0238] The linearization method for the vector is the same as in 2.2.2.3. The p1300 plasmid is linearized using BamHI and XbaI restriction enzymes.

[0239] Thaw 1 µg of pCAMBIA1300-GFP plasmid on ice and prepare on ice.

[0240] Table 2.8 Configuration System

[0241]

[0242] Gently blow the prepared system until it is evenly mixed, and incubate at 37°C for 15 minutes and at 65°C for 20 minutes.

[0243] 2.2.3.3 Seamless Cloning

[0244] The seamless cloning method is the same as in 2.2.2.4. Connect the colonies according to the instructions. Transfer the selected single colonies to LB liquid medium containing 3 mL of kanamycin and incubate overnight at 37°C with shaking at 200 rpm. Perform colony PCR amplification using the bacterial culture as a template. Select PCR products with band sizes matching the expectations and have them sequenced and identified by Sangon Biotech (Shanghai) Co., Ltd.

[0245] The LsRbcS2, LsRbcS6, and LsKN1 gene fragments were inserted into the p1300 vector and recombined into p1300-LsRbcS2, p1300-LsRbcS6, and p1300-LsKN1. These fragments were then transformed into clonal competent cells DH5α, plated on LB medium containing kanamycin, and cultured at 37°C for 16 h.

[0246] Single colonies were picked from plates cultured for 16 h and placed in 4 mL of liquid LB medium supplemented with kanamycin. The culture was incubated at 37°C and 200 rpm for 16 h. Colony PCR was performed on the bacterial culture, and the products were detected by 1% agarose gel electrophoresis. The gel was run at 120 V for about 25 min, and the gel was analyzed using a gel imaging system.

[0247] PCR products were processed by Zhengzhou Branch of Sangon Biotech Co., Ltd. After confirming the sequencing results were correct, p1300-LsRbcS2, p1300-LsRbcS6, and p1300-LsKN1 plasmids were extracted using method 2.2.1.6 and their concentrations were measured.

[0248] 2.2.3.4 Agrobacterium-mediated transformation

[0249] The method for Agrobacterium transformation is the same as in 2.2.2.6. After determining its concentration, it is introduced into Agrobacterium GV3101 competent cells via heat shock. The specific steps are as follows:

[0250] The competent cells, thawed on ice, were gently mixed with 100 ng–1 µg of plasmid DNA, incubated on ice for 5 min, and then flash-frozen in liquid nitrogen for 5 min. They were then heat-shocked in a 37 °C water bath for 5 min, followed by another 5 min on ice. 800 µL of antibiotic-free LB medium was added, and the cells were incubated at 28 °C and 200 rpm for 3 h. The resuscitation solution was centrifuged at 6000 rpm for 1 min, the supernatant was discarded, and the cells were resuspended and plated on LB agar plates containing rifampicin and kanamycin. The plates were incubated upside down at 28 °C for 3 days. Single colonies were picked and incubated in 3 mL of LB liquid medium containing the corresponding antibiotics, with shaking, at 28 °C and 200 rpm for 48 h. Colony PCR was performed using the bacterial culture as a template. PCR products from positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Strains with correct sequencing results were stored at -80 °C after adding glycerol.

[0251] 2.2.3.5 Genetic transformation of Arabidopsis mutants

[0252] Transformation was performed using the Arabidopsis thaliana inflorescence immersion method, with the following specific steps:

[0253] Take 1 mL of Agrobacterium tumefaciens culture that has been verified as positive by PCR and inoculate it into 100 mL of LB liquid medium containing rifampicin (50 µg / mL) and kanamycin (50 µg / mL). Incubate at 28 °C and 200 rpm with shaking.

[0254] When the OD600 value of the bacterial culture reaches 0.4-0.6, centrifuge at 5000 rpm for 5 min to collect the bacterial cells. Discard the supernatant, resuspend the bacterial cells in 5% sucrose solution (containing 0.02% Silwet L-77), and mix thoroughly to prepare the infection solution.

[0255] The day before infection, the Arabidopsis plants to be transformed were thoroughly watered, and the formed pods and fully open flowers were cut off.

[0256] Pour the infection solution prepared in step 2 into a suitable container, and completely immerse the inflorescence of Arabidopsis thaliana in the liquid for 30-60 seconds.

[0257] After infection, the plants were shaded for 18-24 hours, and then cultured under normal light. Repeat infection can be performed 7-10 days after the initial infection to improve transformation efficiency.

[0258] After the Arabidopsis seeds mature, collect them, dry them, and store them in a refrigerator at 4°C.

[0259] 2.2.3.6 Resistance Screening and Molecular Identification in Transgenic Arabidopsis

[0260] Seeds of the T0 generation transgenic Arabidopsis mutants LsRbcS2, LsRbcS6, and LsKN1 were taken from a 4°C freezer and cultured on selection medium. The method is as follows:

[0261] Disinfect an appropriate amount of Arabidopsis thaliana seeds: Disinfect the seeds with 75% ethanol for 30 seconds, then rinse three times with sterile water; disinfect with 2% sodium hypochlorite solution for 60 seconds, then rinse three times with sterile water.

[0262] Suspend the seeds in fresh sterile water, and evenly spread a certain amount of Arabidopsis seed suspension onto MS solid medium (containing 50 mg / mL kanamycin). Seal with sealing film. Incubate for one week in a light incubator at 20°C for 16 / 8 h (light / dark).

[0263] Resistance selection culture: Sterilized seeds were resuspended in sterile water and inoculated onto MS solid medium supplemented with kanamycin (50 mg / L) in an incubator with alternating light and dark cycles of 16 hours and 8 hours at a constant temperature of 20°C for aseptic culture. The culture period was approximately 7 days.

[0264] Plants that maintained a normal green phenotype during the true leaf stage were selected as candidate positive transformants. The selected candidate plants were transplanted into a nutrient substrate for cultivation. When they grew to the stage of 6-8 true leaves, 2-3 tender true leaves were collected for genomic DNA extraction, and further molecular identification was performed using PCR technology.

[0265] Total RNA was extracted from transgenic Arabidopsis thaliana and reverse transcribed into cDNA. The RNA extraction and reverse transcription of cDNA were performed using the same methods as in 1.2.7. The expression of cDNA was then detected using cDNA as a template.

[0266] 2.2.4 Lettuce VIGS silent

[0267] 2.2.4.1 Primer Design

[0268] Based on the CDS sequences of LsRbcS2, LsRbcS6, and LsKN1, BamHI and XbaI restriction sites were designed. Specific primers were designed based on the 15 bp before the BamHI restriction site and the 15 bp after the XbaI site in the TRV2 vector plasmid. The primer sequences were synthesized by Zhengzhou Branch of Sangon Biotech Co., Ltd.

[0269] Table 2.9 Primer sequences for cloning the LsRbcS6, LsRbcS2, and LsKN1 genes.

[0270]

[0271] The PCR reaction system and procedure are the same as in 2.2.1.1. After the reaction, the product is purified using the method in 2.2.1.2.

[0272] 2.2.4.2 Linearized Carrier

[0273] The method for linearizing the vector is the same as in 2.2.2.3. The TRV2 plasmid is linearized using BamHI and XbaI restriction enzymes.

[0274] Thaw 1 µg of pCAMBIA1300-GFP plasmid on ice and prepare the double enzyme digestion reaction system on ice.

[0275] Table 2.10 Reaction System

[0276]

[0277] Gently blow the prepared system until it is evenly mixed, and incubate at 37°C for 15 minutes and at 65°C for 20 minutes.

[0278] 2.2.4.3 Seamless Cloning

[0279] The seamless cloning method is the same as in 2.2.2.4. Connect the colonies according to the instructions. Transfer the selected single colonies to LB liquid medium containing 3 mL of kanamycin and incubate overnight at 37°C with shaking at 200 rpm. Perform colony PCR amplification using the bacterial culture as a template. Select PCR products with band sizes matching the expectations and have them sequenced and identified by Sangon Biotech (Shanghai) Co., Ltd.

[0280] The LsRbcS2, LsRbcS6, and LsKN1 gene fragments were inserted into the p1300 vector and recombined into p1300-LsRbcS2, p1300-LsRbcS6, and p1300-LsKN1. These fragments were then transformed into clonal competent cells DH5α, plated on LB medium containing kanamycin, and cultured at 37°C for 16 h.

[0281] Single colonies were picked from plates cultured for 16 h and placed in 4 mL of liquid LB medium supplemented with kanamycin. The culture was incubated at 37°C and 200 rpm for 16 h. Colony PCR was performed on the bacterial culture, and the products were detected by 1% agarose gel electrophoresis. The gel was run at 120 V for about 25 min, and the gel was analyzed using a gel imaging system.

[0282] PCR products were processed by Zhengzhou Branch of Sangon Biotech Co., Ltd. After confirming the sequencing results were correct, pTRV2-LsRbcS2, pTRV2-LsRbcS6, and pTRV2-LsKN1 plasmids were extracted using method 2.2.1.6 and their concentrations were measured.

[0283] 2.2.4.4 Agrobacterium-mediated transformation

[0284] The method of Agrobacterium transformation is the same as in 2.2.2.6, where the recombinant vector plasmid is introduced into Agrobacterium.

[0285] 2.2.4.5 Instantaneous conversion of lettuce

[0286] Three treatment groups were set up, and the specific inoculation schemes were as follows: the wild-type control group plants were not treated in any way; the empty vector negative control group was inoculated with an equal volume of pTRV1 and pTRV2 empty vectors; the gene silencing treatment group was inoculated with a mixed bacterial solution prepared by mixing pTRV1 and recombinant vectors carrying the target fragment (TRV2-LsRbcS2, TRV2-LsRbcS6, TRV2-LsKN1) at a volume ratio of 1:1.

[0287] Take 1 mL of Agrobacterium tumefaciens bacterial culture that has been identified as positive by PCR, transfer it to LB liquid medium containing kanamycin (total volume 100 mL), and incubate it in a constant temperature shaker at 28℃ and shaken at 200 rpm.

[0288] The absorbance value (OD) of the bacterial culture at a wavelength of 600 nm 600 When the bacterial cells reach a growth rate of 0.6–0.8, centrifuge at 5000 rpm for 5 min to collect the precipitate. Discard the supernatant and resuspend the cells in a buffer solution containing 10 mmol·L⁻¹ MgCl₂, 10 mmol·L⁻¹ MES, and 20 μmol·L⁻¹ acetylsuccinone. Shake well to obtain the bacterial suspension for injection.

[0289] The day before infection, water the lettuce plants to be transformed thoroughly.

[0290] After the injection, the plants were shaded for 22-24 hours, and then normal light was restored for cultivation.

[0291] New leaves that grew after injection were randomly selected and identified by qPCR.

[0292] 2.2.4.6 Expression level detection and statistical analysis

[0293] Wild-type (WT) lettuce was used as a control, pTRV2 empty vector as a negative control, and pTRV2-LsRbcS6, pTRV2-LsRbcS2, and pTRV2-LsKN1 as silenced lines; each line had at least 3 biological replicates (n≥3). Total RNA was extracted from the leaves of each line, reverse transcribed into cDNA, and the relative expression levels of LsRbcS6, LsRbcS2, and LsKN1 were detected by qRT-PCR. Based on the results of Borowski et al., lettuce Tubulin (TUB) was used as an internal reference gene. The 2⁻ΔΔCt method was used for calculation. One-way ANOVA combined with Tukey HSD multiple comparison tests were used for comparisons among multiple groups (≥3 groups); independent samples t-tests were used for comparisons between two groups. Different lowercase letters indicate significant differences (p<0.05).

[0294] 2.2.4.7 Growth index determination

[0295] Two weeks after injection, three plants (n=8) from each strain were randomly selected, and the above-ground parts were collected for fresh weight, dry weight, and leaf area determination. Fresh weight: weighed immediately after collection using an electronic balance; Dry weight: samples were dried in a 60℃ oven to constant weight before weighing. Leaf area: all leaves from each plant were collected, and the area of ​​each leaf was measured using ImageJ. The total leaf area of ​​a single plant was calculated and divided by the number of leaves to obtain the average leaf area. All data are expressed as mean ± standard error (SE).

[0296] 2.2.4.8 Rubisco activity identification

[0297] To verify whether KN1 affects photosynthesis, the Rubisco enzyme content in pTRV2-LsKN1 plants was determined. The Rubisco activity was measured using an enzyme-linked immunosorbent assay (ELISA), and the specific steps are as follows:

[0298] 1) Crude enzyme extraction: Accurately weigh approximately 0.1 g of plant leaf sample, add 1 mL of extraction buffer, and grind thoroughly into a homogenate under ice bath conditions. Sonicate the homogenate (ice bath environment, 200 W power, 3 s working / 7 s intermittent, total processing time 1 min). Then centrifuge at 8000 g for 10 min at 4°C, collect the supernatant (Rubisco crude extract), and transfer it to an ice box for temporary storage until analysis.

[0299] 2) The reaction mixture must be freshly prepared before the experiment: Take reagent 2 and reagent 3 separately and mix thoroughly at a volume ratio of 1:1. Equilibrate to 25℃ and use immediately. Dissolve reagent 4 in 0.5 mL of reagent 1 before use.

[0300] 3) The microplate reader needs to be preheated (≥30 min) and the wavelength adjusted to 340 nm.

[0301] 4) Preparation of reaction system: Add 10 μL of supernatant, 10 μL of reagent IV and 180 μL of working solution to a 96-well plate in sequence, and mix immediately.

[0302] 5) Absorbance measurement: Immediately record the absorbance value A1 at 340 nm for 20 s and the absorbance value A2 at 5 min 20 s, and calculate ΔA = A1 - A2.

[0303] 6) Enzyme activity calculation: Rubisco activity is calculated based on the fresh weight of the sample: Activity (nmol / min / g fresh weight) = 1286×ΔA÷W.

[0304] 2.2.4.9 Chlorophyll content determination

[0305] To verify whether KN1 affects photosynthesis, the chlorophyll content of pTRV2-LsKN1 plants was determined. The specific method followed the instructions of the Solarbio Plant Chlorophyll Content Detection Kit (BC0990). Fresh plant leaves were taken, washed, dried, and the midrib removed. 0.1 g of the solution was accurately weighed and placed in a mortar. 1.5 mL of extraction buffer and a small amount of reagent I were added, and the mixture was thoroughly ground in the dark. The resulting solution was transferred to a 2 mL EP tube and extracted at room temperature in the dark for 30 min, inverting and mixing every 10 min. The mixture was then centrifuged at 4000 r / min for 5 min, and the supernatant was collected.

[0306] Using the extract as a blank control, the absorbance of the supernatant at wavelengths of 663 nm and 645 nm was measured using a UV-Vis spectrophotometer. The contents of chlorophyll a, chlorophyll b, and total chlorophyll were calculated according to the formula:

[0307] Chla(mg / gFW)=0.0015×(12.7×A663−2.69×A645)×V×F / W

[0308] Chlb(mg / gFW)=0.0015×(22.9×A645−4.68×A663)×V×F / W

[0309] TotalChl(mg / gFW)=0.0015×(20.21×A645+8.02×A663)×V×F / W

[0310] Where V is the total volume of the extract (mL), F is the dilution factor, and W is the fresh weight of the plant leaves (g). All experiments were performed in triplicate, and the results were averaged.

[0311] 2.2.5 Yeast mono- and hetero-interactions

[0312] 2.2.5.1 Gene Sequence Cloning

[0313] Primer design was performed using SnapGene software, and specificity was validated using NCBI's Primer-BLAST tool. The validated primers were synthesized at the Zhengzhou branch of Sangon Biotech Co., Ltd., and their sequences are as follows:

[0314] Table 2.11 Primer sequences for LsKN1 gene cloning

[0315]

[0316] The cloning, purification, T-vector ligation, E. coli transformation, colony PCR verification, and sequencing of the LsKN1 gene were all performed according to the method described in "2.2.1". After successful sequencing and confirmation of a positive clone, the plasmid was extracted and stored at -20℃ after amplification culture.

[0317] 2.2.5.2 Primer Design

[0318] Based on the promoter sequences of LsRbcS2 and LsRbcS6, primers containing EcoRI and SmaI restriction sites were designed, respectively. Specific primers were also designed based on the 15 bp before the EcoRI restriction site and the 15 bp after the SmaI restriction site in the pAbAi vector plasmid. Similarly, based on the CDS sequence of LsKN1, primers containing EcoRI and BamHI restriction sites were designed, respectively. Specific primers were also designed based on the 15 bp before the EcoRI restriction site and the 15 bp after the BamHI restriction site in the pGBKT7 vector plasmid. The primer sequences were synthesized by Sangon Biotech Co., Ltd., Zhengzhou Branch.

[0319] Table 2.12 Primer sequences for cloning LsKN1, LsRbcS2, and LsRbcS6 genes

[0320]

[0321] The reaction system and cycling parameters used for PCR amplification were consistent with those described in section 2.2.1.1 of this document. After the reaction, the amplification products were purified and recovered according to the method described in section 2.2.1.2 for subsequent cloning.

[0322] 2.2.5.3 Linearized Carrier

[0323] The pGADTT7 plasmid was double-digested with EcoRI and BamHI, while the pAbAi plasmid was double-digested with EcoI and SmaI. Since the optimal reaction temperatures for NcoI and SmaI restriction enzymes are different (37℃ and 30℃, respectively), a stepwise incubation strategy was adopted when digesting the pAbAi plasmid: the reaction was first carried out at 30℃ for 15 minutes, followed by a further reaction at 37℃ for 15 minutes to ensure that both enzymes could fully exert their cleavage activity. The specific enzyme digestion reaction system and operating procedures are as described in section "2.2.2.3" of this document.

[0324] Thaw 1 µg of pCAMBIA1300-GFP plasmid on ice and prepare the double enzyme digestion reaction system on ice.

[0325] Table 2.13 Reaction System

[0326]

[0327] Gently blow the prepared system until it is evenly mixed, and incubate at 37°C for 15 minutes and at 65°C for 20 minutes.

[0328] 2.2.5.4 Seamless Cloning

[0329] The seamless cloning method is the same as in 2.2.2.4. Connect the colonies according to the instructions. Transfer the selected single colonies to LB liquid medium containing 3 mL of kanamycin and incubate overnight at 37°C and 200 rpm with shaking. Perform colony PCR amplification using the bacterial culture as a template, selecting PCR products with band sizes matching the expectations. Sequencing was performed by Zhengzhou Branch of Sangon Biotech Co., Ltd.

[0330] The LsRbcS6 and LsKN1 gene fragments were inserted into the pAbAi and pADTT7 vectors, respectively, to recombinant them into pAbAi-LsRbcS6 and pGADTT7-LsKN1. These were then transformed into clonal competent cells DH5α. pAbAi-LsRbcS6 was plated on LB medium containing kanamycin, and pGADTT7-LsKN1 was plated on LB medium containing ampicillin. The cells were incubated at 37°C for 16 h.

[0331] Single colonies were picked from plates cultured for 16 h and placed in 4 mL of liquid LB medium containing the corresponding antibiotic. The culture was incubated at 37°C and 200 rpm for 16 h. Colony PCR was performed on the bacterial culture, and the products were detected by 1% agarose gel electrophoresis. The gel was run at 120 V for about 25 min, and the gel was analyzed using a gel imaging system.

[0332] The primer sequences for the PCR products were sequenced by the Zhengzhou branch of Sangon Biotech Co., Ltd. After confirming the sequencing results were correct, the pAbAi-LsRbcS6 and pGADTT7-LsKN1 plasmids were extracted using method 2.2.1.6 and their concentrations were measured.

[0333] 2.2.5.5 Co-conversion

[0334] First, competent yeast cells were prepared. The preparation of competent cells followed a modified version of the classic LiAc / SS Carrier DNA / PEG method. The specific steps are as follows:

[0335] Strain activation and pre-culture: The yeast strain was streaked onto YPDA solid medium (1% Yeast Extract, 2% Peptone, 2% Dextrose, 2% Agar) plates and incubated upside down at 30°C for 3 days until single colonies appeared (approximately 2–3 mm in diameter). Plump single colonies were picked and inoculated into 15 mL sterile centrifuge tubes containing 3 mL of YPDA liquid medium.

[0336] Primary culture: After incubating the above centrifuge tubes in a constant temperature shaker at 30°C and 250 rpm for 12 h, the bacterial culture was taken and the OD600 value was measured.

[0337] Secondary expansion culture: Take 5 μL of primary culture and transfer it to a 250 mL sterile Erlenmeyer flask containing 50 mL of fresh YPDA medium. Incubate at 30°C with shaking at 250 rpm for 16–20 h until OD (dose retardation). 600 The value is 0.15–0.3.

[0338] Bacterial cell collection and resuspension: Collect the bacterial culture into a 50 mL centrifuge tube, centrifuge at 700×g for 5 min at room temperature, and discard the supernatant. Add 100 mL of pre-cooled sterile YPDA medium and gently resuspend the bacterial cells.

[0339] Competent cell growth: Transfer the resuspended bacterial culture back to a sterile Erlenmeyer flask and continue to culture with shaking at 30°C and 250 rpm for 3-5 hours, closely monitoring the bacterial concentration until the OD600 value reaches the optimal transformation window of 0.4-0.5.

[0340] Washing and preparation: Aliquot the bacterial culture again and centrifuge at 700×g for 5 minutes to collect the bacterial cells. Gently wash and resuspend the bacterial cells sequentially with 30 mL of pre-chilled sterile distilled water and 1.5 mL of pre-chilled 1.1× TE / LiAc solution. Finally, resuspend the bacterial cells in an appropriate amount (e.g., 600 μL / tube) of pre-chilled 1.1× TE / LiAc solution to obtain highly efficient competent cells, which can be used immediately for transformation experiments or aliquoted and stored long-term in an ultra-low temperature freezer at -80°C.

[0341] The specific steps for yeast conversion are as follows:

[0342] Take a pre-cooled 1.5 mL centrifuge tube and add approximately 5 μL of linearized target plasmid and 5 μL of salmon sperm vector DNA (10 mg / mL) that has been denatured at 99°C for 5 minutes and then immediately placed on ice.

[0343] Add competent cells: Gently add 50 μL of freshly prepared or thawed yeast competent cell suspension to the tube above, and gently mix with a pipette.

[0344] Add PEG solution: Add 500 μL of pre-cooled PEG / LiAc solution (40% PEG-3350, 100 mM LiAc, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5), and gently vortex or invert the centrifuge tube to ensure thorough mixing.

[0345] Incubation: Place the mixture in a 30°C water bath and incubate for 30 minutes, gently shaking or mixing once every 10 minutes.

[0346] Add DMSO and heat shock: Add 20 µL of DMSO and mix well, then heat shock in a 42°C water bath for 15 min (mix well every 5 min).

[0347] Post-transformation treatment and resuscitation: After heat shock, the centrifuge tubes were immediately placed on ice for a short cooling period, centrifuged at 12,000 × g for 15 s at room temperature, the supernatant was discarded, and the cells were resuspended in 1 mL of fresh YPDA liquid culture medium.

[0348] Phenotypic expression: The resuspension was transferred to a new sterile centrifuge tube and placed in a shaker at 30°C with gentle shaking at approximately 25 rpm for 90 minutes to allow the transformants to express the resistance marker.

[0349] Spreading screening: After resuscitation, centrifuge the bacterial culture at 12,000 × g for 15 s, discard the supernatant, and resuspend in 1 mL of sterile 0.9% NaCl. Spread 200 µL onto the corresponding defective screening plate and incubate upside down at 30°C until transformed clones appear.

[0350] 2.2.6 KN1 overexpression in lettuce

[0351] 2.2.6.1 Plant materials and growth conditions

[0352] Lettuce seeds from the LsKN1 overexpressing line (T2 generation) and its wild-type control (WT) were kindly provided by Professor An Guanghui of Henan Agricultural University. The plants were sown in pots containing nutrient-rich soil and cultured in an artificial climate chamber under the following conditions: temperature 22±2℃, photoperiod of 16h light / 8h darkness, and relative humidity 60%–70%. Samples were taken 6 weeks after sowing for various index measurements.

[0353] 2.2.6.2 Measurement of growth indicators

[0354] Three plants (n=3) were randomly selected from each of the WT and LsKN1 overexpressing lettuce lines (OE), and the above-ground parts were collected for fresh weight, dry weight, and leaf area measurements. The specific methods are the same as in 2.2.4.7.

[0355] 2.2.6.3 Rubisco activity identification

[0356] The Rubisco enzyme content of LsKN1-OE plants was determined by enzyme labeling method, and the Rubisco activity was determined by enzyme labeling method, with the specific steps being the same as in 2.2.4.8.

[0357] 2.2.6.4 Chlorophyll content determination

[0358] The chlorophyll content of LsKN1-OE plants was determined, following the same procedure as in 2.2.4.9.

[0359] 2.3 Results and Analysis

[0360] Bioinformatics analysis, expression pattern analysis under different stresses, and differentially expressed genes analysis of the LsRbcS family members were used to screen LsRbcS2 and LsRbcS6 as major candidate genes regulating lettuce biomass. To further verify and analyze the molecular mechanisms of their high expression, yeast one-hybrid assays, Arabidopsis heterologous overexpression assays, and lettuce VIGS silencing assays were performed to systematically verify the functions of LsRbcS2 and LsRbcS6 and the transcriptional regulatory relationship between LsKN1 and LsRbcS6.

[0361] 2.3.1 Subcellular localization of LsRbcS2 and LsRbcS6

[0362] Subcellular localization experiments showed that the fluorescence signal in plants transformed with the empty vector was localized on chloroplasts. In tobacco plants transformed with the p1300-GFP-LsLsRbcS2 and p1300-GFP-LsLsRbcS6 recombinant vectors, the green fluorescence signal overlapped with the red fluorescence signal, indicating that both LsRbcS2 and LsRbcS6 are localized on chloroplasts, consistent with previous subcellular localization predictions (Table 1.7). Figure 13 ).

[0363] 2.3.2 Functional Validation of Lsrbcs2 and Lsrbcs6 Overexpression

[0364] This study first validated heterologous expression of the homologous RbcS gene deletion mutant (Atrbcs2b) of LsRbcS2 and LsRbcS6 in the model plant Arabidopsis thaliana. Overexpression vectors were constructed, and multiple independent T2 transgenic lines were obtained by transforming the Arabidopsis mutants into the T0 generation using the inflorescence immersion method. Subsequent phenotypic and physiological parameters were measured using the T2 generation lines. The aboveground dry weight, fresh weight, and leaf area of ​​LsRbcS2-OE and LsRbcS6-OE were significantly increased compared to Atrbcs2b (p<0.05). Figure 14 ).

[0365] 2.3.3 VIGS Silencing LsRbcS2 and LsRbcS6 Verification

[0366] 2.3.3.1 Determination of VIGS Silencing Efficiency in Lettuce

[0367] Twenty weeks after VIGS silencing treatment, the transcription levels of the target genes in the corresponding silencing lines were significantly lower than those in the wild type, indicating that the gene expression of LsRbcS6 and LsRbcS2 was effectively silenced. Figure 15 ).

[0368] 2.3.3.2 Determination of phenotype and physiological indicators of lettuce VIGS strains

[0369] Two weeks after VIGS treatment, plant phenotypes were analyzed. The results showed that compared to the wild-type control (WT), the growth vigor of the empty vector control (pTRV2) was slightly weakened, and leaf size, fresh weight, dry weight, and leaf area were all significantly reduced (p<0.05), indicating that the VIGS vector itself had an impact on plant growth. Furthermore, the aboveground fresh and dry weight and leaf area of ​​the pTRV2-LsRbcS2 and pTRV2-LsRbcS6 silenced plants were further significantly reduced. Figure 16 , 17 ).

[0370] 2.3.4 Interaction Analysis of LsRbcS6 and LsKN1

[0371] To investigate the molecular regulatory mechanism of the LsRbcS gene, we analyzed the published lettuce whole-genome ChIP-seq dataset. The results showed a significant enrichment of LsKN1 (Lactuca sativa KNOTTED 1) binding peaks in the LsRbcS6 promoter region, suggesting that LsKN1 may be a direct upstream regulator of LsRbcS6. Based on this, we validated their interaction using a yeast one-hybrid system.

[0372] 2.3.4.1 Screening for self-activation inhibition concentration

[0373] To ensure the reliability of yeast one-hybrid results, a plasmid containing the LsRbcS6 promoter fragment and the reporter gene was first transformed into yeast strain Y1HGold. The strain was then cultured and screened on SD / -Ura medium containing a concentration gradient of abaminibrioside AbA from 0 to 800 ng / mL to exclude the self-activation of the reporter gene promoter. The results showed that when the AbA concentration reached 500 ng / mL, no colonies grew on the medium at all. Figure 18 This confirms that the LsRbcS6 promoter fragment used does not have self-activation activity in this experimental system.

[0374] 2.3.4.2 Yeast one-hybrid verification of interactions

[0375] The results showed that at a concentration of 0 ng / mL AbA, the growth of colonies in each group was basically the same; however, at a concentration of 500 ng / mL AbA, only the yeast colonies transformed with pAbAi-LsRbcS6 and PGADT7-LsKN1 showed the same growth as the positive control. This indicates that LsKN1 can specifically bind to the promoter region of the LsRbcS6 gene, activating the expression of the reporter gene in the yeast system, which verifies the interaction between the two. Figure 19 ).

[0376] 2.3.5 Validation of LsKN1 overexpression

[0377] Phenotypic analysis was performed on the LsKN1 homologous mutant Atstm and LsKN1-OE overexpressing Arabidopsis lines. The results showed that the aboveground dry and fresh weight and leaf area of ​​all LsKN1-OE lines were significantly reduced. This indicates that overexpression of the LsKN1 gene significantly inhibited vegetative growth, leading to a marked limitation on aboveground biomass accumulation and leaf area. Figure 20 ).

[0378] 2.3.6 VIGS Silent LsKN1 Verification

[0379] 2.3.6.1 Determination of VIGS Silencing Efficiency in Lettuce

[0380] The transcription levels of each target gene in the corresponding lines were significantly lower than those in the wild type, confirming that LsKN1 was effectively silenced in the experimental treatment. Figure 21 ).

[0381] 2.3.6.2 Phenotypic, dry and fresh weight and leaf area analysis of lettuce VIGS lines

[0382] In LsKN1-silenced lines, pTRV2-LsKN1-silenced plants showed fewer leaf margin notches compared to the empty vector, consistent with known LsKN1 function. Figure 22 Fresh weight, dry weight, and leaf area were all significantly higher than those of the empty vector (p<0.05). This indicates that LsKN1 may have a negative regulatory effect on leaf size. Figure 23 ).

[0383] 2.3.6.3 Analysis of LsRbcS6 expression level in lettuce VIGS-LsKN1 line

[0384] To investigate the regulatory role of LsKN1 on LsRbcS6, the expression levels of LsRbcS6 in wild-type pTRV2 and pTRV2-LsKN1 were detected by qRT-PCR. Compared with the pTRV2 control, the expression level of LsRbcS6 in the pTRV2-LsKN1 silenced line was significantly increased. Figure 24 ).

[0385] 2.3.6.4 Analysis of Rubisco enzyme and chlorophyll content in the pTRV2-LsKN1 lettuce line

[0386] To investigate the effect of silencing LsKN1 on the photosynthetic function of lettuce, this study measured the Rubisco enzyme activity and chlorophyll content of plants injected with the empty pTRV2 vector and the pTRV2-LsKN1 silencing line. The results showed that, compared with the empty pTRV2 vector plant, the Rubisco enzyme activity of the pTRV2-LsKN1 silencing line was significantly increased (…). Figure 25 Meanwhile, its chlorophyll a, chlorophyll b, and total chlorophyll content were significantly higher than those of the empty vector plant ( Figure 26 ).

[0387] 2.3.7 Validation of LsKN1 overexpression in lettuce

[0388] 2.3.7.1 Phenotypic traits of overexpressed plants

[0389] Morphological observations showed significant differences between the LsKN1-OE strain and the wild-type (WT) plant in overall plant shape and leaf morphology. The LsKN1-OE plant had a compact rosette of leaves and slightly smaller leaves; while the wild-type plant exhibited flat, spreading rosette leaves, larger leaves, and more vigorous overall growth. Figure 27 ).

[0390] 2.3.7.2 Analysis of dry and fresh weight and leaf area of ​​overexpressing plants

[0391] To investigate the effects of LsKN1 overexpression on the vegetative growth of lettuce, this study measured the fresh weight, dry weight, and maximum rosette leaf area of ​​the aboveground parts of each line. The results showed that compared with the wild type (WT), all three growth indicators of the LsKN1-OE overexpression lines exhibited a significant decreasing trend (p<0.05). Specifically, the fresh weight of the OE lines decreased to approximately 84% of the wild type, the dry weight decreased to approximately 90%, and the maximum rosette leaf area decreased to approximately 88% of the wild type. Figure 28 This indicates that overexpression of LsKN1 inhibits biomass accumulation in the aboveground parts of the plant.

[0392] 2.3.7.3 Expression analysis of LsRbcS6 in the OE-LsKN1 lettuce line

[0393] The expression level of LsRbcS6 in wild-type and LsKN1-OE lettuce plants was detected by qRT-PCR. The results showed that the expression level of LsRbcS6 in the LsKN1-OE line was approximately 55% of that in the wild-type (p<0.05). Figure 29 This further supports the negative regulatory role of LsKN1 on photosynthesis-related genes.

[0394] 2.3.7.4 Analysis of Rubisco enzyme and chlorophyll content in the LsKN1-OE lettuce line

[0395] To investigate the photosynthetic characteristics of LsKN1 overexpression lines, this study measured the Rubisco enzyme activity and chlorophyll content of wild-type (WT) and LsKN1-OE lines. The results showed that, compared with WT, the Rubisco enzyme activity of the LsKN1-OE line was significantly reduced (…). Figure 30At the same time, the contents of chlorophyll a, chlorophyll b, and total chlorophyll all decreased significantly. Figure 31 The above results indicate that overexpression of LsKN1 reduces the activity of key photosynthetic enzymes and the content of photosynthetic pigments.

[0396] The results showed that the LsRbcS2 and LsRbcS6 genes positively regulate leaf growth and aboveground biomass accumulation in lettuce, and are major genes regulating biomass traits in lettuce. In rice, OsRbcS2 and OsRbcS4 were expressed at the highest levels in leaves, and co-silenced mutants showed decreased Rubisco content and yield. In tobacco, plants with silenced RbcS showed decreased Rubisco expression, leading to reduced photosynthetic rate, plant growth, and biomass accumulation. In Arabidopsis, RbcS1A and RbcS3B contributed to Rubisco accumulation, and double mutant plants showed significantly inhibited photosynthetic rate and growth. Therefore, there is a close relationship between RbcS expression levels and photosynthetic efficiency and biomass accumulation. LsRbcS2 and LsRbcS6 promote leaf growth and aboveground biomass accumulation, and have great application potential in high-yield lettuce breeding. They can be used as targets for marker-assisted selection, and specific molecular markers (such as SSR markers, InDel markers, or SNP markers) can be developed for the screening and identification of high-yield lettuce varieties. Alternatively, LsRbcS2 and LsRbcS6 can be combined with other identified high-yield, high-quality, and stress-resistant genes (such as nitrogen-efficient, drought-resistant, and disease-resistant genes) to create new lettuce germplasm with excellent comprehensive traits by integrating multiple superior genes into the same genetic background. Molecular markers developed based on major genes can provide more precise screening, significantly shorten the breeding cycle, and improve breeding efficiency. This overcomes the problems of traditional high-yield lettuce breeding, which mainly relies on phenotypic screening, resulting in long cycles, high subjectivity, and low selection efficiency.

[0397] This study found that LsKN1 can limit photosynthetic capacity to some extent by inhibiting the expression of LsRbcS6, thereby prioritizing the allocation of limited resources to morphogenesis. This regulatory model reflects a strategic trade-off in plant resource allocation: during the vegetative growth stage, morphogenesis may take precedence over further enhancement of photosynthetic capacity to avoid over-investment in photosynthetic structures and thus hindering the rapid expansion of leaf morphology. Similar mechanisms have been reported in the Arabidopsis KNOX family, such as KNAT1 / BREVIPEDICELLUS, which can participate in the coordination of leaf development and growth by regulating the expression of downstream target genes (such as KNAT2 / KNAT6 and NCED6 / 9).

[0398] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0399] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A nucleic acid molecule for regulating lettuce biomass, characterized in that, The nucleic acid molecule is a gene as shown in (a), (b), or (c) below: (a) The LsRbcS2 gene with a CDS sequence as shown in SEQ ID NO:3, or the LsRbcS6 gene with a CDS sequence as shown in SEQ ID NO:4; (b) Nucleic acid molecules encoding amino acid sequences as shown in SEQ ID NO:1 or SEQ ID NO:2; (c) A variant sequence that has at least 95% sequence identity with the nucleotide sequence defined in (a) or (b) and is derived from lettuce and encodes a protein with the function of the Rubisco small subunit.

2. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule of claim 1, and a promoter operatively linked upstream of the nucleic acid molecule.

3. A method for increasing the aboveground biomass of lettuce, characterized in that, include: The recombinant expression vector described in claim 2 is introduced into lettuce cells to obtain transgenic lettuce plants that overexpress the LsRbcS2 and / or LsRbcS6 genes, thereby increasing the fresh weight, dry weight, and leaf area of ​​lettuce leaves.

4. A method for increasing the aboveground biomass of lettuce, characterized in that, Inhibiting the expression of the endogenous LsKN1 gene or the activity of its encoded transcription factor in lettuce plants upregulates the expression of the LsRbcS6 gene, thereby increasing the Rubisco enzyme activity and chlorophyll content in lettuce leaves, and ultimately increasing the aboveground biomass. The nucleotide sequence of the LsKN1 gene is shown in SEQ ID NO:5, and the amino acid sequence encoding the transcription factor is shown in SEQ ID NO:

6.

5. The application of the nucleic acid molecule of claim 1 or the recombinant expression vector of claim 2 in the cultivation of high-yield lettuce varieties.

6. A method for screening high-yielding lettuce germplasm, characterized in that, Includes the following steps: (1) Total RNA was extracted from the lettuce leaves to be tested and cDNA was obtained by reverse transcription; (2) The expression levels of LsRbcS2 and / or LsRbcS6 genes in cDNA were detected by qRT-PCR using specific primers. (3) Using wild-type lettuce as a control, plants with significantly higher LsRbcS2 and / or LsRbcS6 gene expression levels than the control were selected as high-yield candidate germplasm.

7. The method according to claim 6, characterized in that, It also includes detecting the expression level of the LsKN1 gene and selecting plants with significantly lower LsKN1 expression levels than the control as high-yield candidate germplasm.

8. A functional module for regulating lettuce biomass, characterized in that, The functional module is composed of the LsKN1 gene and its encoded transcription factor, as well as the LsRbcS6 gene; the transcription factor encoded by the LsKN1 gene specifically binds to the promoter region of the LsRbcS6 gene and negatively regulates the expression of LsRbcS6. The promoter sequence of the LsRbcS6 gene is shown in SEQ ID NO:

7.

9. A functional module for regulating lettuce biomass according to claim 8, characterized in that, The negative regulation is achieved by inhibiting the expression of the LsKN1 gene or inhibiting the activity of its encoded transcription factor, thereby relieving the negative regulation of LsRbcS6 and upregulating the expression of LsRbcS6.

10. A functional module for regulating lettuce biomass according to claim 9, characterized in that, The methods for inhibiting LsKN1 gene expression include gene silencing, gene knockout, gene editing, RNA interference, antisense RNA, or artificial miRNA.

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