LsBOR5b gene and application thereof in regulating and marking bolting time of plant

Abstract

The invention discloses an LsBOR5b gene and application thereof in regulation and marking of plant bolting time, relates to the technical field of biology, and prolongs the bolting and flowering time of lettuce and increases the yield by overexpressing expression of an LsBOR5b homologous gene in a plant. The method has important theoretical research value and wide practical application prospect for cultivating lettuce plants with long bolting and flowering time. Meanwhile, a biosensor can be developed based on the expression specificity of a specific gene in the lettuce in a specific tissue and a development stage disclosed by the research; for example, a biosensor for detecting bolting signals is constructed on the basis of a gene LsBOR5b with obvious expression quantity change in the initial bolting stage, when the gene expression quantity reaches a certain threshold value, the sensor sends out a signal to remind that the lettuce is about to bolt, and harvesting, environment or nutrition supply condition adjustment, test data collection or other corresponding measures are carried out as required at the moment.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an LsBOR5b gene and an application thereof in regulating plant bolting time. Background Art

[0002] Cultivated lettuce (Lactuca sativa L.) belongs to the genus Lactuca L. of the Asteraceae family in taxonomy. It originated in the temperate zone of Asia and is also called lettuce. It has extremely high nutritional value and is rich in inorganic salts, vitamins, polyphenols and lutein. It is a popular edible cultivated vegetable in the world. It has a large cultivation area all over the world, especially in Asia, Europe and North America. According to the main edible part, lettuce can be divided into two types: leaf lettuce (butter lettuce, head lettuce, loose leaf lettuce, romaine lettuce) and lettuce type (stem lettuce). Lettuce is rich in nutrients and minerals needed by the human body and is one of the essential agricultural products consumed by the people. Common cultivated varieties in my country include lettuce, head lettuce, romaine lettuce, American fast lettuce, colorful lettuce, and Roman upright lettuce. According to FAO data in 2022, my country's total output, sown area and gross output value of lettuce and chicory vegetables ranked first in the world, with great economic value.

[0003] Bolting mainly refers to the phenomenon that the stems of vegetative clumping plants begin to elongate rapidly and the plants become taller as the flower buds differentiate, stimulated by environmental changes such as temperature and day length. Lettuce is a facultative long-day plant, and cultivated varieties show great differences in the speed of bolting and flowering. Very early-maturing varieties begin to bolt and bloom 7 weeks after planting under long-day conditions in a greenhouse, while very late-maturing varieties may bloom 4 months after planting. Studies have shown that the content of bitterness-related metabolites in lettuce increases rapidly before and after bolting, which has a great impact on the edible quality of stem lettuce and leaf lettuce, and the leaves are prone to rot (Assefa et al., 2019); in addition, lettuce is prone to premature bolting under heat stress, which seriously affects its yield and causes huge losses in production. Extending the vegetative growth period of lettuce is beneficial to leaf biomass accumulation and quality maintenance.

[0004] In recent years, research teams from Beijing Agricultural College, Beijing Academy of Agricultural and Forestry Sciences and other institutions have explored and functionally identified genes related to bolting of leaf lettuce, and found that genes such as LsARF3, LsRGL1, and LsSTPK have positive or negative regulatory effects on bolting of leaf lettuce (Wang Lu, 2019; Wang et al., 2021; Li et al., 2022), but research related to bolting of stem lettuce has not been reported internationally. The applicant used the latest genome data of cultivated lettuce and combined transcriptome data to conduct whole-genome identification of homologous genes related to flower bud differentiation, bolting and flowering time found in other plants, explored the LsBOR gene family related to bolting in stem lettuce, conducted bioinformatics analysis, and functionally identified key gene family members, establishing a new technical system for transgenic function verification of lettuce, which also provides new important genetic resources for extending the vegetative period of lettuce and increasing yield, and has certain application value and potential economic value. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an LsBOR5b gene and an application thereof in regulating and marking plant bolting time.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] A kit for regulating plant bolting time, the kit comprising molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to plant bolting time; the molecular biological elements may optionally comprise: an element combination for overexpressing the specific genes, and / or an element combination for inhibiting or reducing the expression of the specific genes, and / or an element combination for silencing the expression of the specific genes;

[0008] The specific gene is the LsBOR5b gene, its homologous gene or an equivalent gene having the same plant physiological function as the LsBOR5b gene.

[0009] A recombinant expression vector comprising the LsBOR5b gene.

[0010] A method for prolonging plant bolting time, comprising overexpressing the LsBOR5b homologous gene in the plant to prolong the plant bolting time, wherein the CDS sequence of the gene LsBOR5b is shown in SEQ ID NO:1.

[0011] A method for increasing plant yield comprises overexpressing the LsBOR5b homologous gene in the plant to prolong the plant bolting time, thereby increasing the yield.

[0012] The use of the LsBOR5b gene is used to regulate the bolting time of plants.

[0013] The kit according to claims 1-3, the method according to claim 6, the method according to claim 7, or the use according to claim 8, wherein the plant is lettuce.

[0014] The primer pair for amplifying the nucleotide sequence shown in SEQ ID NO:1 has the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3.

[0015] The invention discloses a genotype molecular marker of lettuce, wherein the genotype molecular marker is the LsBOR5b gene in the BOR gene family of lettuce.

[0016] The uses of the genotype molecular markers described in claim 8 include: ① performing data analysis and planting intervention on lettuce bolting by detecting the expression time and expression level of the genotype molecular marker, and the planting intervention includes harvesting on demand, adjusting the environment or nutrient supply conditions, collecting test data or other corresponding measures; ② screening, preliminary screening, identification, and auxiliary identification of lettuce germplasm resources by detecting the expression time and expression level of the genotype molecular marker; the operation method is: among the natural population of lettuce, lettuce plants from different geographical regions are collected to construct a primary natural population library of lettuce, and the initial screening of potential excellent germplasm resources is achieved by detecting the expression time and expression level and other expression characteristics of the genotype molecular marker.

[0017] According to the use according to claim 9, use ② specifically includes but is not limited to: ②-A, through the expression time analysis of the genotype molecular marker, two types of plant populations of early expression and late expression are screened out respectively, and two secondary lettuce natural population sub-banks T1 and T2 (Time group) are constructed accordingly; ②-B, through the expression quantity analysis of the genotype molecular marker, two types of plant populations of high expression and low expression are screened out respectively, and two secondary lettuce natural population sub-banks Q1 and Q2 (Quantity group) are constructed accordingly; the above two groups of four lettuce natural population sub-banks are used as the initial germplasm resource bank for subsequent lettuce breeding, and hybrid breeding or molecular breeding is carried out within or between sub-banks in T1, T2, Q1, and Q2 respectively to quickly construct superior germplasm.

[0018] A method for developing a lettuce genotype biosensor, using the expression specificity of specific genes in lettuce in specific tissues and developmental stages to develop a biosensor; including but not limited to: ① Based on the gene LsBOR5b whose expression level changes significantly in the early stage of bolting, a biosensor for detecting bolting signals is constructed. When the gene expression level reaches a certain threshold, the sensor sends a signal to remind the lettuce that it is about to bolt. At this time, harvesting, adjustment of environmental or nutrient supply conditions, collection of test data or other corresponding measures are carried out as needed; ② Referring to the equivalent method of ①, other types of biosensors are developed based on the development and tissue expression characteristics of other genes in the lettuce BOR gene family.

[0019] The lettuce genotype biosensor developed in claim 11.

[0020] The beneficial effect of adopting the above technical solution is that the theoretical research and experimental research of the research group of the present invention have confirmed that by overexpressing the expression of the target gene LsBOR5b in lettuce, the bolting time of lettuce can be extended and the yield can be increased, which has important theoretical research value and broad practical application prospects for cultivating lettuce plants with a long bolting time. At the same time, biosensors can also be developed based on the expression specificity of specific genes in lettuce in specific tissues and developmental stages revealed by this study; for example, based on the gene LsBOR5b whose expression level changes significantly in the early stage of bolting, a biosensor for detecting bolting signals is constructed. When the gene expression level reaches a certain threshold, the sensor sends a signal to remind the lettuce that it is about to bolt. At this time, harvesting, adjusting environmental or nutrient supply conditions, collecting test data or other corresponding measures are carried out as needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the gene structure of LsBOR in Example 2, wherein exons and introns are represented by yellow squares and black lines, respectively, and 5' and 3' untranslated regions (UTRs) are represented by blue boxes;

[0022] Figure 2 This is a schematic diagram of the conservative motif analysis of LsBOR in Example 2;

[0023] Figure 3 Schematic diagram of the amino acid sequence of each conserved motif in the lettuce BOR protein in Example 2, wherein the font size indicates the frequency of occurrence of each amino acid;

[0024] Figure 4 The distribution map of the lettuce BOR gene on the chromosome in Example 3;

[0025] Figure 5 This is the colinearity diagram of the lettuce BOR gene in Example 3. Note: the gray line represents the duplication event of all genes in lettuce, and the red line represents the colinear relationship within the LsBOR gene;

[0026] Figure 6 This is a diagram of the colinearity between lettuce, Arabidopsis, and sunflower in Example 3. Note: Different colors represent chromosomes of different species, and chromosome numbers are represented by numbers. The gray lines in the background represent the colinearity between lettuce and other plant genomes, while the red lines represent colinear BOR gene pairs. The black text represents the gene name of the LsBOR gene and the gene ID that has a colinear relationship with the LsBOR gene;

[0027] Figure 7 is the cis-acting element in the promoter of the lettuce BOR gene in Example 4;

[0028] Figure 8 The predicted three-dimensional structure of the lettuce BOR protein in Example 5;

[0029] Fig. 9 The expression profile of the lettuce BOR gene in different tissues and developmental stages in Example 6;

[0030] Fig.10 The expression profile of the lettuce BOR gene under different stress conditions in Example 6;

[0031] Fig.11 The electrophoresis patterns of PCR amplification of LsBOR5b gene and restriction enzyme digestion of recombinant plasmid in Example 7, wherein Figure A is the electrophoresis pattern of amplified product of target gene LsBOR5b, and Figure B is the electrophoresis pattern of PCR restriction enzyme digestion product of recombinant plasmid colony;

[0032] Fig.12 The electrophoresis diagrams of the results of the amplification of the lettuce LsBOR5b gene and the construction of the recombinant vector in Example 8, wherein A is the electrophoresis diagram of the LsBOR5b gene amplification, B is the colony PCR verification diagram of the recombinant plasmid (LsBOR5b), and C is the colony PCR electrophoresis diagram of the pCAMBIA1300-GFP-LsBOR5b recombinant vector;

[0033] Fig.13 This is the subcellular localization map of the pCAMBIA1300-GFP-LsBOR5b fusion protein in Example 8;

[0034] Fig.14 The electrophoresis patterns of the experiment related to LsBOR5b and LsFT genes in Example 9, wherein A is the electrophoresis pattern of the PCR amplification product of the lettuce LsFT gene, B is the electrophoresis pattern of the colony PCR of the pADTT7-LsFT recombinant vector, C is the electrophoresis pattern of the PCR amplification product of the lettuce LsBOR5b gene, and D is the electrophoresis pattern of the colony PCR of the pGBKT7-LsBOR5b recombinant vector;

[0035] Fig.15 This is a diagram showing the analysis of the yeast two-hybrid results of LsBOR5b and LsFT in Example 9;

[0036] Fig.16 The electrophoretic diagrams of the construction results of the pCAMBIA2300-LsBOR5b recombinant vector in Example 10, wherein A is the electrophoretic diagram of the PCR amplification product of the lettuce LsBOR5b gene, and B is the electrophoretic diagram of the colony PCR of the pCAMBIA2300-LsBOR5b recombinant vector;

[0037] Fig.17This is the electrophoresis of the PCR amplification product of the Agrobacterium colony transformed with the pCAMBIA2300-LsBOR5b recombinant vector in Example 10;

[0038] Fig.18 This is a diagram showing the PCR verification results of Arabidopsis thaliana overexpressing LsBOR5b in Example 10.

[0039] Fig.19 This is a graph showing the statistical results of the bolting time of wild-type Arabidopsis thaliana (WT), atbor5 knockout Arabidopsis thaliana (atbor5) and LsBOR5b overexpressing Arabidopsis thaliana (OE-LsBOR5b) in Example 10. DETAILED DESCRIPTION

[0040] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained by purchasing from the market. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0041] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or combinations thereof.

[0042] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0043] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0044] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0045] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1: Identification and physicochemical property analysis of lettuce BOR family genes

[0047] In this study, a total of 14 LsBOR (Lactuca sativa L.BoltingRegulation) genes were identified in lettuce, and their names are shown in Table 1 below. The results of physicochemical property analysis showed that the full length of CDS of LsBOR ranged from 821bp to 1333bp, the length of the encoded protein ranged from 273aa to 444aa, the relative molecular mass ranged from 30.54kDa to 50.10kDa, and the theoretical isoelectric point ranged from 4.71 to 8.63. Except for LsBOR5a, other LsBOR proteins were acidic. Subcellular localization prediction showed that most LsBORs were localized in the nucleus, LsBOR2a, LsBOR4, and LsBOR5b were localized in the cytoplasm, and LsBOR5a and LsBOR16b were localized in the chloroplast.

[0048] Table 1 LsBOR whole genome identification

[0049]

[0050]

[0051] Example 2: Gene structure and protein motif analysis of lettuce BOR

[0052] To explore the structural diversity of BOR genes, we used GSDS to map the exon and intron composition. In general, genes in the same group have similar numbers of exons and introns, and even intron phases, indicating that these genes share a conserved splicing pattern. Figure 1 As shown in the figure, the BOR genes in lettuce are divided into three groups, which is similar to the grouping of Arabidopsis and rice. Among the BOR genes in groups 1 and 2, LsBOR2a contains one exon, while the other genes have two exons. Among the BOR genes in group 3, except LsBOR9b which contains 5 exons, the other members contain 4 exons.

[0053] By analyzing the conserved motifs, we can obtain some information related to the evolution of the gene family. The conserved domains and motifs of the lettuce BOR protein were compared and analyzed using MEME software. Motif 1 to Motif 10 were arranged in ascending order according to the sequence E-value ( Figure 2 , Figure 3 ). The longest of these motifs is 45aa, and the shortest is 6aa. Motif 1 and Motif 2 are the most conserved motifs in all BOR proteins. Motif5 is only found in groups 1, 2, and some members of group 3. In addition, some Motifs only appear in a unique group and are shared by all members of the group, such as Motif4 in group 3. In addition, Motif8 is only present in 2 BOR proteins of group 3, Motif3 and Motif6 are only present in 3 BOR proteins of group 3, and Motif7 and Motif9 are only present in 4 BOR proteins of group 3.

[0054] Example 3. Chromosome distribution and colinearity analysis of BOR family members in lettuce

[0055] To analyze the location of BOR gene family members in the lettuce genome, we performed chromosome mapping and analyzed gene duplication events. According to the mapping results, 14 LsBOR genes were located on 7 different chromosomes, and 1 gene was located on MU039017 ( Figure 4 ). Whole genome duplication analysis is of great significance to the origin, evolution and genome expansion of species. Therefore, we analyzed the BOR gene family duplication events in lettuce to understand the causes of the LsBOR gene duplication events. The results showed that a total of 5 pairs of large fragment duplicated genes (LsBOR2a and LsBOR2b, LsBOR5a and LsBOR5b, LsBOR9a and LsBOR9b, LsBOR9b and LsBOR9c, LsBOR9a and LsBOR9c) were detected ( Figure 5 ). These results suggest that large segment duplication may be the main driving force for the expansion and evolution of BOR genes in the lettuce genome.

[0056] To understand the origin and evolutionary relationships of BOR, we further performed a colinearity analysis and compared the lettuce genome with the Arabidopsis and sunflower genomes. Figure 6 , there are 16 pairs of homologous genes between lettuce and sunflower, which is greater than the 9 pairs between lettuce and Arabidopsis. Among these genes, LsBOR2b, LsBOR9a, LsBOR9b, LsBOR9c, LsBOR13, LsBOR14a and LsBOR16b homologous genes were detected in all three species, LsBOR4, LsBOR5a, LsBOR5b and LsBOR16a homologous genes were only detected between lettuce and sunflower, and other genes were not detected in the duplicated region.

[0057] Through the chromosome distribution and colinearity analysis of lettuce BOR family members, the physical position and distribution pattern of genes in the genome can be clarified. Gene duplication events provide clues for studying gene amplification and evolutionary mechanisms, and colinearity analysis helps trace the origin and evolution of genes.

[0058] Example 4: Analysis of cis-acting elements of the lettuce BOR gene

[0059] Cis-acting elements are binding sites for transcriptional regulatory factors that regulate gene transcription. To investigate the potential function of the LsBOR gene, we analyzed the cis-acting elements at 1500 bp upstream of the LsBOR promoter and excluded elements with unknown functions and general transcriptional regulatory elements (Table 2, Figure 7 ). These cis-acting elements can be roughly divided into four categories, involving light response, hormone response, growth regulation and abiotic stress response. The components involved in light response include G-box, GATA-motif, Box4, TCT-motif, AE-box, GT1-motif, 3-AF1 binding site and MRE. Hormone-related cis-elements include TGACG-motif, CGTCA-motif and ABRE. In addition, there are multiple stress response factors such as LTR and MBS. These results show that most LsBOR genes have light-responsive elements, indicating that LsBOR genes may play a key role in the regulation of light responsiveness.

[0060] Cis-acting elements are key sites for gene transcription regulation. Analyzing their types and distribution can predict which external factors and signal pathways regulate the gene, and further infer the gene function.

[0061] Table 2 Cis-acting elements found in more than three LsBOR genes

[0062]

[0063] Example 5: Prediction of the three-dimensional structure of lettuce BOR protein

[0064] The three-dimensional structure of lettuce BOR protein was predicted using SWISS-MODEL, and the model with the highest QMQE value was selected to construct the protein three-dimensional structure model. Figure 8 ,The secondary structure of lettuce BOR family proteins is mainly composed of random coil, α-helix, and β-turn, and BOR proteins in the same group have similar structures.

[0065] Understanding the spatial structure of a protein helps us infer how it interacts with other molecules and its functional mechanism within the cell, providing a structural basis for subsequent functional verification.

[0066] Example 6: Expression analysis of lettuce BOR gene

[0067] (1) Analysis of the developmental and tissue expression characteristics of the BOR gene in lettuce

[0068] In order to study the tissue-specific expression pattern of BOR genes in lettuce, we analyzed the expression profiles of BOR genes in different tissues and developmental stages. LsBOR2a, LsBOR9a, and LsBOR11 were considered to be non-expressed because their expression levels were too low (cp value greater than 35 or not detected). Fig. 9 , other genes were expressed in all tested tissues and developmental stages of lettuce. Most BOR genes (LsBOR2b, LsBOR4, LsBOR5a, LsBOR5b, LsBOR13, LsBOR16a, LsBOR16b) were highly expressed in lettuce leaves, indicating that these genes may play an important role in the vegetative growth of lettuce. In the early-bolting variety Zixiahong lettuce, LsBOR5b and LsBOR14b were highly expressed in leaves and flowers, and LsBOR9b, LsBOR9c, and LsBOR14a were highly expressed in flowers. These genes may play an important role in the development of lettuce flowers. Fig. 9 In both lettuces, the expression level of LsBOR5a increased from the bolting to the flowering stage, the expression level of LsBOR5b increased from the early bolting to the late bolting stage, and the expression levels of LsBOR4, LsBOR9c, LsBOR13, and LsBOR14a increased from the late bolting to the flowering stage. In white-skinned pointed-leaf lettuce, the expression levels of LsBOR14b, LsBOR16a, and LsBOR16b increased from the early bolting to the late bolting stage.

[0069] Analysis of the expression characteristics of the lettuce BOR gene development and tissues showed that some genes were not expressed, most were highly expressed in leaves, and some had varying expression levels in flowers or bolting and flowering stages, indicating that they play different roles in vegetative growth and reproductive development. Determining the spatiotemporal expression patterns of genes during plant growth and development, and finding genes related to vegetative growth and flower development, will provide clues for studying their role in bolting and flowering.

[0070] (2) Analysis of expression characteristics of lettuce BOR gene under abiotic stress

[0071] In order to explore whether the lettuce BOR gene is involved in the response of lettuce to different abiotic stresses, we further analyzed the expression of the BOR gene in white-skinned pointed-leaf lettuce and purple-red lettuce exposed to salt and drought stress for different periods of time. The analysis results showed that the expression of the lettuce BOR gene changed under different abiotic stress conditions at different times ( Fig.10 ).

[0072] Lettuce BOR genes showed similar expression patterns under salt stress conditions. In the two lettuce varieties, the expression levels of LsBOR9b, LsBOR9c, LsBOR13, LsBOR14a, and LsBOR14b were significantly upregulated at 12 hours of salt stress treatment, and the expression level of LsBOR16a was significantly upregulated at 24 hours of salt stress treatment. LsBOR5b was weakly upregulated in white-skinned pointed-leaf lettuce at 6 hours of salt stress treatment, and was significantly upregulated in purple-red lettuce. Under drought stress conditions, lettuce BOR genes showed significant upregulation in white-skinned pointed-leaf lettuce at 12 hours or 24 hours. Under drought stress conditions, the expression of BOR gene in Zixiahong lettuce did not show strong / obvious up-regulation or down-regulation, LsBOR5b showed weak up-regulation at 6h and 24h, LsBOR16b showed weak up-regulation at 6h and 12h, and LsBOR2b and LsBOR16a showed weak up-regulation at 48h. These results indicate that BOR gene in lettuce may play a key role in the stress process of salt stress and drought stress in lettuce.

[0073] Example 7: Construction of LsBOR5b recombinant vector

[0074] Based on the expression analysis results and the chromosome location information of LsBOR family members was compared with the QTLs affecting the floral transition of lettuce, LsBOR5b, whose expression level increased from the early to late bolting stage of the two lettuces, was selected for subsequent gene function verification. The CDS sequence of gene LsBOR5b is shown in SEQ ID NO:1.

[0075] According to the full-length primers of LsBOR5b gene, the target gene fragment was amplified by PCR using lettuce cDNA as template. The nucleotide sequences of specific primers F1 and R1 required for amplification are shown in SEQ ID NO: 2 and SEQ ID NO: 3 respectively. The amplified band was consistent with the expectation, the fragment length was 1182bp, and the amplified band was single ( Fig.11 -A). After the PCR product is purified, it is connected to the T vector and transformed into E. coli. Single clone colonies are selected for PCR verification ( Fig.11 -B), send the single clones with the correct band size for sequencing, and shake the single clones with sequencing results consistent with the target gene, extract the plasmid, and add glycerol to preserve the bacteria.

[0076] Example 8: Subcellular localization of LsBOR5b

[0077] (1) Construction of pCAMBIA1300-GFP-LsBOR5b recombinant vector

[0078] Based on the cloning primers of LsBOR5b gene containing BamHI and XbaI restriction sites, PCR amplification was performed using the extracted LsBOR5b plasmid as a template ( Fig.12 -A). After the PCR product was purified, it was connected with the pCAMBIA1300-GFP linearized vector by seamless cloning and then transformed into Escherichia coli. Single clone colonies were selected for PCR verification ( Fig.12 -B), send the single clone with the correct band size for sequencing, shake the single clone with the sequencing result consistent with the target gene, extract the plasmid. The extracted pCAMBIA1300-GFP-LsBOR5b plasmid is introduced into Agrobacterium, and the single clone colony is selected for PCR verification ( Fig.12 -C) Single clones with the correct band size were sent for sequencing.

[0079] (2) Subcellular localization of LsBOR5b

[0080] The distribution of proteins in cells is closely related to their functions. Exploring the localization of proteins in cells is helpful to further understand their functions. To verify the subcellular localization of LsBOR5b in lettuce, we constructed a pCAMBIA1300-GFP-LsBOR5b fusion vector to transform tobacco leaves. Using tobacco leaves transformed with the empty vector pCAMBIA1300-GFP as a control, we observed the position of GFP fluorescence under a laser confocal microscope. The results showed that the fluorescence signal in the plants transformed with the empty vector was localized in the nucleus and cell membrane, while the fluorescence signal in the plants transformed with the pCAMBIA1300-GFP-LsBOR5b fusion vector was located in the endoplasmic reticulum ( Fig.13 ), indicating that LsBOR5b protein is mainly distributed in the endoplasmic reticulum.

[0081] Example 9: Interaction analysis between lettuce LsBOR5b and LsFT

[0082] (1) Recombinant plasmid construction

[0083] According to the full-length primers of LsFT gene, the target gene fragment was amplified by PCR using lettuce cDNA as template. The amplified band was consistent with the expectation, the fragment length was 528bp, and the amplified band was single ( Fig.14 -A). After the PCR product is purified, it is connected to the T vector and transferred into E. coli. The single clone colony is selected for PCR verification. The single clone with the correct band size is sent for sequencing. The single clone with the sequencing result consistent with the target gene is shaken and the plasmid is extracted. Using the plasmid as a template, PCR amplification is performed with cloning primers containing EcoRI and BamHI restriction sites. After purification, it is seamlessly cloned with the pADTT7 linearized vector and transferred into E. coli. The single clone colony is selected for PCR verification ( Fig.14 -B), send the single clone with the correct band size for sequencing, shake the single clone with the sequencing result consistent with the target gene, extract the plasmid. Use LsBOR5b recombinant vector as template, use cloning primers containing NcoI and SmaI restriction sites, PCR amplification ( Fig.14-C), and then transformed into E. coli after seamless cloning with the pGBKT7 linearized vector, and single clone colonies were selected for PCR verification ( Fig.14 -D), send the single clone with the correct band size for sequencing, and shake the single clone with the sequencing result consistent with the target gene to extract the plasmid.

[0084] (2) Yeast two-hybrid analysis

[0085] pADTT7-LsFT and pGBKT7-LsBOR5b were co-transformed into Y2HGold competent cells, and single clones were picked for interaction detection. The strain co-transformed with pGBKT7-p53 and pGADT7-T was used as a positive control, the strain co-transformed with pGBKT7-Lam and pGADT7-T was used as a negative control, and the strain co-transformed with pGBKT7-LsBOR5b and pGADT7-T was used as a control group. Fig.15 , Y2HGold[pADTT7-LsFT+pGBKT7-LsBOR5b] and Y2HGold[pGADT7-T+pGBKT7-LsBOR5b] strains grew equally on SD / -Leu / -Trp plates, Y2HGold[pADTT7-LsFT+pGBKT7-LsBOR5b] strains grew normally on SD / -Leu / -Trp / -His / -Ade / X-α-gal / AbA plates and could decompose X-α-gal to show blue color, while Y2HGold[pGADT7-T+pGBKT7-LsBOR5b] strains did not grow or show color on SD / -Leu / -Trp / -His / -Ade / X-α-gal / AbA plates. This result indicates that LsBOR5b interacts with LsFT.

[0086] Example 10: Arabidopsis transformation

[0087] (1) Construction of pCAMBIA2300-LsBOR5b recombinant vector

[0088] Based on the cloning primers of LsBOR5b gene containing BamHI and XbaI restriction sites, PCR amplification was performed using the extracted LsBOR5b plasmid as a template ( Fig.16 -A), and then ligated with the pCAMBIA2300 linearized vector after seamless cloning and transformed into E. coli, and single clone colonies were selected for PCR verification ( Fig.16 -B), send the single clones with the correct band size for sequencing, and shake the single clones with sequencing results consistent with the target gene to extract plasmids.

[0089] (2) Agrobacterium transformation

[0090] The pCAMBIA2300-LsBOR5b plasmid extracted in step (1) of Example 11 was introduced into Agrobacterium GV3101, and a single clone was selected for PCR verification ( Fig.17 ) The single clones with the correct band size were sent for sequencing, and the single clones with sequencing results consistent with the target gene were shaken and preserved with glycerol.

[0091] (3) Resistance screening of LsBOR5 transgenic Arabidopsis

[0092] The infected Arabidopsis mutant seeds were placed on the screening medium for culture and screening, and then transferred to small pots and placed in a light incubator for culture for one week. 2-3 true leaves were collected to extract DNA, and PCR identification was performed using Arabidopsis DNA as a template ( Fig.18 )

[0093] (4) Bolting time statistics

[0094] like Fig.19 As shown, the bolting time of wild-type Arabidopsis (WT), atbor5 knockout Arabidopsis (atbor5) and LsBOR5b overexpression Arabidopsis (OE-LsBOR5b) was statistically analyzed. The bolting time of OE-LsBOR5b was longer than that of WT and atbor. The bolting time of OE-LsBOR5b was about 4.01 days longer than that of WT.

[0095] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

[0096] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0097] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A kit for regulating plant bolting time, characterized in that: The kit contains molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to plant bolting time; the molecular biological elements may optionally include: an element combination for overexpressing the specific genes, and / or an element combination for inhibiting or reducing the expression of the specific genes, and / or an element combination for silencing the expression of the specific genes; The specific gene is the LsBOR5b gene, its homologous gene or an equivalent gene having the same plant physiological function as the LsBOR5b gene.

2. A recombinant expression vector comprising the LsBOR5b gene.

3. A method for prolonging plant bolting time, characterized in that: The expression of the LsBOR5b homologous gene in the plant is overexpressed to prolong the bolting time of the plant. The CDS sequence of the gene LsBOR5b is shown in SEQ ID NO:

1.

4. A method for increasing plant yield, characterized in that: The expression of the LsBOR5b homologous gene in the plant is overexpressed to prolong the plant bolting time, thereby increasing the yield.

5. The use of LsBOR5b gene is to regulate the bolting time of plants.

6. The kit according to claim 1-3, the method according to claim 6, the method according to claim 7, or the use according to claim 8, characterized in that: The plant is lettuce.

7. A primer pair for amplifying the nucleotide sequence shown in SEQ ID NO: 1, having the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO:

3.

8. A genotypic molecular marker for lettuce, characterized in that: The genotype molecular marker is the LsBOR5b gene in the lettuce BOR gene family.

9. The use of the genotype molecular marker according to claim 8, comprising: ① By detecting the expression time and expression amount of the molecular marker of this genotype, data analysis and planting intervention are performed on lettuce bolting, and the planting intervention includes harvesting as needed, adjusting the environment or nutrition supply conditions, collecting test data or other corresponding measures; ② Screening, preliminary screening, identification and auxiliary identification of lettuce germplasm resources are carried out by detecting the expression time and expression amount of the genotype molecular markers; the operation method is: among the natural lettuce population, lettuce plants from different geographical areas are collected to construct a primary lettuce natural population library, and the initial screening of potential excellent germplasm resources is achieved by detecting the expression time and expression amount and other expression characteristics of the genotype molecular markers.

10. The use according to claim 9, characterized in that: Use ② specifically includes but is not limited to: ②-A, through the expression time analysis of the genotype molecular marker, two types of plant populations of early expression and late expression are screened out respectively, and two secondary lettuce natural population sub-banks T1 and T2 (Time group) are constructed accordingly; ②-B, through the expression quantity analysis of the genotype molecular marker, two types of plant populations of high expression and low expression are screened out respectively, and two secondary lettuce natural population sub-banks Q1 and Q2 (Quantity group) are constructed accordingly; the above two groups of four lettuce natural population sub-banks are used as the initial germplasm resource bank for subsequent lettuce breeding, and hybrid breeding or molecular breeding is carried out within or between sub-banks in T1, T2, Q1, and Q2 respectively to quickly construct superior germplasm.

11. A method for developing a lettuce genotype biosensor, characterized in that: Develop biosensors using the expression specificity of specific genes in lettuce in specific tissues and developmental stages; Including but not limited to: ① Based on the gene LsBOR5b whose expression level changes significantly in the early stage of bolting, a biosensor for detecting bolting signals is constructed. When the gene expression level reaches a certain threshold, the sensor sends a signal to remind the lettuce that it is about to bolt. At this time, harvesting, adjustment of environmental or nutrient supply conditions, collection of experimental data or other corresponding measures are carried out as needed; ② Referring to the equivalent method of ①, other types of biosensors are developed based on the development and tissue expression characteristics of other genes in the lettuce BOR gene family.

12. The lettuce genotype biosensor developed according to claim 11.

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