Lsbor5b gene and its application in regulating and marking the time of bolting of plants

Abstract

The application discloses an LsBOR5b gene and application thereof in regulating and marking a plant bolting time, relates to the field of biotechnology, and prolongs the bolting and flowering time of the lettuce and improves the yield by overexpressing the expression of the LsBOR5b homologous gene in the plant, has important theoretical research value and wide practical application prospect for cultivating the lettuce plant with long bolting and flowering time. Meanwhile, the biological sensor can be developed based on the expression specificity of the specific gene in the lettuce in the specific tissue and development stage disclosed by the present application; for example, the biological sensor for detecting the bolting signal is constructed based on the gene LsBOR5b with obvious expression change in the initial bolting stage, the sensor sends a signal when the expression amount of the gene reaches a certain threshold value, the lettuce is about to bolt, and the harvesting, adjustment of the environmental or nutrient supply conditions, collection of test data or other corresponding measures are carried out as required.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to LsBOR5b gene and its application in regulating the bolting time of plants. BACKGROUND

[0002] Lactuca sativa L. belongs to the family Asteraceae and genus Lactuca L. in taxonomy, and is originated from the temperate regions of Asia. It is also known as lettuce, and has very high nutritional value, rich in inorganic salts, vitamins, polyphenols and lutein. It is a widely popular edible cultivated vegetable in the world, and has a large cultivation area in various parts of the world, among which Asia, Europe and North America have a wider cultivation. According to the main edible parts, lettuce can be divided into two types: leaf type (butter lettuce, head lettuce, loose-leaf lettuce, romaine lettuce) and asparagus 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 nation. Common cultivated varieties in China include asparagus lettuce, head lettuce, lettuce, American large lettuce, colored lettuce, and Roman upright lettuce. According to the data of FAO in 2022, China's total production, sowing area and total production value of lettuce and chicory vegetables rank first in the world, and have great economic value.

[0003] Bolting mainly refers to the phenomenon that the stems of clump-type plants in vegetative growth are stimulated by environmental changes such as temperature and day length, and the stems begin to elongate rapidly with the differentiation of flower buds, and the plants become taller. Lettuce is a facultative long-day plant, and the cultivated varieties show great differences in the speed of bolting and flowering. Very early-maturing varieties begin to bolt and flower after 7 weeks of planting under long-day conditions in a greenhouse, while very late-maturing varieties may flower after 4 months of planting. Studies have shown that the content of bitter-related metabolites increases rapidly before and after bolting in lettuce, which greatly affects 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 early bolting under heat stress, which seriously affects its yield and causes great losses in production, and prolonging the vegetative growth period of lettuce is beneficial to leaf biomass accumulation and quality maintenance.

[0004] In recent years, research teams such as Beijing Agricultural University and Beijing Academy of Agriculture and Forestry Sciences have mined and functionally identified genes related to leaf lettuce bolting, and found that LsARF3, LsRGL1, LsSTPK, etc. Genes have positive or negative regulatory effects on leaf lettuce bolting (Wang L, 2019; Wang et al., 2021; Li et al., 2022), but the research on bolting of stem lettuce has not been reported internationally. The applicant uses the latest cultivated lettuce genome data, combined with transcriptome data to identify homologous genes related to flower bud differentiation, bolting and flowering time in other plants, mine the LsBOR gene family related to bolting in stem lettuce, perform bioinformatics analysis, and functionally identify key gene family members, establish a new technical system for functional verification of transgenic lettuce, and provide new important genetic resources for extending the vegetative period of lettuce and improving yield, which has certain application value and potential economic value. SUMMARY

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

[0006] To solve the above technical problems, the technical solutions adopted by the present application are as follows.

[0007] A kit for regulating plant bolting time, the kit comprising a molecular biology element capable of regulating the expression amount of a specific gene; the specific gene is a gene related to the bolting time of the plant; the molecular biology element can be selected from the group consisting of: a combination of overexpression elements of the specific gene, and / or a combination of elements that inhibit or reduce the expression amount of the specific gene, and / or a combination of elements that silence the expression of the specific gene.

[0008] The specific gene is LsBOR5b gene, its homologous gene or equivalent gene with equivalent plant physiological function.

[0009] A recombinant expression vector comprising LsBOR5b gene.

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

[0011] A method for improving the yield of a plant, overexpressing the expression of LsBOR5b homologous gene in the plant to prolong the bolting time of the plant, thereby improving the yield.

[0012] Use of LsBOR5b gene for regulating the bolting time of a plant.

[0013] The kit according to claims 1-3 or the method according to claim 6 or 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 sequence shown in SEQ ID NO: 2, SEQ ID NO: 3.

[0015] The genotypic molecular marker of lettuce is the LsBOR5b gene in the LsBOR gene family of lettuce.

[0016] The use of the genotypic molecular marker of claim 8, comprising: ① analyzing and intervening the bolting of lettuce by detecting the expression time and expression amount of the genotypic molecular marker, and the intervening includes harvesting on demand, adjusting environmental or nutritional supply conditions, collecting test data or other corresponding measures; ② screening, preliminary screening, identifying, and assisting in identifying the germplasm resources of lettuce by detecting the expression time and expression amount of the genotypic molecular marker; the operation method is: collecting lettuce plants from different geographical regions to construct a primary lettuce natural population library, and realizing the preliminary screening of potential excellent germplasm resources by detecting the expression time and expression amount of the genotypic molecular marker and other expression characteristics.

[0017] According to the use of claim 9, in use ②, specifically including but not limited to: ②-A, screening out two groups of plant populations with early and late expression respectively by analyzing the expression time of the genotypic molecular marker, and corresponding to construct two secondary lettuce natural population sub-libraries T1 and T2 (Time group); ②-B, screening out two groups of plant populations with high and low expression respectively by analyzing the expression amount of the genotypic molecular marker, and corresponding to construct two secondary lettuce natural population sub-libraries Q1 and Q2 (Quantity group); the above two groups of four lettuce natural population sub-libraries are used as the initial germplasm resource library for subsequent lettuce breeding, and the initial germplasm resource library is used for hybrid breeding or molecular breeding within or between the sub-libraries in T1, T2, Q1, and Q2 to quickly construct superior germplasm.

[0018] The development method of the lettuce genotypic biosensor, which develops the biosensor by using the expression specificity of specific genes in specific tissues and development stages of lettuce; including but not limited to: ① based on the gene LsBOR5b with obvious expression amount change in the early stage of bolting, a biosensor for detecting bolting signal is constructed, when the expression amount of the gene reaches a certain threshold, the sensor sends a signal to remind that the lettuce will bolt, at this time, harvesting on demand, adjusting environmental or nutritional supply conditions, collecting test data or other corresponding measures are performed; ② based on the development and tissue expression characteristics of other genes in the LsBOR gene family of lettuce, other types of biosensors are developed by referring to the equivalent method of ①.

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

[0020] The beneficial effects produced by the above technical solutions are that the theoretical research and experimental research of the subject group of the present application have confirmed that, by overexpressing the target gene LsBOR5b in lettuce, the bolting time of lettuce is prolonged and the yield is improved, which has important theoretical research value and broad practical application prospect for cultivating lettuce plants with long bolting time. At the same time, biosensors can also be developed based on the expression specificity of specific genes in specific tissues and development stages of lettuce disclosed in the present application; for example, based on the gene LsBOR5b with obvious expression change at the early stage of bolting, a biosensor for detecting bolting signal is constructed, when the expression amount of the gene reaches a certain threshold, the sensor sends a signal to remind that the lettuce will bolt, at which time the corresponding measures such as harvesting, adjusting the environmental or nutrient supply conditions, collecting experimental data or other corresponding measures are taken. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a schematic diagram of the gene structure of LsBOR in Example 2, in which the exons and introns are represented by yellow squares and black lines respectively, and the 5' and 3' untranslated regions (UTR) are represented by blue frames;

[0022] Figure 2 It is a schematic diagram of the analysis of the conserved motifs of LsBOR in Example 2;

[0023] Figure 3 It is a schematic diagram of the amino acid sequences of each conserved motif in the lettuce BOR protein in Example 2, in which the font size represents the frequency of occurrence of each amino acid;

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

[0025] Figure 5 It is a 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 colinearity relationship within the LsBOR gene;

[0026] Figure 6 It is a colinearity relationship diagram of lettuce and Arabidopsis thaliana, Helianthus annuus in Example 3, note: different colors represent different species of chromosomes, and the chromosome number is represented by numbers. The gray line in the background represents the colinearity relationship between lettuce and other plant genomes, and the red line represents the colinear BOR gene pairs. The black text represents the gene name of LsBOR gene and the gene ID with colinearity relationship with LsBOR gene;

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

[0028] Figure 8 Figure for three-dimensional structure prediction of lettuce BOR protein in Example 5;

[0029] Figure 9 Expression profile of lettuce BOR gene in different tissues and developmental stages in Example 6;

[0030] Figure 10 Expression profile of lettuce BOR gene under different stress conditions in Example 6;

[0031] Figure 11 Figure for PCR amplification of LsBOR5b gene and enzyme electrophoretogram of recombinant plasmid in Example 7, wherein Figure A is an electrophoretogram of amplification product of target gene LsBOR5b, and Figure B is an electrophoretogram of PCR enzyme digestion product of recombinant plasmid bacterial colony;

[0032] Figure 12 Figure for amplification of lettuce LsBOR5b gene and electrophoretogram of recombination vector construction results in Example 8, wherein A is an electrophoretogram of LsBOR5b gene amplification, B is an electrophoretogram of recombinant plasmid bacterial colony PCR verification (LsBOR5b), and C is an electrophoretogram of pCAMBIA1300-GFP-LsBOR5b recombinant vector bacterial colony PCR;

[0033] Figure 13 Figure for subcellular localization of pCAMBIA1300-GFP-LsBOR5b fusion protein in Example 8;

[0034] Figure 14 Figure for electrophoretogram of lettuce LsBOR5b and LsFT gene related experiments in Example 9, wherein A is an electrophoretogram of PCR amplification product of lettuce LsFT gene, B is an electrophoretogram of pADTT7-LsFT recombinant vector bacterial colony PCR, C is an electrophoretogram of PCR amplification product of lettuce LsBOR5b gene, and D is an electrophoretogram of pGBKT7-LsBOR5b recombinant vector bacterial colony PCR;

[0035] Figure 15 Figure for analysis of yeast two-hybrid results of LsBOR5b and LsFT in Example 9;

[0036] Figure 16 Figure for electrophoretogram of pCAMBIA2300-LsBOR5b recombinant vector construction results in Example 10, wherein A is an electrophoretogram of PCR amplification product of lettuce LsBOR5b gene, and B is an electrophoretogram of pCAMBIA2300-LsBOR5b recombinant vector bacterial colony PCR;

[0037] Figure 17Figure for electrophoresis of PCR amplification product for transforming Agrobacterium colony with pCAMBIA2300-LsBOR5b recombinant vector in Example 10;

[0038] Figure 18 Figure for PCR verification result of overexpressing LsBOR5b in Arabidopsis in Example 10.

[0039] Figure 19 Figure for statistical result of bolting time of wild type Arabidopsis (WT), atbor5 knock-out Arabidopsis (atbor5) and LsBOR5b overexpressing Arabidopsis (OE-LsBOR5b) in Example 10. DETAILED DESCRIPTION

[0040] The following examples illustrate the present application. The various materials and equipment used in these examples are conventional and available from commercial sources. The experimental methods used in the following examples are conventional unless otherwise stated.

[0041] It should be understood that the term "comprises" when used in this specification and the appended claims, specifies the presence of stated features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0042] It should also be understood that the term "and / or" when used in this specification and the appended claims, means any one or more of the associated listed items, and that includes one or more of the associated listed items.

[0043] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, the appearances of the phrases "in one embodiment" or "in some embodiments" or "in other embodiments" or "in still other embodiments" or similar phrases in various places throughout this specification are not necessarily all referring to the same embodiment, unless otherwise indicated. The terms "including," "comprising," "having," and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0044] In addition, the terms "first," "second," "third," etc. are used herein only to describe different instances of the same item, and do not imply relative importance.

[0045] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

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

[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, GSDS was used to map the composition of exons and introns. Generally, genes within 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, the BOR genes in lettuce are divided into three groups, similar to the grouping in Arabidopsis and rice. In groups 1 and 2, LsBOR2a contains one exon, while the other genes have two exons. In group 3, all BOR genes except LsBOR9b, which contains five exons, contain four exons.

[0053] Analyzing conserved motifs can provide information related to the evolutionary process of gene families. MEME software was used to compare and analyze the conserved domains and motifs of the lettuce BOR protein. Motifs 1 to 10 were arranged in ascending order of sequence E-value.Figure 2 、 Figure 3 The longest motif is 45 aa and the shortest is 6 aa. Motif 1 and Motif 2 are the most conserved motifs among all BOR proteins. Motif 5 is only located in group 1 and part of group 2 and group 3 members. In addition, some motifs only appear in one unique group and are shared by all members within the group, such as Motif 4 in group 3. In addition, Motif 8 only exists in 2 BOR proteins of group 3, Motif 3 and Motif 6 only exist in 3 BOR proteins of group 3, and Motif 7 and Motif 9 only exist in 4 BOR proteins of group 3.

[0054] Example 3, Chromosomal distribution and collinearity analysis of lettuce BOR family members

[0055] To analyze the location of BOR gene family members in the lettuce genome, we performed chromosomal mapping and analyzed gene duplication events. According to the mapping results, 14 LsBOR genes were mapped on 7 different chromosomes, and 1 gene was mapped 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 duplication events of the BOR gene family in lettuce to understand the reasons for the duplication events of LsBOR genes. The results showed that 5 pairs of large fragment duplicated genes were detected (LsBOR2a and LsBOR2b, LsBOR5a and LsBOR5b, LsBOR9a and LsBOR9b, LsBOR9b and LsBOR9c, LsBOR9a and LsBOR9c) Figure 5 These results suggest that large fragment duplication may be the main driving force for the amplification and evolution of BOR genes in the lettuce genome.

[0056] To understand the origin and evolutionary relationship of BOR, we further performed intergroup collinearity analysis and compared the lettuce genome with the Arabidopsis and sunflower genomes. As shown in Figure 6 Among these genes, LsBOR2b, LsBOR9a, LsBOR9b, LsBOR9c, LsBOR13, LsBOR14a and LsBOR16b homologous genes were detected in all 3 species, LsBOR4, LsBOR5a, LsBOR5b and LsBOR16a homologous genes were only detected between lettuce and sunflower, and other genes were not detected in the duplication region.

[0057] The physical location and distribution of genes in the genome can be determined by analyzing the chromosomal distribution and collinearity of the lettuce BOR family members. Gene duplication events provide clues for studying gene amplification and evolution mechanisms. Collinearity analysis helps trace the origin and evolution of genes.

[0058] Example 4, cis-acting element analysis of lettuce BOR genes

[0059] Cis-acting elements are binding sites for transcriptional regulators that regulate gene transcription. To study the potential functions of LsBOR genes, we analyzed the cis-acting elements in the 1500bp upstream sequence of the LsBOR promoter, excluding functionally unknown elements 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 sites, 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 indicate that most LsBOR genes have light-responsive elements, suggesting that LsBOR genes may play a key role in light-responsive regulation.

[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 genes, and further infer the gene functions.

[0061] Table 2 Cis-acting elements found in more than 3 LsBOR genes

[0062]

[0063] Example 5, three-dimensional structure prediction of lettuce BOR proteins

[0064] SWISS-MODEL was used to predict the three-dimensional structure of lettuce BOR proteins, and the model with the highest QMQE value was selected to construct the three-dimensional structure model of the protein. As Figure 8 , the secondary structure of lettuce BOR family proteins is mainly composed of random coils, alpha-helices, and beta-turns. BOR proteins in the same group have similar structures.

[0065] Understanding the spatial structure of proteins helps to infer their interaction modes with other molecules and functional mechanisms in cells, providing a structural basis for subsequent functional verification.

[0066] Example 6, expression analysis of lettuce BOR genes

[0067] (1) Developmental and tissue expression profiling of LsBOR genes

[0068] To investigate the tissue-specific expression patterns of LsBOR genes, we analyzed the expression profiles of LsBOR genes in different tissues and developmental stages. LsBOR2a, LsBOR9a, and LsBOR11 were considered not to be expressed because of their low expression levels (cp values greater than 35 or undetectable). As shown in Table 1, other genes were expressed in all tissues and developmental stages of lettuce. Figure 9 Most of the LsBOR genes (LsBOR2b, LsBOR4, LsBOR5a, LsBOR5b, LsBOR13, LsBOR16a, and LsBOR16b) were highly expressed in leaves, suggesting that these genes might play important roles 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, indicating that these genes might play important roles in the development of flowers in lettuce. Figure 9 In both varieties, the expression levels of LsBOR5a increased from bolting to flowering, LsBOR5b increased from the early bolting stage to the late bolting stage, and LsBOR4, LsBOR9c, LsBOR13, and LsBOR14a increased from the late bolting stage to the flowering stage. In the Bai Pijian lettuce variety, the expression levels of LsBOR14b, LsBOR16a, and LsBOR16b increased from the early bolting stage to the late bolting stage.

[0069] Through the developmental and tissue expression profiling of LsBOR genes, we found that some genes were not expressed, most were highly expressed in leaves, and some showed changes in expression levels during the bolting and flowering stages, indicating that they might play different roles in vegetative growth and reproductive development. Determining the spatiotemporal expression patterns of genes during plant growth and development and identifying genes related to vegetative growth and flower development can provide clues for studying their roles in bolting and flowering.

[0070] (2) Expression profiling of LsBOR genes under abiotic stress

[0071] To investigate whether LsBOR genes are involved in the response of lettuce to different abiotic stresses, we further analyzed the expression levels of LsBOR genes in Bai Pijian lettuce and Zixiahong lettuce exposed to different times of salt and drought stress. The results showed that the expression of LsBOR genes changed under different abiotic stresses at different times. Figure 10

[0072] ​The lettuce BOR genes showed similar expression patterns under salt stress treatment. In the two lettuce varieties, LsBOR9b, LsBOR9c, LsBOR13, LsBOR14a, and LsBOR14b were significantly up-regulated at 12 h of salt stress treatment, and LsBOR16a was significantly up-regulated at 24 h of salt stress treatment. LsBOR5b was weakly up-regulated in White Tip Lettuce Sprout at 6 h of salt stress treatment, and was significantly up-regulated in Purple Xia Red Lettuce Sprout. Under drought stress conditions, the lettuce BOR genes were significantly up-regulated in White Tip Lettuce Sprout at 12 h or 24 h. In Purple Xia Red Lettuce Sprout, the expression of the lettuce BOR genes did not show strong / significant up-regulation or down-regulation under drought stress treatment, LsBOR5b was weakly up-regulated at 6 h and 24 h, LsBOR16b was weakly up-regulated at 6 h and 12 h, and LsBOR2b and LsBOR16a were weakly up-regulated at 48 h. These results indicate that the lettuce BOR genes may play a key role in the stress response of lettuce to salt stress and drought stress.

[0073] Example 7, Construction of LsBOR5b Recombinant Vector

[0074] According to the expression analysis results and comparing the chromosomal localization information of the LsBOR family members with the QTLs affecting the flowering transition of lettuce, LsBOR5b, which was up-regulated at the early stage to the late stage of bolting in two lettuce varieties, was selected for subsequent gene function verification. The CDS sequence of the gene LsBOR5b is shown as SEQ ID NO: 1.

[0075] According to the full-length primer of the LsBOR5b gene, the cDNA of lettuce was used as a template to amplify the target gene fragment by PCR. The nucleotide sequences of the specific primers F1 and R1 required for amplification are shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The amplified band is consistent with the expected one, the fragment length is 1182 bp, and the amplified band is single Figure 11 -A). After purification of the PCR product, the T vector was connected, and the E. coli was transformed. The single colony was picked for PCR verification Figure 11 -B). The single colony with correct band size was sent for sequencing, and the single colony with consistent sequencing results with the target gene was shaken, and the plasmid was extracted and stored in glycerol.

[0076] Example 8, Subcellular Localization of LsBOR5b

[0077] (1) Construction of pCAMBIA1300-GFP-LsBOR5b Recombinant Vector

[0078] According to the cloning primer of the LsBOR5b gene containing the BamHI and XbaI enzyme digestion sites, the extracted LsBOR5b plasmid was used as a template for PCR amplificationFigure 12 -A). After purification, the PCR product was connected with the linearized pCAMBIA1300-GFP vector by seamless cloning, and then transformed into E. coli. Single colony PCR verification was performed on the E. coli, and the single colony with correct band size was sent for sequencing. The single colony with consistent sequencing results was shaken, and the plasmid was extracted. The extracted pCAMBIA1300-GFP-LsBOR5b plasmid was introduced into Agrobacterium, and single colony PCR verification was performed on the E. coli. Figure 12 -B). After purification, the PCR product was connected with the linearized pCAMBIA1300-GFP vector by seamless cloning, and then transformed into E. coli. Single colony PCR verification was performed on the E. coli, and the single colony with correct band size was sent for sequencing. The single colony with consistent sequencing results was shaken, and the plasmid was extracted. The extracted pCAMBIA1300-GFP-LsBOR5b plasmid was introduced into Agrobacterium, and single colony PCR verification was performed on the E. coli. Figure 12 -C). After purification, the PCR product was connected with the linearized pCAMBIA1300-GFP vector by seamless cloning, and then transformed into E. coli. Single colony PCR verification was performed on the E. coli, and the single colony with correct band size was sent for sequencing. The single colony with consistent sequencing results was shaken, and the plasmid was extracted. The extracted pCAMBIA1300-GFP-LsBOR5b plasmid was introduced into Agrobacterium, and single colony PCR verification was performed on the E. coli.

[0079] (2) Subcellular localization of LsBOR5b

[0080] The distribution of proteins in cells is closely related to their functions. To further understand the function of LsBOR5b, we constructed a pCAMBIA1300-GFP-LsBOR5b fusion vector and transformed tobacco leaves. The transformed tobacco leaves with the empty vector pCAMBIA1300-GFP were used as controls. The GFP fluorescence position was observed under a laser confocal microscope. The results showed that the fluorescence signal was located in the nucleus and cell membrane in the plants transformed with the empty vector, and the fluorescence signal was located in the endoplasmic reticulum in the plants transformed with the pCAMBIA1300-GFP-LsBOR5b fusion vector, indicating that LsBOR5b protein was mainly distributed in the endoplasmic reticulum. Figure 13

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

[0082] (1) Recombinant plasmid construction

[0083] According to the full-length primer of LsFT gene, the target gene fragment was amplified by PCR using lettuce cDNA as the template. The amplified band was consistent with the expected one, and the fragment length was 528 bp. The amplified band was single ( Figure 14 -A). After purification, the PCR product was connected with the linearized pCAMBIA1300-GFP vector by seamless cloning, and then transformed into E. coli. Single colony PCR verification was performed on the E. coli, and the single colony with correct band size was sent for sequencing. The single colony with consistent sequencing results was shaken, and the plasmid was extracted. The extracted pCAMBIA1300-GFP-LsBOR5b plasmid was introduced into Agrobacterium, and single colony PCR verification was performed on the E. coli. Figure 14 -B). After purification, the PCR product was connected with the linearized pCAMBIA1300-GFP vector by seamless cloning, and then transformed into E. coli. Single colony PCR verification was performed on the E. coli, and the single colony with correct band size was sent for sequencing. The single colony with consistent sequencing results was shaken, and the plasmid was extracted. The extracted pCAMBIA1300-GFP-LsBOR5b plasmid was introduced into Agrobacterium, and single colony PCR verification was performed on the E. coli. Figure 14 ​-C), after purification, the linearized vector pGBKT7 was ligated by seamless cloning, and then transformed into E. coli, and single colony PCR was used to verify the transformation Figure 14 -D), the single colony with correct band size was sequenced, and the single colony with consistent sequence with the target gene was used for shaking culture and plasmid extraction.

[0084] (2) Yeast two-hybrid analysis

[0085] pADTT7-LsFT and pGBKT7-LsBOR5b were co-transformed into Y2HGold competent cells, and single colonies 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. As shown in Figure 15 , the growth of Y2HGold[pADTT7-LsFT+pGBKT7-LsBOR5b] and Y2HGold[pGADT7-T+pGBKT7-LsBOR5b] strains on SD / -Leu / -Trp plates was the same, the Y2HGold[pADTT7-LsFT+pGBKT7-LsBOR5b] strain grew normally on SD / -Leu / -Trp / -His / -Ade / X-α-gal / AbA plates and could decompose X-α-gal to blue, while the Y2HGold[pGADT7-T+pGBKT7-LsBOR5b] strain did not grow on SD / -Leu / -Trp / -His / -Ade / X-α-gal / AbA plates and did not show color. The results showed that LsBOR5b interacted with LsFT.

[0086] Example 10, Arabidopsis Transformation

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

[0088] According to the cloning primer of LsBOR5b gene containing BamHI and XbaI restriction sites, the extracted LsBOR5b plasmid was used as a template for PCR amplification Figure 16 -A), after purification, the linearized vector pGBKT7 was ligated by seamless cloning, and then transformed into E. coli, and single colony PCR was used to verify the transformation Figure 16 -B), the single colony with correct band size was sequenced, and the single colony with consistent sequence with the target gene was used for shaking culture and plasmid extraction.

[0089] (2) Agrobacterium transformation

[0090] The pCAMBIA2300-LsBOR5b plasmid extracted from step (1) of Example 11 was introduced into Agrobacterium GV3101, and single colonies were picked for PCR verification Figure 17 ), and the single colonies with correct band sizes were sequenced. The single colonies with consistent sequencing results with the target gene were shaken and preserved with glycerol.

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

[0092] The Arabidopsis mutant seeds at the end of the infection were placed on the screening medium for screening and culture, and then placed in a light incubator for one week. The 2-3 true leaves were collected for DNA extraction, and the Arabidopsis DNA was used as a template for PCR identification Figure 18

[0093] (4) Statistical analysis of bolting time

[0094] As shown in Figure 19 , the bolting time of wild-type Arabidopsis (WT), atbor5 knockout Arabidopsis (atbor5), and LsBOR5b overexpression Arabidopsis (OE-LsBOR5b) was counted. 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] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0096] In the above embodiments, the description of each embodiment has its own focus, and the parts not described or recorded in a certain embodiment can be referred to the related description of other embodiments.

[0097] The above-described embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirits and scopes of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.​

Claims

1. A method of prolonging the time of bolting of a plant, characterized in that: The expression of a LsBOR5b gene is overexpressed in the plant to prolong the bolting time of the plant, wherein the CDS sequence of the LsBOR5b gene is shown as SEQ ID NO:1, and the plant is Arabidopsis thaliana.

2. Use of a LsBOR5b gene to prolong the bolting time of Arabidopsis thaliana, wherein the CDS sequence of the LsBOR5b gene is shown as SEQ ID NO:1.

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