Promoter of separated BnaA06.UBC2 gene and application thereof

Abstract

The invention discloses a separated promoter of a BnaA06. UBC2 gene and an application of the separated promoter, two promoter sequences pUBC2SWU47 and pUBC2NY12 of the BnaA06. UBC2 gene are separated from an early flowering strain SWU47 and a late flowering strain NY12 of rape, large fragment insertion, deletion and mononucleotide substitution exist in the two promoter sequences, the pUBC2NY12 deletes two continuous ABRE motifs in a region from-456bp to-171bp relative to the pUBC2SWU47, and the promoter sequences pUBC2SWU47 and pUBC2NY12 are different from the promoter sequences pUBC2SWU47 in the region from-456bp to-171bp. The expression of the BnaA06. UBC2 gene is changed, so that the regulation and control capability of a transcription factor BnaA05. ABI5 on the promoter is changed, the expression difference of the BnaA06. UBC2 gene is caused, and the flowering traits are different in the morning and evening.

Description

Technical Field

[0001] The present invention relates to the technical field of plant transgenic technology, and particularly relates to a promoter of a separated BnaA06.UBC2 gene and its application. Background Art

[0002] Rapeseed is an important oil crop in China, providing more than 50% of the edible vegetable oil in China. Flowering is a sign of the transformation of rapeseed from vegetative growth to reproductive growth. The flowering time (the time from sowing to the first flower) is an important agronomic trait of rapeseed. If rapeseed flowers too early, it may be affected by low temperature during flowering, resulting in insufficient fertilization and reduced yield. If it flowers too late, it will affect the subsequent planting of rice and affect the main food security. Studying the flowering regulation mechanism of rapeseed can provide gene resources for the subsequent creation of new rapeseed varieties with appropriate flowering time and suitable for rice-rapeseed rotation. The flowering regulation of the model plant Arabidopsis thaliana mainly includes the photoperiod pathway, GA pathway, autonomous pathway, age pathway, and vernalization pathway. FLC is a key regulatory factor for regulating flowering in the vernalization pathway. Plants can regulate the expression level of FLC by transcriptional regulation, epigenetic modification, and post-translational modification of FLC, and ultimately regulate flowering.

[0003] UBC2 (UBIQUITIN CARRIER PROTEIN2) is an E2 ubiquitin-conjugating enzyme. In the early stage of the laboratory, 120 stable DH lines were created using the materials with significant differences in flowering time, SWU47 and NY12, as parents. Based on the 4-year flowering time data collected in the early stage and the genetic map constructed by previous sequencing, a stable QTL locus was mapped on A06. Combining transcriptome data, haplotype analysis, and gene function annotation, we identified a key candidate gene, BnaA06.UBC2, at this locus. We extracted the BnaA06.UBC2 gene sequence from the SWU47 and NY12 materials respectively, and found that there were large fragment insertions, deletions, and single nucleotide substitutions in the BnaA06.UBC2 promoter in the SWU47 and NY12 materials. At present, the role of the BnaA06.UBC2 promoter in the flowering regulation of rapeseed has not been clarified. Summary of the Invention

[0004] In view of this, one of the purposes of the present invention is to provide a separated promoter of the BnaA06.UBC2 gene; the second purpose of the present invention is to provide the application of the promoter in regulating the differential expression of the BnaA06.UBC2 gene; the third purpose of the present invention is to provide the application of pUBC2 NY12 in delaying plant flowering; the fourth purpose of the present invention is to provide a method for cultivating late-flowering rapeseed lines.

[0005] To achieve the above purposes, the present invention provides the following technical solutions:

[0006] 1. A promoter of a separated BnaA06.UBC2 gene, wherein the promoter contains at least one natural variation of a poly ABRE motif, and the variation is selected from:

[0007] a) The promoter sequence shown in SEQ ID NO.15, named pUBC2 SWU47 ;

[0008] b) The promoter sequence shown in SEQ ID NO.16, named pUBC2 NY12 , compared with pUBC2 SWU47 , the number of ABRE motifs contained in pUBC2 NY12 is reduced.

[0009] In some embodiments of the present invention, the pUBC2 NY12 is missing 2 consecutive ABRE motifs relative to pUBC2 SWU47 , resulting in a weakened binding ability of the transcription factor BnaA05.ABI5 to the promoter.

[0010] 2. Application of the promoter in regulating differential expression of the BnaA06.UBC2 gene.

[0011] In some embodiments of the present invention, when the promoter is pUBC2 SWU47 , the expression level of the BnaA06.UBC2 gene is relatively low; when the promoter is pUBC2 NY127 , the expression level of the BnaA06.UBC2 gene is relatively high.

[0012] 3. Application of pUBC2 NY12 in delaying plant flowering. By using pUBC2 NY12 to drive overexpression of the BnaA06.UBC2 gene in plant cells, the expression level of the BnaC02.FLC gene can be up-regulated, thereby delaying the flowering time of the plant. The plant is Arabidopsis thaliana or rapeseed.

[0013] 4. A method for cultivating a late-flowering rapeseed line, using pUBC2 NY12 to drive overexpression of the BnaA06.UBC2 gene in rapeseed cells, so that the expression level of the BnaA06.UBC2 gene is increased to obtain a late-flowering rapeseed line.

[0014] In some embodiments of the present invention, the sequence of the BnaA06.UBC2 gene is as shown in SEQ ID NO.6.

[0015] The beneficial effects of the present invention are as follows: The coding sequence and promoter sequence of BnaA06.UBC2 were amplified in the parental materials SWU47 and NY12. Sequence comparison revealed large fragment insertions, deletions, and single nucleotide substitutions. To determine whether the sequence variation of BnaA06.UBC2 affects the flowering time, an expression vector pUBC2 driven by its own promoter was constructed. SWU47 :UBC2 SWU47 and pUBC2 NY12 :UBC2 NY12 were genetically transformed into Arabidopsis thaliana (Col-0). Observation of the phenotypes of transgenic plants showed that the flowering time of the pUBC2 SWU47 :UBC2 SWU47 and pUBC2 NY12 :UBC2 NY12 transgenic lines was delayed compared to Col-0, and the flowering time of the pUBC2 NY12 :UBC2 NY12 line was significantly later than that of the pUBC2 SWU47 :UBC2 SWU47 .

[0016] In addition, observation of the phenotypes of overexpressing Arabidopsis plants showed that the CDS sequence variation of BnaA06.UBC2 in the parental materials did not affect the flowering time of the plants. In this study, GUS gene expression vectors driven by the promoter sequences of BnaA06.UBC2 in the parental materials, namely pUBC2 SWU47 :GUS and pUBC2 NY12 :GUS, were constructed and genetically transformed into Col-0. GUS chemical tissue staining showed that the staining of the pUBC2 NY12 :GUS transgenic plants before bolting was significantly darker than that of the pUBC2 SWU47 :GUS transgenic plants. At the same time, quantitative detection of the expression level of BnaA06.UBC2 in the parental materials showed that the expression level of BnaA06.UBC2 in the NY12 material was significantly higher than that in the SWU47 material. In summary, the variation of the BnaA06.UBC2 promoter sequence led to differential expression of BnaA06.UBC2 in the parental materials, ultimately affecting the regulation of rapeseed flowering.

[0017] The sequence variation of BnaA06.UBC2 in the parental materials led to variation in the transcriptional regulation of BnaA06.UBC2 by the upstream transcription factor BnaA05.ABI5, resulting in an increase in the expression level of BnaA06.UBC2 in the NY12 material. BnaA06.UBC2 interacted with BnaA05.HUB1, and then promoted the expression of the flowering inhibitor BnaC02.FLC, ultimately inhibiting rapeseed flowering. Description of the Drawings

[0018] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0019] Figure 1 For BnaA06.UBC2 SWU47 、BnaA06.UBC2 NY12 Overexpression vector construction; A: BnaA06.UBC2 SWU47 、BnaA06.UBC2 NY12 Cloning (1 is BnaA06.UBC2 SWU47 , 2 is BnaA06.UBC2 NY12 ); B: BnaA06.UBC2 SWU47 、BnaA06.UBC2 NY12 Overexpression vector colony PCR (1-12 are BnaA06.UBC2 SWU47 overexpression vector colony PCR, 13-24 are BnaA06.UBC2 NY12 overexpression vector colony PCR).

[0020] Figure 2 For 35S:UBC2 SWU47 、35S:UBC2 NY12 Investigation of agronomic traits of transgenic Arabidopsis thaliana; A: 35S:UBC2 SWU47 、35S:UBC2 NY12 、Col-0 Arabidopsis thaliana plant pictures at flowering stage; B: 35S:UBC2 SWU47 、35S:UBC2 NY12 Expression level of BnaA06.UBC2 in transgenic Arabidopsis thaliana; C: 35S:UBC2 SWU47 、35S:UBC2 NY12 Flowering time statistics of Col-0; p < 0.01, indicated by "**", p < 0.05, indicated by "*".

[0021] Figure 3 For pUBC2 SWU47 :UBC2 SWU47 、pUBC2 NY12 :UBC2 NY12 Construction of expression vectors; A: BnaA06.UBC2 SWU47、 BnaA06.UBC2 NY12 Full-length gene fragment amplification of BnaA06.UBC2 (1 is BnaA06.UBC2 SWU47 , 2 is BnaA06.UBC2 NY12 ); B: pUBC2 SWU47 :UBC2 SWU47 、pUBC2 NY12: UBC2 NY12 Colony PCR of the expression vector (1 - 12 are pUBC2 SWU47 : UBC2 SWU47 ; 13 - 24 are pUBC2 NY12 : UBC2 NY12 ).

[0022] Figure 4 are pUBC2 SWU47 : UBC2 SWU47 , pUBC2 NY12 : UBC2 NY12 Phenotype map of transgenic Arabidopsis; A: pUBC2 SWU47 : UBC2 SWU47 , pUBC2 NY12 : UBC2 NY12 Picture of transgenic Arabidopsis; B: pUBC2 SWU47 : UBC2 SWU47 , pUBC2 NY12 : UBC2 NY12 Expression level of BnaA06.UBC2 in transgenic Arabidopsis; C: pUBC2 SWU47 : UBC2 SWU47 , pUBC2 NY12 : UBC2 NY12 Flowering time statistics of transgenic Arabidopsis; p < 0.01, indicated by "**", p < 0.05, indicated by "*".

[0023] Figure 5 are pCAMBIA1305.1 - proBnaA06.UBC2 SWU47 , pCAMBIA1305.1 - proBnaA06.UBC2 NY12 Construction of the expression vector; A: BnaA06.UBC2 SWU47、 BnaA06.UBC2 NY12 Amplification of the promoter fragment (1 is proBnaA06.UBC2 SWU47 , 2 is proBnaA06.UBC2 NY12 ); B: Colony PCR of pCAMBIA1305.1 - proBnaA06.UBC2 SWU47 , pCAMBIA1305.1 - proBnaA06.UBC2 NY12 (1 - 12 are pCAMBIA1305.1 - proBnaA06.UBC2 SWU47 ; 13 - 24 are pCAMBIA1305.1 - proBnaA06.UBC2 NY12 ).

[0024] Figure 6 is pCAMBIA1305.1-proBnaA06.UBC2 SWU47 、pCAMBIA1305.1-proBnaA06.UBC2 NY12 GUS chemical staining results of transgenic Arabidopsis thaliana; Note: pUBC2 SWU47 : GUS is pCAMBIA1305.1-proBnaA06.UBC2 SWU47 Transgenic positive Arabidopsis thaliana; pUBC2 NY12 : GUS is pCAMBIA1305.1-proBnaA06.UBC2 NY12 Transgenic positive Arabidopsis thaliana; seeding is the time from sowing to GUS staining.

[0025] Figure 7 is the expression level of BnaA06UBC2 in various tissues of SWU47 and NY12 materials; Note: p < 0.01, indicated by "**", p < 0.05, indicated by "*".

[0026] Figure 8 is BnaA06.UBC2 SWU47 Results of screening for the core fragment of the BnaA06.UBC2 promoter (A: Schematic diagram of BnaA06.UBC2 SWU47 promoter truncation; B: Results of the dual-luciferase assay for promoter truncation).

[0027] Figure 9 is the binding of BnaA05.ABI5 to pBnaA06.UBC2 (A: Binding of BnaA05.ABI5 to pBnaA06.UBC2 SWU47(C) and pBnaA06.UBC2 NY12(C) yeast one-hybrid results; B: Activation of pBnaA06.UBC2 by BnaA05.ABI5 NY12 expression).

[0028] Figure 10 is the change in the expression levels of BnaA05.ABI5 and BnaA06.UBC2 in SWU47 and NY12 after spraying ABA.

[0029] Figure 11 is the change in the expression levels of BnaA06.UBC2, BnaC02.FLC, BnaA02.FT, BnaC02.FT, and BnaC06.FT in SWU47 and NY12 materials at different stages; A is BnaC02.FLC; B is BnaA02.FT; C is BnaC02.FT; D is BnaC06.FT; E is the change in the expression level of BnaA06.UBC2 in SWU47 and NY12 at different stages.

[0030] Figure 12 Haplotype analysis of the BnaA06.UBC2 promoter in the natural population.

[0031] Figure 13 Model for BnaA06.UBC2 regulating rapeseed flowering. Specific implementation manners

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0033] The primer sequences involved in the present invention are shown in Table 1.

[0034] Table 1 Sequence list

[0035]

[0036]

[0037] Example 1. Construction of overexpression vector

[0038] Using the high-quality cDNA of the leaves of SWU47 and NY12 materials as templates respectively, and using the primer pairs OE-BnaA06.UBC2 SWU47 -F / R (SEQ ID No.1~2), OE-BnaA06.UBC2 NY12 -F / R (SEQ ID No.3~4) for PCR amplification to obtain fragments of about 500bp (SEQ ID No.5~6) ( Figure 1 , A). After purifying and recovering the target fragments, they were ligated to the Gateway entry vector according to the instructions. The positive identification of the bacterial liquid was carried out using universal primers. The bacterial liquid with PCR bands meeting the expectations was sent to the company for sequencing. After the correctly sequenced bacteria were expanded and cultured, the plasmids were extracted and the target fragments in the entry vector were transferred to the overexpression vector pEarleyGate101 through the LR reaction. After the reaction was completed, the reaction products were transformed into Escherichia coli DH5α. Using the primers F35SND+OE-BnaA06.UBC2 SWU47 -R and F35SND+OE-BnaA06.UBC2 NY12 -R for positive identification of the Escherichia coli monoclonal colonies by colony PCR, and the monoclonal colonies with PCR product lengths of about 500bp were sent to BGI for sequencing ( Figure 1 , B). The sequencing results were compared using Geneious. The homology between the cloned gene sequence and the reference sequence reached 100%, indicating that the overexpression vector had been successfully constructed, and they were named 35S:UBC2 SWU47 ​NY12 。

[0039] To further determine whether the variation of the CDS of BnaA06.UBC2 in the two parents affects the regulation of flowering, in this study, the overexpression vector constructed was transformed into Col-0. By observing and recording the flowering time of transgenic Arabidopsis thaliana, it was found that the flowering time of 35S:UBC2 SWU47 、35S:UBC2 NY12 transgenic plants was significantly later than that of Col-0, and there was no significant difference in the flowering time between the two overexpression transgenic plants, indicating that BnaA06.UBC2 has the function of inhibiting the flowering of Arabidopsis thaliana, and the variation of the CDS sequence of BnaA06.UBC2 in the parental materials does not affect its regulation of the flowering of Arabidopsis thaliana( Figure 2 ).

[0040] Example 2. Construction of expression vector driven by its own promoter

[0041] The full-length (2000 bp upstream of ATG + coding region) sequences of BnaA06.UBC2 SWU47 、BnaA06.UBC2 NY12 were respectively extracted from the sequencing data of SWU47 and NY12 materials in the early stage as template sequences. Using the gDNA of SWU47 and NY12 leaves as templates respectively, the primer pairs 1305.1-BnaA06.UBC2 SWU47 -F / R (SEQ ID No.7~8), 1305.1-BnaA06.UBC2 NY12 -F / R (SEQ ID No.9~10) were used to amplify the full-length fragments (promoter plus coding region) of BnaA06.UBC2 SWU47 and BnaA06.UBC2 NY12 , and the target fragments of about 3000 bp were purified and recovered( Figure 3 , A). The recovered target fragments were recombinantly ligated with the pCAMBIA1305.1 linearized vector digested with SalⅠ and NheⅠ, and after recombination, they were transformed into Escherichia coli DH5α. The primer pairs M13PF+1305-BnaA06.UBC2 SWU47 -R and M13PF+1305-BnaA06.UBC2 NY12 -R were used to perform colony PCR positive identification on Escherichia coli monoclonal, and the monoclonal with a PCR product length of about 3000 bp was selected and sent to BGI for sequencing( Figure 3 , B). The Geneious was used to compare the sequencing results. The homology between the cloned gene sequence and the reference sequence reached 100%, indicating that pCAMBIA1305.1-BnaA06.UBC2 SWU47, pCAMBIA1305.1 - BnaA06.UBC2 NY12 Expression vectors, named pUBC2 SWU47 :UBC2 SWU47 , pUBC2 NY12 :UBC2 NY12 and the plasmids were extracted for backup.

[0042] To explore the effect of BnaA06.UBC2 promoter sequence variation on the difference in flowering time between the two parental materials, in this study, the constructed pUBC2 SWU47 :UBC2 SWU47 , pUBC2 NY12 :UBC2 NY12 expression vectors were transformed into Col - 0. Observing the phenotypes of transgenic Arabidopsis thaliana, it was found that the flowering of pUBC2 SWU47 :UBC2 SWU47 , pUBC2 NY12 :UBC2 NY12 transgenic Arabidopsis thaliana was significantly later than that of Col - 0. At the same time, pUBC2 SWU47 :UBC2 SWU47 was earlier than pUBC2 NY12 :UBC2 NY12 transgenic plants. Identification of the BnaA06.UBC2 expression level found that the expression level of pUBC2 SWU47 :UBC2 SWU47 transgenic plants was about 2 times that of Col - 0, and the expression level of pUBC2 NY12 :UBC2 NY12 transgenic plants was about 4 times that of Col - 0 ( Figure 4 ).

[0043] The results showed that the full - length fragment of BnaA06.UBC2 was involved in the regulation of Arabidopsis thaliana flowering, and the sequence variation of BnaA06.UBC2 in the two parental materials led to changes in the function of BnaA06.UBC2 in flowering regulation, ultimately resulting in variations in the flowering time of pUBC2 SWU47 :UBC2 SWU47 , pUBC2 NY12 :UBC2 NY12 transgenic Arabidopsis thaliana.

[0044] Example 3. Construction of an expression vector for promoter - driven GUS

[0045] From the results of Example 1 and Example 2, it can be seen that BnaA06.UBC2 is involved in the regulation of Arabidopsis flowering, and sequence variations in its parental materials will affect its regulation of flowering. At the same time, sequence variations in the CDS of BnaA06.UBC2 do not affect its regulation of flowering. Therefore, we speculate that sequence variations in the promoter of BnaA06.UBC2 affect its transcriptional activity, resulting in variations in the expression level of BnaA06.UBC2 in the parental materials, and ultimately leading to differences in flowering time between the two materials. To verify this conjecture, we designed the following experiment.

[0046] Extract BnaA06.UBC2 from the sequencing data of SWU47 and NY12 materials in the early stage SWU47 and BnaA06.UBC2 NY12 The 2000 bp sequence upstream of the start codon (ATG) was used as the promoter sequence. Using the leaf gDNA of SWU47 and NY12 materials as templates respectively, primer pairs 1305.1-proUBC2 SWU47 -F / R (SEQ ID No.11~12) and 1305.1-proUBC2 NY12 -F / R (SEQ ID No.13~14) were used to amplify the promoter sequences of BnaA06.UBC2 SWU47 and BnaA06.UBC2 NY12 ( Figure 5 , A). The target fragment of about 2000 bp (SEQ ID No.15 - 16) was purified and recovered. The recovered target fragment was recombinantly ligated with the pCAMBIA1305.1 linearized vector linearized with SalⅠ and NocⅠ using recombinase. After recombination, it was transformed into Escherichia coli DH5α. Colony PCR positive identification of Escherichia coli monoclonal was performed using the universal primers M13PF + M13PR, and the monoclonal with a PCR product length of about 2000 bp was selected and sent to BGI for sequencing ( Figure 5 , B). The sequencing results were compared using Geneious. The homology between the cloned gene sequence and the reference sequence reached 100%, indicating that the pCAMBIA1305.1-proBnaA06.UBC2 SWU47 and pCAMBIA1305.1-proBnaA06.UBC2 NY12 expression vectors were successfully constructed, and were named pUBC2 SWU47 :GUS and pUBC2 NY12 :GUS respectively, and the plasmids were extracted for standby.

[0047] The constructed pUBC2 SWU47 :GUS and pUBC2 NY12: GUS expression vector, transformed into Col-0, and the T2 generation homozygous plants were screened. By observing the results of GUS chemical tissue staining, it was found that, as Figure 6 shown, pUBC2 NY12 : The staining of GUS transgenic plants in the leaves of Arabidopsis seedlings and before bolting was significantly darker than that of pUBC2 SWU47 : GUS transgenic Arabidopsis. It indicated that pUBC2 NY12 had stronger transcriptional activity than pUBC2 SWU47 , suggesting that sequence variation in the BnaA06.UBC2 promoter would lead to different transcriptional activities.

[0048] From the above results, it was known that sequence variation in the BnaA06.UBC2 promoter would affect its transcriptional activity. To further understand whether sequence variation in the BnaA06.UBC2 promoter would cause changes in the transcriptional level of BnaA06.UBC2. Therefore, in this study, the expression levels of BnaA06.UBC2 were quantitatively detected in the parental materials, and the results were as Figure 7 shown. In the leaves before bolting and flower bud tissues, the expression levels of BnaA06.UBC2 in the NY12 material were significantly higher than those in the SWU47 material. It indicated that sequence variation in the BnaA06.UBC2 promoter would lead to changes in its transcriptional activity, ultimately resulting in changes in the transcriptional level of BnaA06.UBC2.

[0049] Example 4. The poly ABRE cis element affects the transcriptional regulation of ABI5 on target genes

[0050] Through the experimental results of Example 3, it was confirmed that sequence variation in the BnaA06.UBC2 promoter would lead to changes in its transcriptional activity. We hypothesized that the changes in the transcriptional activities of the promoters in the two BnaA06.UBC2 materials might be due to sequence variation in the promoter, which caused some cis - acting elements to change, resulting in changes in the binding and regulation patterns of some upstream trans - acting factors to the two promoters, ultimately leading to changes in the transcriptional activities of the two promoters. To verify this hypothesis, we conducted the following experiments.

[0051] I. Analysis of differences in cis - acting elements of the BnaA06.UBC2 promoter in parental materials

[0052] We respectively extracted the BnaA06.UBC2 promoter sequences from the previous sequencing data of parental materials, and named them pUBC2 SWU47 and pUBC2 NY12, the cis - acting element prediction website plant care (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) was used for cis - acting element prediction. The results showed that in pUBC2 SWU47 and pUBC2 NY12 there are various cis - acting elements such as light response and hormone response. By comparison, it was found that there are 9 different cis - acting elements in pUBC2 SWU47 and pUBC2 NY12 .

[0053] II. Screening of the core sequence of pUBC2 SWU47 In order to clarify which cis - acting elements are responsible for the change in the transcriptional activity of pUBC2

[0054] , we truncated pUBC2 SWU47 and pUBC2 NY12 successively according to the different cis - acting elements, truncated them at - 839bp, - 456bp, and - 171bp respectively, constructed pGREENⅡ0800 - LUC vectors, and performed transient expression in tobacco. The transcriptional activity of the truncated promoter was judged by detecting the ratio of the fluorescence intensity of firefly luciferase to that of Renilla luciferase in the transiently expressed tobacco leaves. The experiment found that when the promoter was truncated from - 456bp to - 171bp, the transcriptional activity of the promoter decreased significantly ( SWU47 ), indicating that the core sequence of pUBC2 Figure 8 is located at - 456~ - 171bp. This sequence was named pUBC2 SWU47 , and the homologous fragment in the BnaA06.UBC2 SWU47(C) promoter was named pUBC2 NY12 by sequence alignment. Through comparison, we found that there are 2 mutations in the ABRE motif in pUBC2 NY12(C) and pUBC2 SWU47(C) . NY12(C)

[0055] III. ABI5 targets and regulates the transcriptional expression of BnaA06.UBC2

[0056] In order to find the trans - acting elements that have different regulatory effects on the promoters of the parental materials of BnaA06.UBC2, we first used pUBC2 SWU47(C) as the bait sequence and searched for sequences that bind to pUBC2 SWU47(C)As for the trans-acting factors, we screened a total of 10 transcription factors, one of which is BnaA05.ABI5. Referring to the literature, it was found that BnaA05.ABI5, as a transcription factor responsive to ABA, binds to the ABRE motif. Combining the above results, namely pUBC2 SWU47(C) and pUBC2 NY12(C) There are differences in 2 ABRE motifs, so we used BnaA05.ABI5 (BnaA05G0087100ZS) as the key candidate transcription factor for subsequent experimental verification.

[0057] Yeast one-hybrid point-to-point verification found that BnaA05.ABI5 can bind to both pUBC2 SWU47(C) and pUBC2 NY12(C) fragments ( Figure 9 , A). Further dual-luciferase experiments found that BnaA05.ABI5 can activate the expression of pUBC2 NY12(C) but cannot activate the expression of pUBC2 SWU47(C) ( Figure 9 , B).

[0058] Since BnaA05.ABI5 is an abscisic acid-responsive gene and its expression is induced by ABA, in order to verify the transcriptional regulation of BnaA05.ABI5 on BnaA06.UBC2 in rapeseed, we sprayed SWU47 and NY12 plants with ABA and further determined the binding and regulatory relationship of BnaA05.ABI5 to BnaA06.UBC2 by quantitatively detecting the expression levels of BnaA05.ABI5 and BnaA06.UBC2. The results are as Figure 10 shown. When ABA was sprayed on SWU47 and NY12, the expression levels of BnaA05.ABI5 in SWU47 and NY12 materials both increased significantly, but only the expression level of BnaA06.UBC2 in NY12 increased significantly, indicating that BnaA05.ABI5 only activates the expression of BnaA06.UBC2 NY12 and has no regulatory effect on the expression of BnaA06.UBC2 SWU47 .

[0059] Previous studies have shown that the ABA-responsive factor ABI5 is involved in flowering regulation. The abi5 mutant shows early flowering, and overexpression of ABI5 results in delayed flowering (Wang et al., 2013). The research of the present invention shows that ABI5 can regulate gene expression by binding to the ABRE (ABSCISIC ACID RESPONSE ELEMENT) motif in the promoter. This study found that there are 4 consecutive tandem ABRE motifs in the pUBC2 SWU47 sequence, but due to sequence variation, pUBC2NY12 There are only 2 consecutive ABRE motifs in the sequence. Experiments have found that BnaA05.ABI5 can bind to pUBC2 simultaneously SWU47 and pUBC2 NY12 , but BnaA05.ABI5 can only activate the expression of pUBC2 NY12 and has no obvious regulatory effect on pUBC2 SWU47 . This is consistent with previous studies that poly-ABRE motif inhibits gene expression. That is to say, the sequence variation in the promoter region of BnaA06.UBC2 in SWU47 and NY12 materials affects the transcriptional level of BnaA06.UBC2 by influencing the binding and activation ability of ABI5, ultimately resulting in a significant difference in the flowering time of the two materials

[0060] Example 5. Detection of the expression levels of flowering marker genes in parental materials

[0061] To further verify that BnaA06.UBC2 can regulate rapeseed flowering by regulating FLC expression, we quantitatively detected the expression levels of BnaA06.UBC2, BnaA10.FLC, BnaC02.FLC, BnaA02.FT, BnaC02.FT, and BnaC06.FT in the leaf tissues of SWU47 and NY12 materials at the 1st, 3rd, 5th, 7th, 9th, 10th, and 11th weeks after vernalization. The results are as follows Figure 11 shown. The expression level of BnaA06.UBC2 at each stage in NY12 was significantly higher than that in SWU47( Figure 11 , E). During the growth and development of SWU47 and NY12, the expression levels of BnaA10.FLC and BnaC02.FLC both decreased slowly. However, in the NY12 material, the decreasing rate of the expression level of BnaC02.FLC was lower than that in SWU47, and the expression levels at the 9th, 10th, and 11th weeks were significantly higher than those in SWU47( Figure 11 , A). With the growth and development of rapeseed, the expression levels of BnaA02.FT, BnaC02FT, and BnaC06.FT gradually increased in both SWU47 and NY12. However, the increasing rate of the expression level in SWU47 was significantly higher than that in NY12. At the same time, at the 9th, 10th, and 11th weeks of vernalization, the expression levels of BnaA02.FT, BnaC02FT, and BnaC06.FT in the SWU47 material were significantly higher than those in NY12( Figure 11 , B, C, D). The above results indicate that BnaA06.UBC2 can inhibit rapeseed flowering by promoting the expression of BnaC02.FLC

[0062] Example 6. Haplotype analysis of the BnaA06.UBC2 promoter in the natural population

[0063] This study shows that sequence variation in BnaA06.UBC2 leads to differences in flowering time between the parental materials of rapeseed SWU47 and NY12. To clarify the role of BnaA06.UBC2 promoter sequence variation in improving flowering time, we performed haplotype analysis using the resequencing data of a natural population consisting of 588 materials and the phenotypic data of flowering time. The results are as Figure 12 shown in Figure A. A total of 8 major haplotypes were identified, among which Hap1 had the highest proportion (38.90%), followed by Hap2 (22.40%). This study found that Hap2 was the earliest flowering haplotype with a flowering time of approximately 162 days, Hap5 was the latest flowering haplotype with a flowering time of approximately 170 days, and the flowering times of Hap1 / 3 / 4 / 6 / 7 / 8 were between the two ( Figure 12 , Figure B). It was found that the late-flowering parent NY12 belonged to Hap5, and the early-flowering parent belonged to Hap4. Through the calculation of evolutionary selection using the coefficient of kurtosis (Fst) and nucleotide diversity (π), it was found that the BnaA06.UBC2 promoter was located in the selection interval of different ecotypes of rapeseed ( Figure 12 , Figure C).

[0064] Example 7. BnaA06.UBC2 Regulatory Model for Rapeseed Flowering

[0065] Based on the above experimental results, we propose the following BnaA06.UBC2 regulatory model for rapeseed flowering ( Figure 13 ). Due to the sequence variation of the BnaA06.UBC2 promoter, two ABRE motifs are deleted in the promoter of BnaA06.UBC in the NY12 material. The change in the ABRE motif leads to variation in the regulation of the two promoters by the upstream transcription factor BnaA05.ABI5, resulting in a significantly higher expression level of BnaA06.UBC2 in the NY12 material than in SWU47. BnaA06.UBC2 interacts with the E3 ubiquitin ligase BnaA05.HUB1, provides ubiquitin molecules for BnaA05.HUB1 to modify the FLC H2B histone, and ultimately leads to an increase in the expression level of FLC. Since FLC is a flowering inhibitor, the increase in its expression level inhibits rapeseed flowering, ultimately resulting in significantly later flowering of the NY12 material than SWU47.

[0066] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A promoter of a separated BnaA06.UBC2 gene, characterized in that, The promoter contains at least one natural variation of the poly-ABRE motif, wherein the variation is selected from: a) The promoter sequence shown in SEQ ID NO.15, named pUBC2 SWU47 ; b) The promoter sequence shown in SEQ ID NO.16, named pUBC2 NY12 , compared with pUBC2 SWU47 , pUBC2 NY12 contains a reduced number of ABRE motifs.

2. The promoter according to claim 1, wherein The pUBC2 NY12 Relative to pUBC2 SWU47 Two consecutive ABRE motifs are missing, resulting in a weakened binding ability of the transcription factor BnaA05.ABI5 to the promoter.

3. Use of the promoter according to claim 1 in regulating differential expression of the BnaA06.UBC2 gene.

4. The application according to claim 3, wherein The promoter is pUBC2 SWU47 When, the expression level of the BnaA06.UBC2 gene is low; The promoter is pUBC2 NY12 When, the expression level of the BnaA06.UBC2 gene is relatively high. 5.pUBC2 NY12 Use in delaying plant flowering, characterized in that, Using pUBC2 NY12 Driving the overexpression of the BnaA06.UBC2 gene in plant cells can up-regulate the expression level of the BnaC02.FLC gene, thereby delaying the flowering time of plants. The plants are Arabidopsis thaliana or rapeseed.

6. A method for cultivating a late-flowering rapeseed line, characterized in that, Use pUBC2 NY12 Drive the overexpression of the BnaA06.UBC2 gene in rapeseed cells, increase the expression level of the BnaA06.UBC2 gene, and obtain a late-flowering rapeseed line.

7. The method according to claim 6, characterized in that, The sequence of the BnaA06.UBC2 gene is as shown in SEQ ID NO. 6.

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