Use of a bcDET2 gene

Abstract

The application relates to the field of plant genetic breeding technology, and particularly relates to application of a BcDET2 gene, a base sequence of the BcDET2 gene is shown as SEQ ID NO. 1, and an amino acid sequence of an expression protein is shown as SEQ ID NO. 2; overexpression of the BcDET2 gene in Arabidopsis thaliana can make the Arabidopsis thaliana bloom early; overexpression of the BcDET2 gene in Brassica campestris can significantly improve the blooming time of the transgenic plants; overexpression of the BcDET2 gene in Brassica campestris can change the expression levels of FUL1, SOC1, FLC1 and the like, and affect the blooming time of the plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetics and breeding technology, specifically to the application of the BcDET2 gene. Background Technology

[0002] *Brassica campestris* L. syn. B. rapa L. ssp. chinensis var. rosularis Tsen, a variety belonging to the Brassica genus of the Brassicaceae family, is widely grown in the Yangtze-Huaihe River basin of my country. It is highly favored by consumers for its high nutritional value and excellent flavor. Chinese cabbage requires low-temperature vernalization and photoperiod induction to flower and set seeds; therefore, cultivation often requires avoiding environmental conditions conducive to vernalization to prevent premature bolting. In recent years, research on the late bolting characteristics and utilization of Chinese cabbage has gradually become a new hot topic in China, leading to the development of many new varieties. Internationally, research on how to control or mitigate the environmental conditions for premature bolting has been more detailed. As a major winter and spring vegetable, late bolting breeding materials for *Brassica campestris* are gradually receiving attention.

[0003] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of how to regulate the bolting and flowering time of Brassica oleracea through gene regulation, and to provide an application of the BcDET2 gene.

[0005] To achieve the above objectives, this invention discloses the application of BcDET2 gene overexpression in promoting early bolting and flowering in transgenic Arabidopsis and Brassica rapa plants, wherein the base sequence of the BcDET2 gene is shown in SEQ ID NO.1.

[0006] The amino acid sequence of the expressed protein of the BcDET2 gene is shown in SEQ ID NO.2.

[0007] The overexpression vector for the BcDET2 gene is pCAMBIA1305-BcDET2.

[0008] The present invention also discloses the application of the BcDET2 gene in promoting the flowering process of cruciferous plants, wherein the BcDET2 gene regulates the expression of FUL1, SOC1, FLC1 and FT1 genes.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention clones the BcDET2 gene, and overexpression of this BcDET2 gene in Arabidopsis thaliana can cause it to bolt and flower earlier; overexpression in spinach can significantly increase the bolting and flowering time of transgenic plants; overexpression in spinach can change the expression levels of genes such as FUL1, SOC1, and FLC1, and affect the bolting and flowering time of plants. Attached Figure Description

[0010] Figure 1 A clone of the BcDET2 gene;

[0011] Figure 2 Functional analysis of the BcDET2 gene in Arabidopsis thaliana: Overexpression of the BcDET2 gene in Arabidopsis thaliana. A: Protein abundance of the BcDET2 gene at different vernalization times. B: Transcriptional level of the BcDET2 gene at different vernalization times. C: Relative expression level of BcDET2 at different vernalization times as analyzed by quantitative real-time analysis.

[0012] Figure 3 For the functional analysis of the BcDET2 gene in Arabidopsis thaliana, A: Overexpression of the BcDET2 gene in Arabidopsis thaliana can significantly advance flowering; B: Analysis of the number of rosette leaves in BcDET2 transgenic Arabidopsis thaliana and wild-type plants at flowering; C: Analysis of the number of flowering days in BcDET2 transgenic Arabidopsis thaliana and wild-type plants; D: Relative expression level of the AtFUL1 gene in BcDET2 transgenic Arabidopsis thaliana and wild-type plants; E: Relative expression level of the AtSOC1 gene in BcDET2 transgenic Arabidopsis thaliana and wild-type plants; F: Relative expression level of the AtFLC1 gene in BcDET2 transgenic Arabidopsis thaliana and wild-type plants.

[0013] Figure 4 For the functional analysis of the BcDET2 gene in *Brucea javanica*, the following are the results: A: Overexpression of the BcDET2 gene in *Brucea javanica* significantly advances flowering; B: Analysis of the number of rosette leaves in BcDET2 transgenic *Brucea javanica* and wild-type plants at flowering time; C: Analysis of the number of flowering days in BcDET2 transgenic *Brucea javanica* and wild-type plants; D: Relative expression level of the BcFUL1 gene in BcDET2 transgenic *Brucea javanica* and wild-type plants; E: Relative expression level of the BcSOC1 gene in BcDET2 transgenic *Brucea javanica* and wild-type plants; F: Relative expression level of the BcFLC1 gene in BcDET2 transgenic *Brucea javanica* and wild-type plants. Detailed Implementation

[0014] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0015] I. Cloning of the BcDET2 gene

[0016] 1. Place 1-month-old seedlings of *Brucea javanica* at 4℃ for 30 days of low-temperature vernalization, extract total RNA from tender leaves, and reverse transcribe it into cDNA.

[0017] 2. Using the BraA10g023600.3C gene sequence of Chinese cabbage from the BRAD database (http: / / brassicadb.cn) as a template, upstream and downstream specific primers BcDET2-F1 and BcDET2-R1 were designed.

[0018] BcDET2-F1:ATGGAGATGGTGACGAG

[0019] BcDET2-R1:CTAGAACACAAAGGGAATCAG

[0020] 3. Using cDNA template, BcDET2-F1 as the upstream primer and BcDET2-R1 as the downstream primer, a PCR reaction was performed; the purified PCR product was ligated into the pMD-19 vector to obtain the pMD-BcDET2 recombinant vector; this vector was transformed into DH5α Escherichia coli, and after positive PCR verification of the bacterial culture, sequencing was performed to confirm the result.

[0021] 4. For example Figure 1 As shown, the full-length BcDET2 gene sequence, 825 bp, encoding 274 amino acids, was cloned. Its base sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2.

[0022] II. Detection of BcDET2 gene expression level

[0023] 1. Using tender leaf tissues from 1-month-old *Brassica oleracea* seedlings after vernalization at 4℃ for 0, 15, and 30 days, transcriptome and proteome sequencing were performed using full-length transcriptome sequencing and TMT-labeled LC-MS / MS techniques to analyze the transcriptional and protein levels of the BcDET2 gene.

[0024] 2. Take tender leaf tissues from 1-month-old seedlings of *Brucea javanica* and vernalize them at 4℃ for 0, 15, and 30 days. Extract RNA and reverse transcribe it into cDNA.

[0025] 3. Using the BcDET2 gene sequence as a template, design specific upstream and downstream primers BcDET2-F2 and BcDET2-R2 for real-time PCR.

[0026] BcDET2-F2:TCGAGGTTCTGTTCATTCACAAG

[0027] BcDET2-R2:AACGCCGTGGATGAGAAGTA

[0028] 4. Using the BcActin gene as an internal reference (F: TGGGTTTGCTGGTGACGAT, R: TGCCTAGGACGACCAACAATACT), quantitative real-time PCR analysis was performed.

[0029] 5. Results are as follows Figure 2 As shown, the transcription and protein levels of the BcDET2 gene were significantly higher during vernalization in January-aged seedlings than at the beginning of vernalization. Quantitative real-time PCR analysis revealed that the gene expression level gradually increased with vernalization time. This indicates that the BcDET2 gene may be involved in the vernalization pathway to regulate the timing of bolting and flowering in plants.

[0030] III. Construction of BcDET2 gene overexpression vector

[0031] 1. Using software such as CE Design V and Snap Gene, homologous recombination primer sequences BcDET2-F2 and BcDET2-R2, and BcDET2-F3 and BcDET2-R3 were designed for the BcDET2 gene.

[0032] BcDET2-F2: gacgatgacgataagggatccATGGAGATGGTGACGAG

[0033] BcDET2-R2: tgcctgcaggtcgactctagaGAACACAAAGGGAATCAG

[0034] 2. Using the pMD-BcDET2 recombinant vector template, PCR amplification was performed using BcDET2-F2 / BcDET2-R2, and the PCR product BcDET2 fragment was purified.

[0035] 3. The pCAMBIA1305 vector (containing the 35S-MYC-nos multiple cloning expression cassette) was double-digested using BamHI and XbaI restriction endonucleases, and the digested fragments were recovered.

[0036] 4. Use The plus One step PCR Cloning Kit ligates the BcDET2 fragment, a purified PCR product of the BcDET2-F2 / BcDET2-R2 primer pair, with a pCAMBIA1305 double-digested fragment. The ligation products are then transformed into E. coli DH5α, and bacterial PCR is performed for verification. Positive bacterial cultures are selected for sequencing confirmation, ultimately yielding the p1305-BcDET2 overexpression vector.

[0037] 5. The p1305-BcDET2 overexpression vector was transformed into Agrobacterium GV3101 to finally obtain the GV3101-p1305-BcDET2 engineered strain.

[0038] IV. Genetic transformation of Arabidopsis thaliana using the BcDET2 gene

[0039] 1. The engineered bacteria GV3101-p1305-BcDET2 were used to infect Arabidopsis thaliana (Col-1) using the inflorescence infection method to obtain T0 generation transgenic seeds 35S:BcDET2.

[0040] 2. The obtained T0 generation transgenic seeds were screened for hygromycin resistance to identify resistant plants, and positive plants were obtained by PCR.

[0041] 3. After obtaining T1 seeds from positive plants, the same method described above is used until stable T3 generation transgenic plants are obtained.

[0042] 4. Col-1 and 35S:BcDET2 transgenic plants were cultured under normal conditions, and the expression levels of genes such as DET2, FLC1, SOC1, and FUL1 were analyzed. Simultaneously, the bolting and flowering time of each plant, and the number of rosette leaves when the first flower opened, were observed. Results are as follows: Figure 3 As shown, the expression levels of genes such as SOC1 and FUL1 in the 35S:BcDET2 transgenic plants were significantly higher than those in the wild-type Col-1 plants, while the expression level of FLC1 gene was significantly lower than that in the wild-type plants. In addition, the bolting and flowering time and the number of rosette leaves of the 35S:BcDET2 transgenic plants were significantly lower than those of the wild-type Col-1 plants.

[0043] V. Genetic transformation of *Brucea javanica* plants using the BcDET2 gene

[0044] 1. The engineered bacteria GV3101-p1305-BcDET2 obtained was used to infect the flower buds of *Brucea javanica* using the inflorescence infection method. After the infected flower buds opened, they were self-pollinated to obtain T0 generation transgenic seeds.

[0045] 2. After the p1305-BcDET2 transgenic seeds germinated, positive transgenic plants were obtained by hygromycin and BcDET2 target gene PCR identification. The expression level of BcDET2 gene in the positive plants was detected by real-time PCR, and plants with BcDET2 gene overexpression were screened.

[0046] 3. One-month-old wild-type *Brucea javanica* WT and BcDET2 transgenic seedlings were vernalized at 4℃ for 30 days, then transferred to normal culture conditions. The timing of bolting and flowering, as well as the number of rosette leaves at the time of bolting and flowering, were observed. Results are as follows: Figure 4As shown, plants overexpressing the BcDET2 gene had a shorter bolting and flowering time than wild-type plants, a significantly reduced number of rosette leaves, a significantly reduced expression level of the FLC1 gene, and significantly upregulated genes such as SOC1 and FUL1.

[0047] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. The application of BcDET2 gene overexpression in promoting early bolting and flowering in transgenic Arabidopsis and Brassica rapa plants, characterized in that, The base sequence of the BcDET2 gene is shown in SEQ ID NO.

1.

2. The application of BcDET2 gene overexpression as described in claim 1 in promoting early bolting and flowering in transgenic Arabidopsis and Brassica rapa plants, characterized in that... The amino acid sequence of the expressed protein of the BcDET2 gene is shown in SEQ ID NO.

2.

3. The application of BcDET2 gene overexpression as described in claim 1 in promoting early bolting and flowering in transgenic Arabidopsis and Brassica rapa plants, characterized in that... The overexpression vector for the BcDET2 gene is pCAMBIA1305-BcDET2.

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