Application of rapeseed EOD3 gene in increasing rapeseed plant height, branch number and biomass

Abstract

The present invention discloses the application of rapeseed EOD3 gene in improving rapeseed plant height, branch number and biomass, and belongs to the field of plant genetic engineering. The nucleotide sequence of the rapeseed EOD3 gene is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2. Overexpression of the gene in Arabidopsis can significantly increase plant height and biomass, and overexpression of the gene in rapeseed can significantly increase plant height, branch number and biomass. The rapeseed EOD3 gene has the application of improving plant height, branch number and biomass, and also has the application of improving plant varieties or preparing transgenic plants. The present invention has a good application prospect in the breeding of crops such as rapeseed with improved plant height, branch number and / or biomass.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and more specifically relates to the application of rapeseed EOD3 gene in improving rapeseed plant height, branch number and biomass. Background Art

[0002] Rapeseed plant height, branch number, and biomass have always attracted the attention of rapeseed researchers, and they have a certain impact on rapeseed yield. Many rapeseed plant height regulatory QTL loci have been discovered, but only a few rapeseed plant height genes have been identified, such as BnaMAX1 (Zheng et al., 2020) and BnaC04.BIL1 (Yang et al., 2021). So far, only a few genes may affect the number of branches after mutation, such as AP1 (Shah et al., 2018) and TFL1 (Sriboon et al., 2020), and the genes that can truly regulate the number of branches have yet to be identified. In addition, there are few reports on the regulatory genes of rapeseed biomass. Therefore, in-depth research on the regulatory mechanisms of plant height, branch number, and biomass in rapeseed is of great significance for plant type improvement and yield increase.

[0003] Rapeseed is used as feed, and its production efficiency is higher than that of Gramineae. For example, the daily growth rate of feed rapeseed is about 2 to 2.5 times that of ryegrass (Bell et al., 2020). Mixing silage with corn can also significantly improve the overall nutritional quality and fermentation quality of feed (Yin Tingfang and Zhang Fenghua 2018). Therefore, feed rapeseed has the advantages of low planting cost, short growth cycle, fast growth rate, high yield, good quality, high feeding value, and good feeding effect, which is conducive to promoting the development of animal husbandry (Fu Tingdong et al. 2004). At the same time, rapeseed can also be used as a vegetable. The rapeseed stalks can be collected for fresh consumption and pickling. The larger the biomass, the higher the stalk value, and thus the higher its economic value.

[0004] The accumulation of rapeseed biomass is a concrete manifestation of its vegetative growth. Its photosynthesis, respiration, and the transport and distribution of substances determine the size of the biomass, which is also the basis for the final formation of grain yield (Zhang Hongzhi et al. 2020). Genes related to biomass modulation affect the growth, development, and synthesis pathways of related substances of cell structures. For example, overexpression of AtGA2ox8 reduces the accumulation of Arabidopsis biomass (Zhao et al. 2010). In addition, genes that modify cell walls such as CesA, GH9, GH10, GT61, and GT43 have a certain effect on the biomass accumulation of plants. The genetic analysis of high-biomass hybrid Arabidopsis by Yang et al. showed that candidate genes with high biomass are involved in the regulation of the cell cycle and then in the regulation of the immune response to biological and non-biological stimuli (Yang et al. 2017). Wang Yanhua et al. (2020) found through genetic association analysis and WGCNA of natural population materials of rapeseed that HCEF1 encodes chloroplast-1,6-bisphosphate (FBPase), thereby coordinating the metabolism of carbon and nitrogen and affecting biomass. Summary of the invention

[0005] The present invention finds that overexpression of the cytochrome P450 protein gene EOD3 of rapeseed (referred to as BnaA4.EOD3 gene in the present invention) in Arabidopsis can significantly increase plant height and biomass, and overexpression in rapeseed can increase plant height, branch number and biomass. The function of the gene in regulating plant height, branch number and biomass has not been reported yet. The gene is a new gene for regulating plant height, branch number and biomass of rapeseed, and can be applied to crop breeding to increase plant height, branch number and biomass.

[0006] The object of the present invention is to provide an application of rapeseed EOD3 gene in increasing rapeseed plant height, branch number and biomass. Genetic transformation in Arabidopsis thaliana and rapeseed confirmed that BnaA4.EOD3 gene mainly increases total biomass by increasing plant height and branch number.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A rapeseed BnaA4.EOD3 gene, the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2. The nucleotide sequence of the rapeseed BnaA4.EOD3 gene is preferably shown in SEQ ID NO.1.

[0009] Based on the function of the rapeseed BnaA4.EOD3 gene discovered by the present invention, it has at least one of the following applications: application of the rapeseed BnaA4.EOD3 gene in increasing the height of plants, application of the rapeseed BnaA4.EOD3 gene in increasing the number of plant branches, and application of the rapeseed BnaA4.EOD3 gene in increasing plant biomass.

[0010] The BnaA4.EOD3 gene is also used in plant variety improvement or preparation of transgenic plants. The plant variety improvement is to improve the plant height, branch number and / or biomass of the plant, and the preparation of transgenic plants is to prepare transgenic plants with increased plant height, branch number and / or biomass.

[0011] The plants include rapeseed, Arabidopsis thaliana and the like.

[0012] The above application can be achieved by overexpressing the BnaA4.EOD3 gene in plants using conventional methods in the art.

[0013] Compared with the prior art, the present invention has the following advantages: the present invention discloses for the first time the application of rapeseed BnaA4.EOD3 gene in improving rapeseed plant height, branch number and biomass. The transgenic results using Arabidopsis thaliana and rapeseed as receptors confirm that the plant height and biomass of plants overexpressing the BnaA4.EOD3 gene are significantly improved compared with wild-type receptors, and overexpression in rapeseed can also increase the branch number. Therefore, the BnaA4.EOD3 gene has a good application prospect in the breeding of crops such as rapeseed for improving plant height, branch number and / or biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The phenotypic statistics of the positive strains of BnaA4.EOD3 transgenic Arabidopsis thaliana are shown in Figure 1. A is the relative expression level of BnaA4.EOD3 in different transgenic individual strains of BnaA4.EOD3. BC is the phenotypic data of plant height and biomass of each positive transgenic strain and negative control (Col); the data in the figure are the mean ± SD of at least 3 biological replicates, b indicates significant difference at the P < 0.01 level, and c indicates significant difference at the P < 0.001 level.

[0015] Figure 2 The phenotypic statistics of the positive transgenic rapeseed lines of BnaA4.EOD3 gene are shown in Figure 4. Among them, AC is the phenotypic data of plant height, branch number and biomass of each positive transgenic line and negative control (Westar).

[0016] Figure 3 The spatial structure of the rapeseed EOD3 protein of the present invention and the one that has been published. A and B: the spatial structure of the rapeseed EOD3 protein of the present invention, C and D: the spatial structure of the rapeseed BnaEOD3 protein that has been published online. Note: The spatial structure of the protein is automatically generated by the software, and the arrows point to the positions with large differences in spatial structure. DETAILED DESCRIPTION

[0017] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention.

[0018] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the reagents or materials used are all from commercial channels.

[0019] Example 1: Cloning of the coding region of the rapeseed BnaA4.EOD3 gene

[0020] The BnaA4.EOD3 gene was amplified by PCR using the first chain of cDNA of Double 11 in Brassica napus L. as a template, the forward primer of BnaA4.EOD3 was: 5'-cgcgagctcggtaccATGGCTACCGAACTCGCAAG-3', the reverse primer was: 5'-ggatccccgggtaccctaAGTACGTCTACGGCGCAGTTTAG-3', and finally the nucleotide sequence shown in SEQ ID NO.1 was obtained, which encoded a protein with the amino acid sequence shown in SEQ ID NO.2.

[0021] In the above scheme, in order to achieve connection with the vector, corresponding homologous recombination arm sequences were added to the 5' end of the forward and reverse primers.

[0022] Example 2: Construction of overexpression vector

[0023] The plasmid expression vector pCAMBIA2300S (provided by Wuhan Tianwen Biotechnology Co., Ltd., based on the commercial pCAMBIA2300 vector backbone, with 35S promoter added to its multiple cloning site) was linearized, and the reaction system was as follows: BamHI 2μL, 10*cut smart buffer 5μL, vector 25μL, sterile water 18μL; total 50μL. Reaction at 37℃ for 2h.

[0024] Then, the double-enzyme-digested plasmid and PCR product were purified and recovered according to the instructions of the agarose gel recovery kit (Axygen, AP-96-GX-1, USA), and the recovery results were detected by 1% agarose gel.

[0025] The cloned PCR product of rapeseed BnaA4.EOD3 and the linearized plasmid expression vector pCAMBIA2300S were homologously recombined. The reaction system was as follows: pCAMBIA2300S (50ng / μL) 2μL, PCR product 4μL, 2×one step reaction mix 6μL. React at 50℃ for 15min, and then transform DH5α. The subsequent operations are as follows:

[0026] (1) Take 10 μL of the above reaction product and place it in DH5α competent cells on ice for 20 min;

[0027] (3) After heating at 42°C for 90 seconds, place in an ice water bath for 3 minutes;

[0028] (3) Add 400 μL LB liquid medium and culture at 37°C and 150 rpm / min for 60 min;

[0029] (4) Cultivate overnight on LB solid medium containing kanamycin (Kan, 50 mg / L);

[0030] (5) Pick a single colony and culture it on LB solid medium (Kan, 50 mg / L) for about 12 h. At the same time, perform PCR colony detection to identify positive clones.

[0031] Example 3: Transformation of Agrobacterium tumefaciens GV3101

[0032] The plasmid was extracted using a plasmid extraction kit, and the specific instructions were followed. The sequence of the target fragment recombined into the vector was confirmed to be accurate by further first-generation sequencing. The above recombinant plasmid was then transformed into Agrobacterium GV3101, and the method steps were as follows:

[0033] (1) Add 1 μg of purified plasmid to 100 μL competent Agrobacterium GV3101 and gently shake to mix;

[0034] (2) Place on ice for 5 min and immediately place in liquid nitrogen for 5 min;

[0035] (3) 37°C water bath for 5 min;

[0036] (4) Add 350 μL of LB medium and culture on a shaker at 200 rpm / min at 28°C for 1 h;

[0037] (5) Centrifuge to remove most of the supernatant, precipitate, mix gently by pipetting, take 100 μL of the bacterial solution, and apply it on an LB plate containing 50 mg / L kanamycin and 12.5 mg / L rifampicin;

[0038] (6) After culturing at 28°C for 48 h, resistant colonies were observed to grow. A single colony was inoculated into 2 mL of LB medium (containing kanamycin and rifampicin) and cultured at 28°C with shaking overnight.

[0039] (7) PCR identification of positive colonies.

[0040] Example 4: Agrobacterium-mediated transformation of Arabidopsis thaliana by floral dip

[0041] Dyeing solution preparation: 5% sucrose + 0.015% surfactant.

[0042] YEP medium preparation: yeast extract 10 g / L, peptone 10 g / L, NaCl 5 g / L; high pressure sterilization at 121°C, 18 min.

[0043] The specific steps of conversion are as follows:

[0044] (1) Pick a single colony and place it in 10 mL YEP medium (Kan + :50mg / L,Gen + :25mg / L,Rif + : 25 mg / L), 28°C, 200 rpm / min shaking culture for 8 h;

[0045] (2) Take 500 μL of bacterial solution from (1) and add 200 mL of YEP medium (Kan: 50 mg / L, Gen: 25 mg / L, Rif + :25mg / L), and cultured at 28℃, 200rpm / min with shaking until OD 600 =1.5-2.0;

[0046] (3) Centrifuge the bacterial solution in (2) at 6000 rpm / min for 10 min. Suspend the precipitate with transformation solution and adjust the final OD 600 Between 1.5-2.0;

[0047] (4) Before transformation, cut off the siliques that have bloomed;

[0048] (5) Dip the inflorescence for 30 seconds to 1 minute, culture in the dark for 24 hours, and then switch to normal culture;

[0049] (6) One week later, the inflorescences were transformed again and cultured normally until they bloomed and bore fruit. The harvested seeds were recorded as the T0 generation.

[0050] Example 5: Screening of positive transgenic Arabidopsis

[0051] Preparation of 1 / 2MS solid culture medium (1 / 2MS + 24% sucrose + 0.8% agar (special for microbial culture)): weigh 2.215g MS (Murashing & Skoog basal mediun w / vitamins), dissolve in pure water (a small amount); weigh 24g sucrose, dissolve, and make up to 1L; weigh 8g Phytagel; adjust the pH to 5.8; divide into portions, and sterilize under high pressure (120°C, 20min).

[0052] The specific steps for sterilizing Arabidopsis seeds are as follows: wash once with 75% ethanol for 3-5 minutes; wash three times with ddH2O; wash once with 10% sodium hypochlorite for 3-5 minutes; wash three times with ddH2O; and suspend with a small amount of 0.1% agar.

[0053] Resistance screening of transgenic Arabidopsis and PCR positive identification: After the harvested T0 Arabidopsis seeds were disinfected, they were screened on resistance plates containing kanamycin (50 mg / L) and Meropen (the concentration of the antibacterial working solution was 25 mg / L). After 7-10 days, the untransformed Arabidopsis seeds did not take root, turned yellow and died, while the transformed Arabidopsis seedlings were able to grow normally. After flowering and seeding, the seeds of the individual plants were collected and recorded as the T1 generation. The T1 seeds produced by the T1 generation Arabidopsis resistant seedlings were screened for resistance again to obtain the T2 generation resistant seedlings. At least 7 individuals were selected from each T2 generation line (a total of 13 lines were selected and named EOD3-2, EOD3-3, EOD3-4, EOD3-5, EOD3-6, EOD3-7, EOD3-9, EOD3-11, EOD3-12, EOD3-13, EOD3-14, EOD3-16, and EOD3-18), the whole genome DNA of the leaves was extracted, and then these plants were PCR positive identified.

[0054] The identification primers are as follows: vector reverse primer: pC2300s-R: 5'-gagaaactcgagcttgcatgc-3', target fragment forward primer EOD3-F: 5'-GGATTGTATCCGAGCATCGTAATC-3'. The amplification system and method are as follows: 20 μL reaction system is shown in Table 1.

[0055] Table 1 PCR reaction system of positive plants

[0056]

[0057] PCR reaction procedure: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 59℃ annealing for 30s, 72℃ annealing extension for 1min, 34 cycles; 72℃ extension for 10min, 4℃ storage for future use. Then the above PCR products were tested by 1% agarose gel electrophoresis, and the results showed that the T2 generation of the 13 strains screened by the resistance medium were all positive.

[0058] Example 6: Real-time fluorescence quantitative PCR detection of transformed gene expression

[0059] After all the individual plants of the 13 T2 generation lines transformed with the overexpression vector in Example 5 were positively identified, mixed leaves of the positive individual plants were selected from each line for RNA extraction and reverse transcription and real-time fluorescence quantitative PCR were performed to detect the expression of the gene.

[0060] Arabidopsis β-actin1 (at2g37620) (Rus et al., 2006) was used as the internal reference gene. The forward primer of BnaA4.EOD3 gene was 5'-GCCATAGTATTCGTATGGCTCG-3', and the reverse primer was 5'-GTGAGCTAGAGTGCTGGACATG-3'.

[0061] Arabidopsis leaf RNA extraction, reverse transcription of the first strand of cDNA, and fluorescence quantitative PCR were all performed using the kit according to the instructions. Figure 1 As shown in A, BnaA4.EOD3 was highly expressed in these positive transgenic plants.

[0062] Example 7: Phenotypic identification of T2 transgenic Arabidopsis lines

[0063] For the character examination of the T2 generation Arabidopsis plants used in Example 6, eight mature plants were selected from each line to measure plant height and whole plant dry weight (biomass).

[0064] The results are as follows Figure 1 As shown: the plant height of the transgenic lines EOD3-2, EOD3-3, EOD3-4, EOD3-5, EOD3-6, EOD3-7, EOD3-9, EOD3-11, EOD3-12, EOD3-13, EOD3-14, EOD3-16, and EOD3-18 increased significantly ( Figure 1 The biomass of these strains also increased significantly compared to the control ( Figure 1 In C), the increase ranged from 36% to 68%, indicating that the BnaA4.EOD3 gene increased plant height and biomass at the same time.

[0065] Example 8: Agrobacterium-mediated transformation of rapeseed Westar with overexpression vector

[0066] The genetic transformation experiment of rapeseed infected with Agrobacterium tumefaciens GV3101 containing the BnaA4.EOD3 gene overexpression vector pCAMBIA2300S was commissioned to Wuhan Boyuan Biotechnology Co., Ltd. The genetic transformation process and positive identification were based on the company's experimental methods (https: / / plant.biorun.com / single / 20).

[0067] Example 9: Phenotypic identification of T0 generation transgenic rapeseed

[0068] In Example 9, 19 T0 generation BnaA4.EOD3 gene overexpressing positive strains and 4 control group negative plants were used to perform phenotypic measurements after normal growth to maturity in the greenhouse, mainly including plant height, number of branches and dry weight of the whole plant at maturity (total biomass).

[0069] The results are as follows Figure 2 As shown: the plant height of transgenic lines of ox-EOD3-1, ox-EOD3-2, ox-EOD3-3, ox-EOD3-4, ox-EOD3-5, ox-EOD3-6, ox-EOD3-7, ox-EOD3-8, ox-EOD3-9, ox-EOD3-10, ox-EOD3-11, ox-EOD3-12, ox-EOD3-13, ox-EOD3-15, ox-EOD3-16, ox-EOD3-17, ox-EOD3-18, ox-EOD3-19, and ox-EOD3-20 was increased compared with the control group, among which there were 12 positive lines with an increase of more than 20% (accounting for 63% of the total positive lines, Figure 2 In addition, among the 19 positive strains, 14 strains had more than 20% more branches than the control group ( Figure 2 At the same time, the total biomass of 14 positive strains (74% of the positive strains) increased by more than 20% compared with the control group ( Figure 2 The results showed that overexpression of EOD3 in rapeseed could increase the plant height and branch number, and ultimately lead to an increase in the total biomass of rapeseed.

[0070] Example 10: Comparative analysis of the amino acid sequence and protein structure of BnaA4.EOD3 in the present invention and the amino acid sequence and protein structure of BnaEOD3 in rapeseed published

[0071] Through sequence comparison analysis, it was found that the amino acid sequence SEQ ID NO.2 of the protein encoded by BnaA4.EOD3 in the present invention and the protein sequence SEQ ID NO.3 (derived from different rapeseed varieties) encoded by the reported gene BnaA04g27100D had a similarity of 99%. The spatial structures of the two proteins were predicted using the website http: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index, and the predicted protein structures were observed using the website http: / / www.sbg.bio.ic.ac.uk / ~ezmol / . The results showed that although the two sequences differed by only one amino acid, their protein spatial structures were quite different: Figure 2As shown (A, B: the spatial structure of the BnaA4.EOD3 protein of the present invention, C, D: the spatial structure of the rapeseed BnaEOD3 protein published online). This analysis indicates that they have different functions. Muhammad et al. (2020) used gene editing to delete the functions of three homologous copies of BnaA04g27100D, resulting in shorter siliques and smaller seed weights, indicating that the gene can affect seed development. No protein function similar to that discovered in the present invention was found, which further confirmed that the differences in the two sequences led to different protein functions.

[0072] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A type of rapeseed BnA . EOD3 A gene characterized by: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The rapeseed according to claim 1 BnA . EOD3 A gene characterized by: Its nucleotide sequence is shown in SEQ ID NO.

1.

3. The rapeseed according to claim 1 or 2 BnA . EOD3 The use of genes in improving plant strains is characterized by: By BnA . EOD3 The application is achieved by overexpressing the gene in plants; the plants are rapeseed and Arabidopsis thaliana.

4. The rapeseed according to claim 1 or 2 BnA . EOD3 The application of a gene in increasing the number of plant branches is characterized by: By BnA . EOD3 The application is achieved by overexpressing the gene in a plant; the plant is rapeseed.

5. The rapeseed according to claim 1 or 2 BnA . EOD3 The application of genes in increasing plant biomass is characterized by: By BnA . EOD3 The application is achieved by overexpressing the gene in plants; the plants are rapeseed and Arabidopsis thaliana.

6. The rapeseed according to claim 1 or 2 BnA . EOD3 The application of genes in plant variety improvement is characterized by: The plant variety improvement is to improve the plant height and / or branch number and / or biomass of the plant by BnA . EOD3 The application is achieved by overexpressing the gene in plants; the plants are rapeseed and Arabidopsis thaliana.

7. The rapeseed according to claim 1 or 2 BnA . EOD3 The use of a gene in preparing a transgenic plant is characterized by: The preparation of transgenic plants is to prepare transgenic plants with increased plant height and / or branch number and / or biomass, by BnA . EOD3 The application is achieved by overexpressing the gene in plants; the plants are rapeseed and Arabidopsis thaliana.

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