Gene for regulating and controlling plant seed size, overexpression vector, agrobacterium tumefaciens and application

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene for regulating and controlling the size of a plant seed, an overexpression vector, agrobacterium and application. The gene is a BnaA03. MKK6 gene, the nucleotide sequence of the gene is as shown in SEQ ID NO: 1, the coding sequence of the gene is as shown in SEQ ID NO: 2, and the protein sequence of the gene is as shown in SEQ ID NO: 3. The plants comprise brassica napus and / or arabidopsis thaliana. According to the invention, a BnaA03.MKK6 transgenic material is constructed, agronomic traits are investigated, and basic functions and molecular mechanisms of the BnaA03.MKK6 transgenic material are preliminarily explored. Researches find that the BnaA03. MKK6 protein is expressed on a cell nucleus and a cell membrane and has tissue expression specificity; and the BnaA03. MKK6 can positively regulate and control the size of the plant seeds and the elongation of primary roots. The invention provides a new thought and direction for high-quality molecular breeding of brassica napus.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a gene for regulating plant seed size, an overexpression vector, Agrobacterium and applications. Background Art

[0002] Brassica napus is one of the three major types of rapeseed, which is a natural interspecific hybridization between Brassica rapa and Brassica oleracea. As one of the important oil crops in my country and the only winter oil crop in my country, Brassica napus has high economic and nutritional value. It can not only be used to produce edible oil, but also is an important industrial raw material for high-protein feed, high-energy fuel, etc. However, due to my country's large population, limited planting area, complex terrain, changeable climate, frequent diseases and insect pests, etc., the quality and yield of rapeseed are seriously limited. The increase in rapeseed yield is of great significance to solving the problems of shortage of vegetable oil resources in my country, and it helps to ensure the security of my country's oil supply. Thousand-grain weight is an important factor in rapeseed yield, and seed size is a key phenotype affecting thousand-grain weight. Research on the mechanism of regulating seed size can provide an important theoretical basis for crop breeding, and genes that regulate seed size can also be used to increase crop yield. Studying the molecular regulation mechanism of seed size of Brassica napus is of great significance to increasing rapeseed yield per unit area and thus increasing oil yield per unit area.

[0003] MAPK (mitogen-activated protein kinase) cascade plays an important role not only in plant growth and development, but also in the response to biotic and abiotic stresses. MAPK cascade signal amplification is achieved through three phosphorylation kinases. Initially, MAPKKKs (mitogen-activated protein kinase kinasekinase) phosphorylates the Ser / Thr domain (Ser / Thr-x) on the A-loop of MKKs (mitogen-activated protein kinase kinase). 3-5 -Ser / Thr motif), while phosphorylated MKKs phosphorylate the Thr and Tyr residues in the Thr-x-Yyr structure of MAPKs to amplify the signal. Phosphorylated MAPKs regulate various downstream proteins to control plant growth, development, and stress resistance.

[0004] At present, there have been many studies on regulating seed size. For example, patent CN117402895A reported the Brassica napus BnaSAP.A04 gene and its application in regulating rapeseed seed size. This patent preliminarily studied the regulatory effect of SAP gene on rapeseed seed size. The SAP homologous gene BnaSAP.A04 was cloned from Brassica napus by homologous cloning method; then the overexpression vector pCambina1302-BnaSAP.A04 was constructed, and the recombinant was transformed into the hypocotyl callus of Westar rapeseed by Agrobacterium GV3101-mediated genetic transformation method to obtain positive transgenic plants. Its research confirmed that overexpression of Brassica napus BnaSAP.A04 gene can significantly increase the size and weight of rapeseed seeds.

[0005] MAPK cascades play an important role not only in plant growth and development, but also in generating responses to biotic and abiotic stresses. In previous studies, BnaMPK3 promoted organ size by interacting with BnaARF2 in Brassica napus. MAPK4 / 5, MAPK3 / 6 (MPK3 / 6), DA1, and ubiquitin-specific protease 15 (UBP15) were shown to act downstream of the ER and regulate seed size. The OsMKKK10-OsMKK4-OsMAPK6 signaling pathway was shown to positively regulate rice grain size and weight. The above studies all prove that the MAPK cascade is closely related to plant seed development.

[0006] Therefore, it is of great significance to study the regulatory mechanism of MAPK cascade on seed size. Summary of the invention

[0007] The present invention has carried out an in-depth exploration of the regulatory mechanism of MAPK cascade reaction on seed size. The present invention has discovered a core gene BnaA03.MKK6 (BnaA03G0115500ZS) that regulates seed size. BnaA03.MKK6 is a member of the MKK family in Brassica napus. The present invention has conducted a preliminary exploration of its basic functions and molecular mechanisms by constructing transgenic materials of BnaA03.MKK6 and conducting agronomic trait investigations. The present invention has confirmed that BnaA03.MKK6 can regulate the seed size of Brassica napus and / or Arabidopsis thaliana. The present invention not only improves the regulatory network of MAPK cascade reaction regulating seed development, but also provides new ideas for high-quality molecular breeding of Brassica napus.

[0008] In view of this, one of the objectives of the present invention is to provide a BnaA03.MKK6 gene for promoting plant seed growth, which can regulate seed size.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The BnaA03.MKK6 gene promotes plant seed growth, the nucleotide sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO: 1; the plant includes Brassica napus and / or Arabidopsis thaliana.

[0011] Furthermore, the coding sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO:2.

[0012] Furthermore, the BnaA03.MKK6 gene encodes a protein having an amino acid sequence as shown in SEQ ID NO:3.

[0013] Furthermore, BnaA03.MKK6 protein is expressed in the cell nucleus and cell membrane, and has tissue expression specificity.

[0014] Furthermore, the BnaA03.MKK6 positively regulates plant seed size and primary root elongation.

[0015] A second object of the present invention is to provide an overexpression vector containing the aforementioned BnaA03.MKK6 gene.

[0016] A third object of the present invention is to provide an Agrobacterium containing the above-mentioned overexpression vector.

[0017] A fourth object of the present invention is to provide a method for obtaining high-yield, well-grown Brassica napus.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] A method for obtaining high-yield and well-grown Brassica napus, comprising the following steps:

[0020] (1) constructing an overexpression vector containing the Brassica napus BnaA03.MKK6 gene;

[0021] (2) Transforming the overexpression vector obtained in step (1) with Agrobacterium to screen transgenic plants, thereby obtaining high-yield Brassica napus with excellent growth and development.

[0022] Furthermore, step (1) specifically includes: using Brassica napus cDNA as a template, using primers p101-BnaA03.MKK6-F and p101-BnaA03.MKK6-R for PCR amplification; recombining the amplified BnaA03.MKK6 gene cDNA into the pENTR / D-TOPO entry vector, and transforming the Escherichia coli competent DH5α; identifying positive clones and sequencing to obtain positive plasmids; and performing LR reaction on the obtained positive plasmids and the pEarleyGate101 expression vector to obtain an overexpression vector of the BnaA03.MKK6 gene.

[0023] Furthermore, the nucleotide sequence of the p101-BnaA03.MKK6-F is shown in SEQ ID NO:4, and the nucleotide sequence of the p101-BnaA03.MKK6-R is shown in SEQ ID NO:5.

[0024] A fourth object of the present invention is to provide an application of the aforementioned BnaA03.MKK6 gene, the aforementioned overexpression vector and / or the aforementioned Agrobacterium in promoting plant seed growth and primary root growth.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] The use of the aforementioned BnaA03.MKK6 gene, the aforementioned overexpression vector and / or the aforementioned Agrobacterium in promoting the seed growth and primary root growth of plants, wherein the plants include Brassica napus and / or Arabidopsis thaliana.

[0027] A fifth object of the present invention is to provide a use of the aforementioned BnaA03.MKK6 gene, the aforementioned overexpression vector and / or the aforementioned Agrobacterium in increasing plant yield.

[0028] To achieve the above object, the present invention adopts the following technical solutions:

[0029] The use of the aforementioned BnaA03.MKK6 gene, the aforementioned overexpression vector and / or the aforementioned Agrobacterium in improving plant yield, wherein the plants include Brassica napus and / or Arabidopsis thaliana.

[0030] The sixth object of the present invention is to provide an application of BnaA03.MKK6 protein in regulating plant seed size and primary root growth by interacting with BnaC03.MPK4 protein and / or BnaC03.MPK6 protein.

[0031] To achieve the above object, the present invention adopts the following technical solutions:

[0032] The application of BnaA03.MKK6 protein in regulating plant seed size and primary root growth by interacting with BnaC03.MPK4 protein and / or BnaC03.MPK6 protein; the plants include Brassica napus and / or Arabidopsis thaliana; the BnaA03.MKK6 protein positively regulates plant seed size and primary root growth; the amino acid sequence of the BnaA03.MKK6 protein is shown in SEQ ID NO:3.

[0033] Furthermore, the BnaA03.MKK6 protein is used to regulate plant seed size and primary root growth by interacting with the BnaC03.MPK4 protein.

[0034] Furthermore, the pseudo-phosphorylated BnaA03.MKK6 protein was able to phosphorylate the TEY domain of the BnaC03.MPK4 protein.

[0035] The beneficial effects of the present invention are:

[0036] 1. The present invention conducted transcriptome sequencing analysis on materials with significant differences in seed size of six species in Yu's Triangle, and found a core gene BnaA03.MKK6 (BnaA03G0115500ZS) that may regulate seed size. GUS staining results showed that BnaA03.MKK6 had a higher expression level in the two true leaf stage, inflorescence development, mid-stage, and 0-14 days of silique development, indicating that BnaA03.MKK6 had tissue-specific expression, and the BnaA03.MKK6 protein was subcellularly localized in the nucleus and cell membrane.

[0037] 2. The present invention studies have confirmed that heterologous overexpression of BnaA03.MKK6 positively regulates seed size and primary root growth of Arabidopsis. Specifically, heterologous overexpression of BnaA03.MKK6 in Arabidopsis will lead to larger cotyledons, earlier growth and development, larger seeds, and increased root length in the seedling stage of Arabidopsis. Compared with the wild type, the cotyledons of Arabidopsis T-DNA insertion mutants at the seedling stage become smaller, the seeds become smaller, and the root system becomes shorter. Knockout of AtMKK6 in Arabidopsis results in smaller cotyledons, smaller seeds, or even deformed development, and shorter root systems in the seedling stage of the knockout plants, among which cr-atmkk6-3-9 even shows abnormal root development. This indicates that AtMKK6 is involved in biological development processes such as Arabidopsis seed size regulation and root development.

[0038] 3. The present invention found that BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 have physical interactions. In vitro phosphorylation experiments verified that BnaA03.MKK6 can phosphorylate BnaC03.MPK4 in vitro, and the phosphorylation site is located in the TEY domain of BnaC03.MPK4. It is speculated that BnaA03.MKK6 phosphorylates the TEY domain in BnaC03.MPK4 and thus participates in the regulation of the cell cycle, further affecting seed size. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the result of identifying the positive transgenic Arabidopsis plants; Figure 1 -A is the result of positive plant identification of pEarleyGate101-BnaA03.MKK6 using two pairs of primers: F35S3ND+p101-BnaA03.MKK6-R (left) and p101-BnaA03.MKK6-F+OCS5ND (right); Figure 1 -B is the result of expression identification of OV-MKK6-4 and OV-MKK6-9 strains, **** in the figure indicates significant difference of p<0.0001, using t-test, the bar graph represents the SD of the mean, mean ± SD, n = 3; Figure 1 -C is the result of positive plant identification of pCambia1305.1-BnaA03.MKK6 using M13pF+Pro-BnaA03.MKK6-R; Figure 1 -D is the result of positive plant identification of pCambia1305.1-BnaA03.MKK6 by Pro-BnaA03.MKK6-F+M13pR;

[0040] Figure 2 This is the result of identifying Arabidopsis mutant plants; Figure 2 -A is the T-DNA insertion site analysis of the SALK-084332C mutant; Figure 2 -B is the T-DNA insertion site analysis of the SALK-016750C mutant; Figure 2 -C is the result of identifying Col-0 using LP+RP+LB (as a control); Figure 2 -D is the result of identifying atmkk6 using LP+RP+LB; Figure 2 -E is the result of identifying atmpk6 using LP+RP+LB; Figure 2 -F is the result diagram of atmkk6 expression level identification; Figure 2-G is the result of atmpk6 expression level identification; **** in the figure indicates significant difference of p<0.0001, using t-test, the bar graph represents the SD of the mean, mean±SD, n=3;

[0041] Figure 3 This is the result of Arabidopsis knockout plant identification; Figure 3 -A is the result of positive plant identification of pYLCRISPR / Cas9-AtMKK6 using PB-L and PB-R; Figure 3 -B is the result of the knockout site analysis of Arabidopsis plants;

[0042] Figure 4 This is the result of identifying rapeseed overexpression plants; Figure 4 -A is the positive plant identification of pEarleyGate101-BnaA03.MKK6; Figure 4 -B is the result of the expression level identification of the overexpression strains, **** indicates a significant difference of p < 0.0001, using t-test, the bar graph represents the SD of the mean, mean ± SD, n = 3;

[0043] Figure 5 This is the result of the rapeseed knockout plant identification; Figure 5 -A is the result of positive plant identification using PB-L+PB-R (right) and cas9-F+cas9-R (left); Figure 5 -B is the result of knockout site analysis in rapeseed plants;

[0044] Figure 6 The results of GUS tissue staining analysis of BnaA03.MKK6, wherein a~j are control groups, which are respectively GUS staining driven by the 35S strong promoter at two true leaves stage, four true leaves stage, leaf, inflorescence, 5th d silique, 5th d seed, 12th d silique, 12th d seed, 19th d silique, and 19th d seed, and k~s are GUS staining driven by the BnaA03.MKK6 promoter at two true leaves stage, four true leaves stage, leaf, inflorescence, 5th d silique, 5th d seed, 12th d silique, 12th d seed, 19th d silique, and 19th d seed;

[0045] Figure 7 The results of the observation of subcellular localization of BnaA03.MKK6 are shown in the figure, where Chloroplast-RFP represents the state of red light excitation, 35S::BnaA03.MKK6-GFP represents the state of green light excitation, Bright represents the bright field, Merge represents the superposition state, and the scale length is 5μm;

[0046] Figure 8This is a summary diagram of the results of seed phenotype observation and root phenotype observation; Figure 8 -A is a graph comparing the seed sizes of Col-0 and atmkk6; Figure 8 -B is a bar graph comparing the seed sizes of Col-0 and atmkk6, **** indicates a significant difference of p<0.0001, using t-test, the bar graph represents the SD of the mean, mean ± SD, n = 3; Figure 8 -C is a graph comparing the seed sizes of Col-0, cr-mkk6-9, and cr-mkk6-10;

[0047] Figure 8 -D is a bar graph comparing the seed sizes of Col-0, cr-mkk6-9 and cr-mkk6-10, ns indicates no significant difference, **** indicates significant difference with p<0.0001, t-test was used, the bar graph indicates the SD of the mean, mean ± SD, n = 3; Figure 8 -E is the seed size picture of Col-0, OV-MKK6-4 and OV-MKK6-9, ns indicates no significant difference, **** indicates significant difference with p<0.0001, t-test was used, the bar graph indicates the SD of the mean, mean ± SD, n = 3; Figure 8 -F is a bar graph comparing the seed sizes of Col-0, OV-MKK6-4 and OV-MKK6-9, ns indicates no significant difference, **** indicates significant difference with p<0.0001, t-test was used, the bar graph indicates the SD of the mean, mean ± SD, n = 3; Figure 8 -G is the comparison of the root systems of Col-0 and heterologous transformed plants; Figure 8 -H is the comparison of hypocotyls between Col-0 and heterologous transformed plants; Figure 8 -I is a bar graph comparing the root lengths of Col-0, OV-MKK6-4 and OV-MKK6-9, ns indicates no significant difference, * indicates significant difference at p < 0.01, using t-test, the bar graph indicates the SD of the mean, mean ± SD, n = 3;

[0048] Fig. 9 is a graph showing phenotypic observations during the growth phase; Fig. 9 -A is a graph showing comparison of seedling phenotypes; Fig. 9 -B is a graph comparing the phenotypes of Col-0 and atmkk6 in the late growth and development period; Fig. 9 -C is a graph comparing the phenotypes of Col-0, OV-MKK6-4, and OV-MKK6-9 during growth and development; Fig. 9 -D is a graph comparing the silique lengths of Col-0, OV-MKK6-4, and OV-MKK6-9; Fig. 9-E is a bar graph comparing the silique length of Col-0, OV-MKK6-4 and OV-MKK6-9, in which ns indicates no significant difference using t-test, and the bar graph indicates the SD of the mean, mean ± SD, n = 3;

[0049] Fig.10 This is the experimental result diagram for protein interaction verification; among them, Fig.10 -A is the result of yeast two-hybrid verification of the interaction between BnaC03.MPK4, BnaC03.MPK6, BnaA03.MPK6 and BnaA03.MKK6 proteins; Fig.10 -B is the result of BiFC dual luciferase verification of the interaction between BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 proteins;

[0050] Fig.11 This is the result of protein sequence analysis, where Fig.11 -A is the analysis result diagram of BnaA03.MKK6 protein sequence; Fig.11 -B is the analysis result diagram of BnaC03.MPK4 and BnaC03.MPK6 protein sequences;

[0051] Fig.12 Figure 2 is the result of in vitro phosphorylation experiment; Fig.12 -A is a schematic diagram of mutating all Ser / Thr structures in the A-loop of BnaA03.MKK6 protein to Asp; Fig.12 -B is His-BnaA03.MKK6 DD Structural diagram; Fig.12 -C is the structure diagram of GST-BnaC03.MPK4; Fig.12 -D is the structure diagram of GST-BnaC03.MPK6; Fig.12 -E is the detection result of purified protein, where 1 represents GST, 2 represents GST-BnaC03.MPK4, 3 represents GST-BnaC03.MPK6, and 4 represents His-BnaA03.MKK6 DD ; Fig.12 -F is a diagram showing BnaA03.MKK6 phosphorylating BnaC03.MPK4 in vitro. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0053] In the embodiment of the present invention:

[0054] (1) It was confirmed that heterologous expression of BnaA03.MKK6 regulates seed size and primary root growth in Arabidopsis thaliana.

[0055] BnaA03.MKK6 is a member of the MKK family of Brassica napus. In order to study the function of BnaA03.MKK6, the present invention heterologously overexpresses BnaA03.MKK6 in Arabidopsis thaliana, and the results show that the transgenic plants are larger than the wild-type seeds, the length of the siliques has no significant change, and the root system is better developed. The phenotypes of the T-DNA insertion mutant and the knockout mutant of AtMKK6 show that the seeds are smaller and the root system is shorter than the wild-type seeds. Among them, the knockout mutant seeds become smaller more obviously, and even have symptoms of seed deformity, and the root system is shorter and even has a phenotype of abnormal root development. According to the above phenotypic observations, it is speculated that the heterologous overexpression of BnaA03.MKK6 will positively regulate the seed size of Arabidopsis thaliana and have a positive effect on the rooting of seeds. At the same time, AtMKK6 in Arabidopsis thaliana positively regulates the seed development of Arabidopsis thaliana. Knocking out AtMKK6 in Arabidopsis thaliana will seriously affect the seed size and seed morphology, and even cause the root system to fail to develop normally after seed sowing. For more details, see the embodiment section.

[0056] (2) Clarify the mechanism by which BnaA03.MKK6 regulates seed size

[0057] In order to study the pathway of BnaA03.MKK6 regulating seed size, the present invention screened the homologous genes BnaC03.MPK4 and BnaC03.MPK6 of MPK4 and MPK6 in Arabidopsis thaliana as candidate genes for point-to-point verification. Yeast two-hybrid and BiFC dual luciferase experiments both showed that BnaA03.MKK6 interacted with BnaC03.MPK4 and BnaC03.MPK6.

[0058] Analysis of the protein sequence of BnaA03.MKK6 revealed that BnaA03.MKK6 has a highly conserved A-loop and Ser / Thr domain. Analysis of the protein sequences of BnaC03.MPK4 and BnaC03.MPK6 revealed that both have the same conserved TEY (Thr-Glu-Tyr) domain as the human p44 / 42 protein. Therefore, we chose Anti-Phospho-44 / 42 antibody as the phosphorylation antibody to detect the in vitro phosphorylation of BnaC03.MPK4 and BnaC03.MPK6.

[0059] In order to make BnaA03.MKK6 have phosphorylation activity, the present invention first site-directedly mutated two Ser / Thr residues on the Ser / Thr domain of BnaA03.MKK6 into Asp with pseudophosphorylation activity. Subsequent in vitro phosphorylation results showed that BnaA03.MKK6 could phosphorylate the TEY domain on BnaC03.MPK4, which was consistent with the expected results, but surprisingly, we did not observe BnaA03.MKK6 phosphorylating BnaC03.MPK6. The reason for this is that BnaA03.MKK6 may require more stringent conditions to phosphorylate BnaC03.MPK6.

[0060] In the embodiments of the present invention, Arabidopsis thaliana Col-0 ecotype, Nicotiana benthamiana and Brassica napus conventional species Zhongshuang 11 (ZS11) were all provided by the Rapeseed Bioengineering Technology Center; Arabidopsis thaliana T-DNA insertion mutants were purchased from the AraShare website (https: / / www.arashare.cn / index / Product / index.htmL).

[0061] In the embodiment of the present invention, experimental materials such as Arabidopsis thaliana and tobacco are planted in a constant temperature incubator, and transgenic rapeseed is planted in a school transgenic test site. The planting conditions of the constant temperature incubator are: Arabidopsis thaliana seeds are disinfected and sown on 1 / 2MS culture plates after vernalization, and cultured in a greenhouse for 10 to 14 days. The Intellus controller controls the light experiment for 16 hours, darkness for 8 hours, temperature at 20 to 22°C, and humidity at 70% to 75%. After 10 to 14 days, the seedlings are transplanted into the soil and continue to be cultured in the greenhouse. Tobacco seeds are directly sown in the soil, and the culture conditions are the same as Arabidopsis thaliana. Transgenic rapeseed is cultured under natural conditions.

[0062] In the examples of the present invention, the experimental strains DH5α, GV3101, GV3101 (pSoup), BL21 and Y2H Gold were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0063] In the examples of the present invention, the pEASY-Blunt vector was purchased from Quanshijin Biotechnology Co., Ltd.; other experimental vectors, including pEarlyGate101, pCambia1305.1, pCambia1302, pGBKT7, pGBKT7-53, pGBKT7-Lam, pGADT7, pET-28a, pGEX-4T1, pNC-BiFC-Ecc and pNC-BiFC-Enn, were all stored in the laboratory.

[0064] In the embodiment of the present invention, the BnaA03.MKK6 gene sequence is shown as SEQ ID NO:1, the coding sequence is shown as SEQ ID NO:2, and the protein sequence is shown as SEQ ID NO:3.

[0065] In the present embodiment, the full-length CDS sequence of BnaA03.MKK6 found on the BnaPIR website was used as a template, and the primers were designed using the Primer 5 software. The primer sequences are shown in Tables 1 to 3.

[0066] Table 1. List of primers used for vector construction

[0067]

[0068]

[0069] Table 2. List of primers used for quantitative PCR

[0070]

[0071] Table 3. List of primers used for site-directed mutagenesis

[0072]

[0073] In the embodiment of the present invention, the plant identification primers are shown in Table 4.

[0074] Table 4. Primers for plant identification

[0075]

[0076] In the embodiments of the present invention, if the relevant experimental scheme is unclear, reference may be made to the relevant paper "Molecular Mechanism of Brassica napus BnaA03.MKK6 Regulating Seed Size", which will be published later.

[0077] Example 1. BnaA03.MKK6 gene cloning and vector construction

[0078] ZS11 leaves were used as templates to extract total RNA from the samples in strict accordance with the instructions of the EZ-10 DNAaway RNA Mini-Preps Kit. The quality and concentration of the sample RNA were tested by agarose gel electrophoresis, and cDNA was synthesized using a reverse transcription kit from US EVERBRIGHT INC.

[0079] The chromosomal location information of MKK6 family members was obtained using the Brassica napus database (http: / / www.genoscope.cns.fr / brassicanapus / ), and primers were designed based on the ORF sequence of the BnaA03.MKK6 gene.

[0080] The extracted cDNA was used as a template for PCR amplification, and the amplified product was sequenced to obtain the correct cDNA sequence, as shown in SEQ ID NO. 2. The amplified BnaA03.MKK6 gene cDNA was recombined into the pENTR / D-TOPO entry vector using the pENTR / D-TOPO Cloning Kit, and transformed into the competent DH5α of the large intestine. After the positive clones with the correct sequence were expanded, the plasmid was extracted using the EasyPure Plasmid MiniPrep Kit, and the LR reaction was performed with the pEarleyGate101 expression vector to form the pEarleyGate101-BnaA03.MKK6 overexpression vector for overexpression and subcellular localization.

[0081] According to the promoter sequence of the BnaA03.MKK6 gene, specific primers (SEQ ID NO.6 and SEQ ID NO.7) were designed to amplify the 1500bp sequence upstream of the promoter. The specific sequence is shown in SEQ ID NO.45. The vector pCAMBIA1305.1-BnaA03.MKK6 promoter vector was constructed by recombination method. The obtained recombinant plasmid was transformed into Agrobacterium GV3101 and infected wild-type Arabidopsis thaliana to obtain T3 transgenic plants.

[0082] Using the Atu3d vector as a template, the expression cassette was then constructed using primers (SEQ ID NO.46 to SEQ ID NO.51), and the SgRNA expression cassette was assembled into the pYLCRISPR / Cas9 vector using the Golden-Gate method. The recombinant product was transformed into Escherichia coli competent cells, and PB-L+PB-R (SEQ ID NO.52 and SEQ ID NO.53) was used for bacterial testing. The results showed that 12 samples with correct bacterial testing were obtained. The strains with correct bacterial testing were sent to BGI for sequencing. Geneious was used to compare the sequencing results and obtain the pYLCRISPR / Cas9-BnaA03.MKK6 vector. pYLCRISPRCas9-AtA03.MKK6 was constructed in the same way.

[0083] Example 2. Acquisition and identification of transgenic lines

[0084] (1) Obtaining and identifying positive transgenic Arabidopsis plants

[0085] The Agrobacterium carrying the pEarleyGate101-BnaA03.MKK6 overexpression vector was genetically transformed into Arabidopsis, and the T0 generation seeds were harvested, re-sown and cultured to the seedling stage, and sprayed with 0.15 mg / mL Basta for screening. The surviving T1 generation plants were identified as positive plants and the expression level was observed. cDNA was extracted from the surviving plants, and positive plants were identified using two pairs of primers, F35S3ND+p101-BnaA03.MKK6-R and p101-BnaA03.MKK6-F+OCS5ND. The results are shown in Figure 1 -A and Figure 1 -B, a total of 12 positive plants were obtained, and the expression levels of BnaA03.MKK6 in all 12 plants were significantly upregulated. OV-MKK6-4 and OV-MKK6-9 with higher expression levels and differences were selected for subsequent experiments. Agrobacterium carrying the pCambia1305.1-BnaA03.MKK6 vector was transformed into Arabidopsis thaliana in the flowering period, and the T0 generation seeds were harvested and re-sown on 1 / 2MS+Hyg (25 mg / mL) solid culture medium for screening. DNA was extracted from surviving plants, and two pairs of primers, M13p-F+Pro-BnaA03.MKK6-R and Pro-BnaA03.MKK6-F+M13p-R, were used to identify positive plants. The results showed that 9 positive plants were obtained, see for details. Figure 1 -C~ Figure 1 -D, positive plants were used for subsequent experiments.

[0086] (2) Obtaining and identifying Arabidopsis mutant plants

[0087] The T-DNA insertion mutant strains SALK-084332C and SALK-016750C of AtMKK6 (AT5G56580) and AtMPK6 (AT2G43790) were purchased from the ABRC website. The insertion site of SALK-084332C was located in the promoter fragment, and the insertion site of SALK-016750C was located in the 5'UTR. Figure 2 -A~ Figure 2 -B. The mutant strains were cultured to the T2 generation, and total DNA was extracted. The positive plants were identified using primers LP+RP+LB (atmkk6-LP, atmkk6-RP and LBb1.3). Figure 2 -C~ Figure 2-E, Col-0 has a 1000bp band (a), and the SALK-084332C and SALK-016750C mutant lines have only a 700bp band (b and c), indicating that the SALK-084332C and SALK-016750C mutant lines are both homozygous mutants, and the plants were named atmkk6 and atmpk6. qPCR quantitative identification was performed on the homozygous plants, and the results are shown in Figure 2 -F. Figure 2 -G, the expression levels of atmkk6 and atmpk6 were significantly downregulated, and they were used as alternative plants for subsequent experiments.

[0088] (3) Obtaining and identifying Arabidopsis knockout plants

[0089] Agrobacterium carrying the pYLCRISPR / Cas9-AtMKK6 vector was used to transform Arabidopsis thaliana at the flowering stage, and the T1 generation seeds were harvested and re-sown on a culture dish containing 1 / 2MS+Kan (25 mg / mL) for screening. DNA was extracted from the surviving T1 generation plants, and positive plants were identified using PB-L+PB-R (SEQ ID NO.52 and SEQ ID NO.53). The results are as follows: Figure 3 -A. cDNA was extracted from the positive plants, and the target gene fragment was amplified and sent to Chinese rice graduate students for HI-TOM sequencing. The sequencing results were analyzed and it was found that both plants had a single base G deletion mutation, resulting in a frameshift mutation, and cr-mkk6-3-9 had a single base A insertion, see Figure 3 -B and named them cr-mkk6-3-9 and cr-mkk6-3-10 for subsequent experiments.

[0090] (4) Obtaining and identifying rapeseed overexpression plants

[0091] DNA was extracted from overexpressing rapeseed plants, and positive plants were identified using two pairs of primers: F35S3ND+p101-BnaA03.MKK6-R and p101-BnaA03.MKK6-F+OCS5ND. Figure 4 -A, a total of 6 positive plants were obtained. RNA was extracted from the 6 transgenic rapeseeds, and after reverse transcription of cDNA, qPCR was used to identify the expression level. The results are shown in Figure 4 -B, the expression levels of BnaA03.MKK6 in the six transgenic plants were significantly upregulated compared with ZS11 and can be used for subsequent experiments.

[0092] (5) Obtaining and identifying rapeseed knockout plants

[0093] DNA was extracted from the knockout rapeseed plants, and positive plants were identified using PB-L+PB-R (SEQ ID NO.52 and SEQ ID NO.53) and cas9-F+cas9-R (SEQ ID NO.54 and SEQ ID NO.55), and five positive plants were obtained. For details, see Figure 5 -A. cDNA was extracted from the positive plants, and the target gene fragment was amplified and sent to the Rice Research Institute for HI-TOM sequencing. The sequencing results were analyzed and it was found that two plants had long-fragment base mismatch mutations, resulting in the inability to correctly encode proteins, cr-BnaMKK6-3 had GA and TC mutations, and cr-BnaMKK6-5 had T base deletion, resulting in frameshift mutations. For details, see Figure 5 -B, and named them cr-BnaMKK6-3 and cr-BnaMKK6-5 for subsequent experiments.

[0094] Example 3. Analysis of BnaA03.MKK6 expression pattern

[0095] The present invention obtains an expression vector for GUS gene expression driven by the BnaA03.MKK6 promoter. Homozygous plants of the T3 generation were used for GUS staining observation, and a GUS gene expression vector driven by a 35S strong promoter was selected to transform Arabidopsis as a control. The GUS staining results showed that BnaA03.MKK6 had a higher expression level in the two true leaf stage, inflorescence development stage, and on the 5th and 12th days of silique and seed development, while in the four true leaf stage, BnaA03.MKK6 had a higher expression in the roots and a lower expression in the leaves. On the 19th day of leaf, silique and seed development, BnaA03.MKK6 was not expressed, see for details. Figure 6 The results are basically consistent with the tissue expression pattern of BnaA03.MKK6 predicted by the website.

[0096] (2) Subcellular localization of BnaA03.MKK6 protein

[0097] The pEarleyGate101-BnaA03.MKK6 vector with fluorescence signal was further constructed for subcellular localization analysis. Fluorescence signal was observed in the nucleus and cell membrane of protoplasts, indicating that BnaA03.MKK6 protein was localized in both the cell membrane and the nucleus, indicating that BnaA03.MKK6 may play a role in the nucleus and cell membrane. Agrobacterium carrying the pEarleyGate101-BnaA03.MKK6 vector was injected into tobacco leaves. Figure 7 As shown, fluorescence signals were observed in the cell nucleus and cell membrane of tobacco leaves. This experiment once again verified that the BnaA03.MKK6 protein was localized in both the cell membrane and the cell nucleus.

[0098] Example 4. BnaA03.MKK6 affects rapeseed growth and development

[0099] (1) Observation of seed size and root phenotype

[0100] In order to explore the biological function of BnaA03.MKK6, the present invention observed the changes in seeds of wild-type Col-0 and T-DNA inserted mutant atmkk6, transgenic plants OV-MKK6-4 and OV-MKK6-9 that heterologously overexpress BnaA03.MKK6, and Arabidopsis knockout plants cr-mkk6-3-9 and cr-mkk6-3-10 at maturity under an electron microscope. The seed area was calculated using imageJ. The results showed that compared with Col-0, the surface area of ​​atmkk6, cr-mkk6-9, and cr-mkk6-10 seeds was significantly reduced, and the seeds of cr-mkk6-9 and cr-mkk6-10 were malformed, such as Figure 8 -A~ Figure 8 -D; the seed area of ​​OV-MKK6-4 and OV-MKK6-9 was significantly larger than that of Col-0, as shown in Figure 8 -E~ Figure 8 -F. The above seeds were re-sown on 1 / 2MS medium, and the length of primary roots in the seedling stage was counted. The results showed that compared with Col-0, atmkk6 had no significant change in primary roots, cr-mkk6-10 had shortened primary roots, OV-MKK6-4 and OV-MKK6-9 had elongated primary roots compared with Col-0, and cr-mkk6-9 had abnormal root development, see Figure 8 -G~ Figure 8 -I. The experimental results show that BnaA03.MKK6 positively regulates plant seed size and primary root elongation.

[0101] (2) Phenotypic observation during growth stage

[0102] The above plants were transplanted into nutrient soil to observe their phenotypic characteristics during the growth stage. The results showed that compared with Col-0, atmkk6, cr-mkk6-3-9, and cr-mkk6-3-10 had smaller leaf areas and weaker growth during the vegetative growth stage. Fig. 9 -A. Compared with Col-0, OV-MKK6-4 and OV-MKK6-9 have larger leaves and stronger growth, as shown in Fig. 9 -B. During the reproductive growth stage, atmkk6 showed no significant changes compared to Col-0, as shown in Fig. 9 -B. However, compared with Col-0, OV-MKK6-4 and OV-MKK6-9 grew faster after bolting, as shown in Fig. 9-C. The siliques at maturity were observed and found to have a significant increase in length compared to Col-0 in OV-MKK6-4, while there was no significant change in the length of OV-MKK6-9 siliques. Fig. 9 -D and Fig. 9 -E as shown.

[0103] Example 5. Interaction between BnaA03.MKK6 protein and BnaC03.MPK4 and BnaC03.MPK6 proteins

[0104] In Arabidopsis, MKK6 has been reported to interact with MPK4 and MPK6, and MPK4 has been shown to be involved in cell cycle regulation. The phenotype of atmpk4 is similar to that of atmkk6. Therefore, the present invention uses the proteins encoded by the homologous genes BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MPK6 of MPK4 and MPK6 in rapeseed as candidate proteins for BnaA03.MKK6 interacting proteins for yeast two-hybrid verification. The yeast two-hybrid results showed that BnaC03.MPK4, BnaC03.MPK6, BnaA03.MPK6 proteins interacted with BnaA03.MKK6 protein, such as Fig.10 -A. To further verify the experimental results, the present invention conducted a BiFC experiment, where the Ecc-BnaC03.MPK4 and Ecc-BnaC03.MPK6 vectors were co-injected with Enn-BnaA03.MKK6 into tobacco leaf cells, and the fluorescence signal was observed under a laser confocal fluorescence microscope. The results are shown in FIG. Fig.10 -B shows that BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 proteins interact with each other in the nucleus and cell membrane, which is consistent with the yeast two-hybrid results.

[0105] Example 6. His-BnaA03.MKK6 DD In vitro phosphorylation of GST-BnaC03.MPK4

[0106] (1) Protein sequence analysis

[0107] The experiment in Example 5 has proved that BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 proteins interact with each other. To further determine how BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 proteins function, the present invention attempts to verify the phosphorylation relationship between BnaA03.MKK6 and BnaC03.MPK4 and BnaC03.MPK6 proteins. BnaA03.MKK6 protein sequence analysis shows that BnaA03.MKK6 has a highly conserved A-loop and Ser / Thr structure of the MKK family, see Fig.11 -A. Protein structure analysis of BnaC03.MPK4 and BnaC03.MPK6 revealed that both of them have TEY domains. Fig.11 -B, phosphorylation of this domain can be detected by Anti-Phospho-44 / 42 antibody.

[0108] (2) In vitro phosphorylation assay

[0109] In preparation for the subsequent in vitro phosphorylation experiment, the present invention uses the site-directed mutagenesis kit provided by Bio-Tech to mutate the Ser residues and Thr residues in the Ser / Thr structure in the A-loop of the BnaA03.MKK6 protein to Asp, such as Fig.12 -A. BnaA03.MKK6 was obtained by amplifying ZS11 cDNA and site-directed mutagenesis vector as templates. DD 、BnaC03.MPK4、BnaC03.MPK6. DD Recombined in pET-28a, BnaC03.MPK4 and BnaC03.MPK6 were recombined in the vector pGET-4T1. His-BnaA03.MKK6 was successfully obtained DD , GST-BnaC03.MPK4, GST-BnaC03.MPK6 vectors, see Fig.12 -B~ Fig.12 -D.

[0110] The obtained His-BnaA03.MKK6 DD , GST-BnaC03.MPK4, GST-BnaC03.MPK6 vector plasmids were re-transformed into BL21 E. coli competent cells. The cells were collected and purified to obtain purified His-BnaA03.MKK6 DD , GST-BnaC03.MPK4, GST-BnaC03.MPK6 proteins were detected by SDS electrophoresis. Fig.12-E, the present invention obtained His-BnaA03.MKK6 of 50kDa size DD , GST-BnaC03.MPK4 with a size of 70 kDa and GST-BnaC03.MPK6 with a size of 65 kDa. The protein bands were single and clearly visible, indicating that the purification was successful.

[0111] The purified His-BnaA03.MKK6 DD The protein was incubated with GST-BnaC03.MPK4 and GST-BnaC03.MPK6 proteins in an in vitro phosphorylation system. Western Blot was used to detect the in vitro phosphorylated proteins, and it was found that GST-BnaC03.MPK4 was phosphorylated by His-BnaA03.MKK6. DD Phosphorylation in vitro was observed in the TEY domain, while GST-BnaC03.MPK6 was not observed to be phosphorylated by His-BnaA03.MKK6. DD In vitro phosphorylation, see Fig.12 -F.

Claims

1. The BnaA03.MKK6 gene for promoting plant seed growth is characterized in that: The nucleotide sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO: 1; the plant includes Brassica napus and / or Arabidopsis thaliana.

2. The BnaA03.MKK6 gene according to claim 1, characterized in that The coding sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO:2; the BnaA03.MKK6 gene encodes a protein with an amino acid sequence shown in SEQ ID NO:

3.

3. An overexpression vector containing the BnaA03.MKK6 gene according to any one of claims 1 to 2.

4. Agrobacterium containing the overexpression vector according to claim 3.

5. A method for obtaining high-yield, well-grown Brassica napus, characterized in that: The steps include: (1) constructing an overexpression vector containing the Brassica napus BnaA03.MKK6 gene; (2) Transforming the overexpression vector obtained in step (1) with Agrobacterium to screen transgenic plants, thereby obtaining high-yield Brassica napus with excellent growth and development.

6. The method according to claim 5, characterized in that Step (1) specifically includes: performing PCR amplification using Brassica napus cDNA as a template; recombining the amplified BnaA03.MKK6 gene cDNA into the pENTR / D-TOPO entry vector, and transforming the E. coli competent DH5α; identifying positive clones and sequencing to obtain positive plasmids; performing LR reaction on the obtained positive plasmids and the pEarleyGate101 expression vector to obtain an overexpression vector of the BnaA03.MKK6 gene.

7. The method according to claim 6, characterized in that The PCR amplification primers include p101-BnaA03.MKK6-F having a nucleotide sequence as shown in SEQ ID NO:4 and p101-BnaA03.MKK6-R having a nucleotide sequence as shown in SEQ ID NO:

5.

8. Use of the BnaA03.MKK6 gene according to any one of claims 1 to 2, the overexpression vector according to claim 3 and / or the Agrobacterium according to claim 4 in promoting plant seed growth and primary root growth, characterized in that: The plants include Brassica napus and / or Arabidopsis thaliana.

9. Use of the BnaA03.MKK6 gene according to any one of claims 1 to 2, the overexpression vector according to claim 3 and / or the Agrobacterium according to claim 4 in increasing plant yield, characterized in that: The plants include Brassica napus and / or Arabidopsis thaliana.

10. Use of BnaA03.MKK6 protein in regulating plant seed size and primary root growth by interacting with BnaC03.MPK4 protein and / or BnaC03.MPK6 protein, characterized in that: The plant includes Brassica napus and / or Arabidopsis thaliana; the BnaA03.MKK6 protein positively regulates plant seed size and primary root growth; the amino acid sequence of the BnaA03.MKK6 protein is shown in SEQ ID NO:3.

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