Genes, overexpression vectors, agrobacterium and applications for regulating plant seed size

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a gene for regulating plant seed size, an overexpression vector, an agrobacterium and application. The gene is BnaA03.MKK6 gene, the nucleotide sequence of which 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; the plant includes Brassica napus and / or Arabidopsis thaliana. The application constructs the transgenic material of BnaA03.MKK6 and performs agronomic trait investigation, and preliminarily explores the basic function and molecular mechanism. It is found that the BnaA03.MKK6 protein is expressed on the cell nucleus and cell membrane, has tissue expression specificity, and positively regulates the plant seed size and primary root elongation. The application provides a new idea and direction for high-quality molecular breeding of Brassica napus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a gene for regulating plant seed size, an overexpression vector, an agrobacterium and application. BACKGROUND

[0002] Brassica napus is one of three types of oilseed rape, which is formed by natural interspecific hybridization of Brassica rapa and Brassica oleracea. As one of important oil crops in China, Brassica napus has high economic value and nutritional value, and can be used for producing edible oil, and is also important industrial raw material for high-protein feed and high-energy fuel. Thousand kernel weight is an important factor for forming the yield of oilseed rape, and seed size is a key phenotype affecting the thousand kernel weight. The research on the mechanism for regulating seed size can provide important theoretical basis for crop breeding, and can realize the yield increase of crops by using genes for regulating seed size. The research on the molecular regulation mechanism of seed size of Brassica napus has important significance for increasing the yield of oilseed rape per unit area and then increasing the oil yield per unit area.

[0003] The MAPK (mitogen-activated protein kinase) cascade not only plays an important role in plant growth and development, but also plays an important role in the response to biotic and abiotic stresses. The MAPK cascade signal amplification is realized through three kinds of phosphorylation kinases. First, two Ser / Thr residues in the Ser / Thr-x 3-5 -Ser / Thr motif in the A-loop ring of MKKs (mitogen-activated protein kinase kinase) are phosphorylated by MAPKKKs (mitogen-activated protein kinase kinase kinase), then the Thr and Tyr residues in the Thr-x-Yyr structure of the phosphorylated MKKs are phosphorylated by MAPKs to amplify the signal, and the phosphorylated MAPKs regulate various downstream proteins to regulate plant growth and development and stress resistance.

[0004] At present, there are many studies on the regulation of seed size. For example, CN117402895A patent reports Brassica napus BnaSAP.A04 gene and its application in regulating the size of rapeseed. The patent preliminarily studies the regulation of SAP gene on the size of rapeseed, clones the SAP homologous gene BnaSAP.A04 from Brassica napus by homologous cloning method; then constructs the overexpression vector pCambina1302-BnaSAP.A04, and transforms the recombinant into the hypocotyl callus of Westar rapeseed by Agrobacterium GV3101 mediated genetic transformation method, and obtains positive transgenic plants. The study confirms that overexpression of Brassica napus BnaSAP.A04 gene can significantly increase the size and weight of rapeseed.

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

[0006] Therefore, it is of great significance to study the regulation mechanism of MAPK cascade reaction on seed size. SUMMARY

[0007] The present application carries out in-depth exploration on the regulation mechanism of MAPK cascade reaction on seed size. The present application finds a core gene BnaA03.MKK6 (BnaA03G0115500ZS) for regulating seed size. BnaA03.MKK6 is a member of the MKK family in Brassica napus. The present application preliminarily explores the basic function and molecular mechanism of BnaA03.MKK6 by constructing transgenic materials of BnaA03.MKK6 and investigating agronomic traits. The present application confirms that BnaA03.MKK6 can regulate the size of Brassica napus and / or Arabidopsis thaliana seeds. The present application not only perfects the regulation network of MAPK cascade reaction on seed development, but also provides a new idea for high-quality molecular breeding of Brassica napus.

[0008] Therefore, one of the purposes of the present application is to provide a BnaA03.MKK6 gene for promoting plant seed growth, which can regulate seed size.

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

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

[0011] Further, a coding sequence of the BnaA03.MKK6 gene is shown as SEQ ID NO:2.

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

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

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

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

[0016] The third object of the present application is to provide an agrobacterium containing the aforementioned overexpression vector.

[0017] The fourth object of the present application is to provide a method for obtaining Brassica napus with high yield and excellent growth and development.

[0018] To achieve the above-mentioned objects, the present application adopts the following technical solutions:

[0019] The method for obtaining Brassica napus with high yield and excellent growth and development comprises the following steps:

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

[0021] (2) performing agrobacterium transformation on the overexpression vector obtained in step (1), and screening transgenic plants, thereby obtaining Brassica napus with high yield and excellent growth and development.

[0022] Further, step (1) specifically includes: using rapeseed cDNA as a template, PCR amplification is performed using primers p101-BnaA03.MKK6-F and p101-BnaA03.MKK6-R; the amplified BnaA03.MKK6 gene cDNA is recombined into the pENTR / D-TOPO entry vector and transformed into competent DH5α cells in the large intestine; positive clones are identified and sequenced to obtain positive plasmids; the obtained positive plasmids are subjected to LR reaction with the pEarleyGate101 expression vector to obtain the overexpression vector of the BnaA03.MKK6 gene.

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

[0024] The fourth objective of this 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 objectives, the present invention adopts the following technical solution:

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

[0027] The fifth objective of this invention is to provide an application of the aforementioned BnaA03.MKK6 gene, the aforementioned overexpression vector, and / or the aforementioned Agrobacterium in improving plant yield.

[0028] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0030] The sixth objective of this invention is to provide an application of the BnaA03.MKK6 protein in regulating plant seed size and primary root growth through interaction with BnaC03.MPK4 and / or BnaC03.MPK6 proteins.

[0031] To achieve the above objectives, the present invention adopts the following technical solution:

[0032] Application of BnaA03.MKK6 protein in regulating seed size and primary root growth in plants through interaction with BnaC03.MPK4 and / or BnaC03.MPK6 protein; said plants include Brassica napus and / or Arabidopsis thaliana; said BnaA03.MKK6 protein positively regulates seed size and primary root growth in plants; said amino acid sequence of said 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 pseudophosphorylated BnaA03.MKK6 protein can phosphorylate the TEY domain of the BnaC03.MPK4 protein.

[0035] The beneficial effects of this invention are as follows:

[0036] 1. This invention, through transcriptome sequencing analysis of materials from six species of *Dermacentor yuensis* with significant differences in seed size, identified a core gene, BnaA03.MKK6 (BnaA03G0115500ZS), which may regulate seed size. GUS staining results showed that BnaA03.MKK6 had high expression levels during the two-true-leaf stage, inflorescence development, mid-stage, and silique development (0–14 days), indicating that BnaA03.MKK6 exhibits tissue-specific expression, and that the BnaA03.MKK6 protein is subcellularly located in the nucleus and cell membrane.

[0037] 2. This invention confirms that heterologous overexpression of BnaA03.MKK6 positively regulates seed size and primary root growth in Arabidopsis thaliana. Specifically, heterologous overexpression of BnaA03.MKK6 in Arabidopsis thaliana leads to larger cotyledons, earlier growth and development, larger seeds, and increased root length in the seedling stage. Compared to the wild type, the T-DNA insertion mutant in Arabidopsis thaliana has smaller cotyledons, smaller seeds, and shorter roots in the seedling stage. Knockout of AtMKK6 in Arabidopsis thaliana results in smaller cotyledons, smaller seeds (even with malformed development), and shorter roots in the seedling stage; in particular, cr-atmkk6-3-9 even exhibits abnormal root development. This indicates that AtMKK6 participates in the regulation of seed size and root development in Arabidopsis thaliana.

[0038] 3. This invention reveals a physical interaction between BnaC03.MPK4, BnaC03.MPK6, and BnaA03.MKK6. In vitro phosphorylation experiments confirmed that BnaA03.MKK6 can phosphorylate BnaC03.MPK4 in vitro, with the phosphorylation site located in the TEY domain of BnaC03.MPK4. It is speculated that BnaA03.MKK6, through phosphorylation of the TEY domain in BnaC03.MPK4, jointly participates in the regulation of the cell cycle and further influences seed size. Attached Figure Description

[0039] Figure 1 This image shows the results of identifying positive transgenic Arabidopsis thaliana plants; among them, Figure 1 -A shows the results of identifying positive plants using two primer pairs, F35S3ND+p101-BnaA03.MKK6-R (left) and p101-BnaA03.MKK6-F+OCS5ND (right), for pEarleyGate101-BnaA03.MKK6. Figure 1 -B is the result of the identification of expression levels of OV-MKK6-4 and OV-MKK6-9 lines. **** in the figure indicates that the difference is significant at p<0.0001. The t-test was used, and the bar chart represents the SD of the mean. Mean ± SD, n=3; Figure 1 -C shows the results 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 image shows the results of the identification of Arabidopsis thaliana mutant plants; among them, Figure 2 -A represents the T-DNA insertion site analysis of the SALK-084332C mutant; Figure 2 -B represents the T-DNA insertion site analysis of the SALK-016750C mutant; Figure 2 -C is the result graph of the identification of Col-0 using LP+RP+LB (as a control); Figure 2 -D is the result graph of the identification of atmkk6 using LP+RP+LB; Figure 2 -E is the result of identifying atmpk6 using LP+RP+LB; Figure 2 -F represents the results of atmkk6 expression level identification; Figure 2-G represents the results of atmpk6 expression level identification; **** in the figure indicates a significant difference of p<0.0001, using t-test, and the bar chart represents the SD of the mean, mean ± SD, n=3;

[0041] Figure 3 This image shows the results of identification of knockout Arabidopsis thaliana plants; among them, Figure 3 -A shows the results 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 thaliana plants;

[0042] Figure 4 The image shows the results of identification of rapeseed plants overexpressing the gene; among them, Figure 4 -A indicates positive plant identification of pEarleyGate101-BnaA03.MKK6; Figure 4 -B is the result of the identification of expression level of overexpression lines. **** indicates that the difference is significant at p<0.0001. The t-test was used, and the bar chart represents the SD of the mean. Mean ± SD, n=3;

[0043] Figure 5 The image shows the results of identifying rapeseed plants that were knocked out; among them... Figure 5 -A shows the results of positive plant identification using PB-L+PB-R (right) and cas9-F+cas9-R (left); Figure 5 -B is the result of the analysis of knockout sites in rapeseed plants;

[0044] Figure 6 The image shows the results of GUS tissue staining analysis of BnaA03.MKK6. In the image, a~j are the control group, and the images show GUS staining driven by the 35S strong promoter at the two true leaf stage, four true leaf stage, leaf, inflorescence, silique on day 5, seed on day 5, silique on day 12, seed on day 12, silique on day 19, and seed on day 19, respectively. k~s are GUS staining driven by the BnaA03.MKK6 promoter at the two true leaf stage, four true leaf stage, leaf, inflorescence, silique on day 5, seed on day 5, silique on day 12, seed on day 12, silique on day 19, and seed on day 19, respectively.

[0045] Figure 7 The image shows the results of subcellular localization observation of BnaA03.MKK6. Chloroplast-RFP indicates red light excitation, 35S::BnaA03.MKK6-GFP indicates green light excitation, Bright indicates bright field, Merge indicates superposition state, and the scale bar length is 5 μm.

[0046] Figure 8This is a summary chart of the results of seed phenotypic and root phenotypic observations; among them... Figure 8 -A is a graph comparing the seed sizes of Col-0 and atmkk6; Figure 8 -B is a bar chart comparing the seed sizes of Col-0 and atmkk6. **** indicates a significant difference at p < 0.0001. The t-test is used, and the bar chart represents the SD of the mean. The mean ± SD is used, and n = 3. Figure 8 -C is a graph comparing the seed sizes of Col-0 with those of cr-mkk6-9 and cr-mkk6-10; Figure 8 -D is a bar chart comparing the seed sizes of Col-0 with cr-mkk6-9 and cr-mkk6-10. ns indicates no significant difference, **** indicates a significant difference with p < 0.0001. The t-test is used, and the bar chart represents the SD of the mean. The mean ± SD is used, and n = 3. Figure 8 -E represents the seed size images of Col-0 and OV-MKK6-4, OV-MKK6-9, ns indicates no significant difference, **** indicates a significant difference of p<0.0001, t-test is used, and the bar chart represents the SD of the mean, mean ± SD, n=3; Figure 8 -F is a bar chart comparing the seed sizes of Col-0 with OV-MKK6-4 and OV-MKK6-9. ns indicates no significant difference, **** indicates a significant difference with p < 0.0001. The t-test is used, and the bar chart represents the SD of the mean. The mean ± SD is used, and n = 3. Figure 8 -G represents a comparison of the root systems of Col-0 and allogeneic transformed plants; Figure 8 -H represents a comparison of the hypocotyls of Col-0 and allogeneic transformed plants; Figure 8 -I is a bar chart comparing the root length of Col-0 with OV-MKK6-4 and OV-MKK6-9. ns indicates no significant difference, * indicates a significant difference with p<0.01. t-test is used, and the bar chart represents the SD of the mean. Mean ± SD, n=3;

[0047] Figure 9 Figures showing phenotypic observations at different growth stages; among them, Figure 9 -A is a graph comparing seedling phenotypes; Figure 9 -B is a graph comparing the phenotypes of Col-0 and atmkk6 during the later stages of growth and development; Figure 9 -C is a graph comparing the phenotypes of Col-0 with OV-MKK6-4 and OV-MKK6-9 during their growth and development; Figure 9 -D is a graph comparing the length of siliques of Col-0 with those of OV-MKK6-4 and OV-MKK6-9; Figure 9-E is a bar chart comparing the silique length of Col-0 with OV-MKK6-4 and OV-MKK6-9. In the figure, ns indicates that there is no significant difference using the t-test, and the bar chart represents the SD of the mean. The mean ± SD is n=3.

[0048] Figure 10 The figure shows the experimental results for verifying protein-protein interactions; among them, Figure 10 -A is a graph showing the results of yeast two-hybrid verification of the interaction between BnaC03.MPK4, BnaC03.MPK6, BnaA03.MPK6 and BnaA03.MKK6 proteins; Figure 10 -B is the result of BiFC dual-luciferase verification of the interaction relationship between BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 proteins;

[0049] Figure 11 This is a diagram showing the results of protein sequence analysis, in which... Figure 11 -A is the analysis result of the BnaA03.MKK6 protein sequence; Figure 11 -B is the result of the analysis of the protein sequences of BnaC03.MPK4 and BnaC03.MPK6;

[0050] Figure 12 This is a graph showing the results of an in vitro phosphorylation experiment; in which, Figure 12 -A is a schematic diagram showing the mutation of all Ser / Thr structures in the A-loop of the BnaA03.MKK6 protein to Asp; Figure 12 -B represents His-BnaA03.MKK6 DD Structural diagram; Figure 12 -C represents the structure diagram of GST-BnaC03.MPK4; Figure 12 -D represents the structure diagram of GST-BnaC03.MPK6; Figure 12 -E represents the detection results of purified protein, where 1 represents GST, 2 represents GST-BnaC03.MPK4, 3 represents GST-BnaC03.MPK6, and 4 represents His-BnaA03.MKK6. DD ; Figure 12 -F is a graph showing the in vitro phosphorylation of BnaA03.MKK6 and BnaC03.MPK4. Detailed Implementation

[0051] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0052] In the embodiments of the present invention:

[0053] (1) It was confirmed that heterologous expression of BnaA03.MKK6 regulates the seed size and primary root growth of Arabidopsis thaliana.

[0054] BnaA03.MKK6 is a member of the MKK family in Brassica napus. To investigate the function of BnaA03.MKK6, this invention heterologously overexpressed BnaA03.MKK6 in Arabidopsis thaliana. The results showed that transgenic plants had larger seeds compared to wild-type plants, with no significant change in silique length, and better root development. The T-DNA insertion mutant and knockout mutant of AtMKK6 showed smaller seeds and shorter roots compared to wild-type plants. The knockout mutant exhibited a more pronounced decrease in seed size, even showing symptoms of seed malformation, and shorter roots, even showing abnormal root development. Based on these phenotypic observations, it is speculated that heterologous overexpression of BnaA03.MKK6 positively regulates seed size in Arabidopsis thaliana and has a positive effect on root development. Simultaneously, AtMKK6 in Arabidopsis thaliana positively regulates seed development; knocking out AtMKK6 in Arabidopsis thaliana severely affects seed size and morphology, and may even lead to abnormal root development after sowing. For more detailed information, please refer to the Examples section.

[0055] (2) Clarify the mechanism by which BnaA03.MKK6 regulates seed size.

[0056] To investigate the pathway by which BnaA03.MKK6 regulates seed size, this invention screened the homologous genes of MPK4 and MPK6 in Arabidopsis thaliana and Brassica napus, BnaC03.MPK4 and BnaC03.MPK6, as candidate genes for point-to-point verification. Yeast two-hybrid and BiFC dual-luciferase assays both showed that BnaA03.MKK6 interacts with BnaC03.MPK4 and BnaC03.MPK6.

[0057] Analysis of the BnaA03.MKK6 protein sequence revealed that it possesses a highly conserved A-loop and Ser / Thr domain. Analysis of the BnaC03.MPK4 and BnaC03.MPK6 protein sequences showed that both possess the same conserved TEY (Thr-Glu-Tyr) domain as the human p44 / 42 protein. Therefore, we selected the Anti-Phospho-44 / 42 antibody as the phosphorylation antibody to detect the in vitro phosphorylation of BnaC03.MPK4 and BnaC03.MPK6.

[0058] To impart phosphorylation activity to BnaA03.MKK6, this invention first involves site-directed mutation of two Ser / Thr residues in the Ser / Thr domain of BnaA03.MKK6 to Asp, which exhibits pseudophosphorylation activity. Subsequent in vitro phosphorylation results showed that BnaA03.MKK6 could phosphorylate the TEY domain on BnaC03.MPK4, consistent with expectations. However, unexpectedly, we did not observe phosphorylation of BnaC03.MPK6 by BnaA03.MKK6. This is presumably because BnaA03.MKK6 may require more stringent conditions to phosphorylate BnaC03.MPK6.

[0059] In this embodiment of the invention, Arabidopsis thaliana Col-0 ecotype, Nicotiana benthamiana, and ZS11, a conventional rapeseed variety, were all provided by the Rapeseed Biotechnology Center; the Arabidopsis thaliana T-DNA insertion mutant was purchased from the AraShare website (https: / / www.arashare.cn / index / Product / index.htmL).

[0060] In this embodiment of the invention, Arabidopsis thaliana, tobacco, and other experimental materials were grown in a constant-temperature incubator, while transgenic rapeseed was grown in the school's transgenic experimental plot. The incubator growing conditions were as follows: Arabidopsis seeds, after vernalization and sterilization, were sown on 1 / 2 MS culture plates and cultured in a greenhouse for 10-14 days. An Intellus controller controlled the light exposure for 16 hours and darkness for 8 hours, maintaining a temperature of 20-22°C and humidity of 70%-75%. After 10-14 days, the seedlings were transplanted into soil and continued to be cultured in the greenhouse. Tobacco seeds were directly sown in the soil, and the cultivation conditions were the same as for Arabidopsis thaliana. Transgenic rapeseed was cultured under natural conditions.

[0061] In this embodiment of the invention, the experimental strains DH5α, GV3101, GV3101 (pSoup), BL21 and Y2H Gold were all purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0062] In this embodiment of the invention, the pEASY-Blunt vector was purchased from TransGen Biotech 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.

[0063] In this embodiment of the invention, the BnaA03.MKK6 gene sequence is shown in SEQ ID NO:1, the coding sequence is shown in SEQ ID NO:2, and the protein sequence is shown in SEQ ID NO:3.

[0064] In this embodiment of the invention, the full-length CDS sequence of BnaA03.MKK6 found on the BnaPIR website was used as a template, and primers were designed using the software Primer 5. The primer sequences are shown in Tables 1 to 3.

[0065] Table 1. List of primers used for vector construction

[0066]

[0067] Table 2. List of primers used for quantitative PCR

[0068]

[0069] Table 3. List of primers used for site-directed mutagenesis

[0070]

[0071] In this embodiment of the invention, the primers for plant identification are shown in Table 4.

[0072] Table 4. Primer list for plant identification

[0073]

[0074] If any experimental procedures are unclear in the embodiments of this invention, please refer to the relevant paper "Molecular mechanism of seed size regulation by BnaA03.MKK6 in Brassica napus", which will be published later.

[0075] Example 1. Cloning and vector construction of the BnaA03.MKK6 gene

[0076] Total RNA was extracted from ZS11 leaves using the EZ-10 DNA away RNA Mini-Preps Kit instructions. The RNA quality and concentration were determined by agarose gel electrophoresis. cDNA was synthesized using the USEVERBRIGHT INC. reverse transcription kit.

[0077] Chromosomal location information of MKK6 family members was obtained using the rapeseed genome website Brassica napus database (http: / / www.genoscope.cns.fr / brassicanapus / ), and primers were designed based on the ORF sequence of the BnaA03.MKK6 gene.

[0078] Using the extracted cDNA as a template, PCR amplification was performed. 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. This vector was then transformed into competent DH5α cells in the large intestine. Positive and correctly sequenced clones were amplified and plasmids were extracted using the EasyPure Plasmid MiniPrep Kit. These plasmids were then reacted with the pEarleyGate101 expression vector via a logarithmic reaction to form the pEarleyGate101-BnaA03.MKK6 overexpression vector for overexpression and subcellular localization.

[0079] Based on the promoter sequence of the BnaA03.MKK6 gene, specific primers (SEQ ID NO.6 and SEQ ID NO.7) were designed to amplify the 1500 bp sequence upstream of the promoter, as shown in SEQ ID NO.45. The vector pCAMBIA1305.1-BnaA03.MKK6 promoter vector was constructed by recombination. The obtained recombinant plasmid was transformed into Agrobacterium GV3101 and infected wild-type Arabidopsis thaliana to obtain T3 generation transgenic plants.

[0080] Using the Atu3d vector as a template, an expression cassette was constructed using primers (SEQ ID NO. 46 to SEQ ID NO. 51). The SgRNA expression cassette was assembled into the pYLCRISPR / Cas9 vector using the Golden-Gate method. The recombinant product was transformed into competent E. coli cells, and bacterial testing was performed using PB-L+PB-R (SEQ ID NO. 52 and SEQ ID NO. 53). The results showed that 12 samples were correctly tested. The correctly tested strains were sent to BGI Genomics for sequencing. The sequencing results were compared using Geneious to obtain the pYLCRISPR / Cas9-BnaA03.MKK6 vector. pYLCRISPRCas9-AtA03.MKK6 was constructed using the same method.

[0081] Example 2. Obtaining and Identifying Transgenic Lines

[0082] (1) Obtaining and identifying transgenic Arabidopsis thaliana positive plants

[0083] Agrobacterium was used to genetically transform Arabidopsis thaliana with the overexpression vector pEarleyGate101-BnaA03.MKK6. T0 generation seeds were harvested, re-sown, and cultured until the seedling stage. Selection was performed by spraying with 0.15 mg / mL Basta. The surviving T1 generation plants were identified as positive plants and their expression levels were observed. cDNA was extracted from the surviving plants, and positive plants were identified using two primer pairs: F35S3ND + p101-BnaA03.MKK6-R and p101-BnaA03.MKK6-F + OCS5ND. Results are as follows: Figure 1 -A and Figure 1 As shown in -B, a total of 12 positive plants were obtained, and the expression level of BnaA03.MKK6 was significantly upregulated in all 12 plants. OV-MKK6-4 and OV-MKK6-9, with relatively high expression levels and significant differences, were selected for subsequent experiments. Agrobacterium carrying the pCambia1305.1-BnaA03.MKK6 vector was transformed into flowering Arabidopsis thaliana. T0 generation seeds were harvested and re-sown on 1 / 2 MS + Hyg (25 mg / mL) solid medium for screening. DNA was extracted from surviving plants, and positive plants were identified using two primer pairs: M13p-F + Pro-BnaA03.MKK6-R and Pro-BnaA03.MKK6-F + M13p-R. The results showed that 9 positive plants were obtained; see details below. Figure 1 -C~ Figure 1 -D, positive plants are used for subsequent experiments.

[0084] (2) Obtaining and identifying Arabidopsis thaliana mutant plants

[0085] T-DNA insertion mutant lines SALK-084332C and SALK-016750C, representing AtMKK6 (AT5G56580) and AtMPK6 (AT2G43790), were purchased from the ABRC website. The insertion site of SALK-084332C is located in the promoter fragment, while the insertion site of SALK-016750C is located in the 5' UTR. Figure 2 -A~ Figure 2 -B. Total DNA was extracted from the mutant lines after culturing to the T2 generation. Positive plants were identified using primers LP+RP+LB (atmkk6-LP, atmkk6-RP, and LBb1.3). Figure 2 -C~ Figure 2As shown in Figure -E, Col-0 has a 1000bp band (a), while the SALK-084332C and SALK-016750C mutant lines have only one 700bp band (b and c), indicating that the SALK-084332C and SALK-016750C mutant lines are homozygous mutants. These plants were named atmkk6 and atmpk6, respectively. qPCR quantitative identification of the homozygous plants yielded the following results: Figure 2 -F、 Figure 2 As shown in -G, the expression levels of atmkk6 and atmpk6 were significantly downregulated, and these plants were selected as candidate plants for subsequent experiments.

[0086] (3) Obtaining and identifying Arabidopsis thaliana knockout plants

[0087] Agrobacterium carrying the pYLCRISPR / Cas9-AtMKK6 vector was transformed into flowering Arabidopsis thaliana. T1 generation seeds were harvested and re-sown on 1 / 2 MS + Kan (25 mg / mL) culture dishes for screening. DNA was extracted from 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 is shown. cDNA was extracted from the plants identified as positive, and the target gene fragment was amplified and sent to the China Rice Research Institute for HI-TOM sequencing. Analysis of the sequencing results revealed that both plants exhibited a single-base G deletion mutation, leading to a frameshift mutation, and a single-base A insertion in cr-mkk6-3-9. See details... Figure 3 -B, and named them cr-mkk6-3-9 and cr-mkk6-3-10 for subsequent experiments.

[0088] (4) Obtaining and identifying rapeseed overexpression plants

[0089] DNA was extracted from the overexpressing rapeseed plants, and positive plants were identified using two primer pairs: F35S3ND + p101-BnaA03.MKK6-R and p101-BnaA03.MKK6-F + OCS5ND. The results are as follows: Figure 4 As shown in -A, a total of 6 positive plants were obtained. RNA was extracted from the 6 transgenic rapeseed plants, and cDNA was reverse transcribed. The expression level was identified using qPCR. The results are as follows: Figure 4 As shown in -B, the expression level of BnaA03.MKK6 in the six transgenic plants was significantly upregulated compared to ZS11, which can be used for subsequent experiments.

[0090] (5) Obtaining and identifying rapeseed plants by knocking them down

[0091] 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). Five positive plants were obtained, as detailed below. Figure 5 -A. cDNA was extracted from plants identified as positive, and the target gene fragment was amplified and sent to the Rice Research Institute for HI-TOM sequencing. Analysis of the sequencing results revealed long-fragment base mismatch mutations in both plants, resulting in incorrect protein coding. GA and TC mutations were found in cr-BnaMKK6-3, and a T base deletion was found in cr-BnaMKK6-5, leading to a frameshift mutation. See details... Figure 5 -B, named cr-BnaMKK6-3 and cr-BnaMKK6-5, for subsequent experiments.

[0092] Example 3. Analysis of BnaA03.MKK6 expression pattern

[0093] This invention obtained an expression vector for GUS gene expression driven by the BnaA03.MKK6 promoter. Homozygous T3 generation plants were used for GUS staining observation, while Arabidopsis thaliana was transformed with a GUS gene expression vector driven by a strong 35S promoter as a control. GUS staining results showed that BnaA03.MKK6 had high expression levels at the two true leaf stage, inflorescence development stage, and on days 5 and 12 of silique and seed development. At the four true leaf stage, BnaA03.MKK6 showed high expression in the roots and low expression in the leaves. BnaA03.MKK6 was not expressed on day 19 of leaf, silique, and seed development. (See details...) Figure 6 The results are largely consistent with the website's predicted expression pattern of BnaA03.MKK6.

[0094] (2) Subcellular localization of BnaA03.MKK6 protein

[0095] A fluorescent vector, pEarleyGate101-BnaA03.MKK6, was further constructed for subcellular localization analysis. Fluorescent signals were observed on both the cell nucleus and cell membrane of protoplasts, indicating that the BnaA03.MKK6 protein is simultaneously localized to both the cell membrane and nucleus, suggesting that BnaA03.MKK6 may function in both the nucleus and cell membrane. Agrobacterium carrying the pEarleyGate101-BnaA03.MKK6 vector was injected into tobacco leaves, and... Figure 7 As shown, fluorescent signals were observed on both the nucleus and cell membrane of tobacco leaf cells. This experiment further confirms that the BnaA03.MKK6 protein is simultaneously localized on both the cell membrane and the cell nucleus.

[0096] Example 4. The effect of BnaA03.MKK6 on rapeseed growth and development

[0097] (1) Observation of seed size and root phenotype

[0098] To investigate the biological function of BnaA03.MKK6, this invention used wild-type Col-0 and the T-DNA insertion mutant atmkk6, transgenic plants OV-MKK6-4 and OV-MKK6-9 that heterologously overexpressed BnaA03.MKK6, and Arabidopsis knockout plants cr-mkk6-3-9 and cr-mkk6-3-10 that knocked out BnaA03.MKK6 at maturity. Seed surface area was calculated using ImageJ. The results showed that compared to Col-0, the seed surface area of ​​atmkk6, cr-mkk6-9, and cr-mkk6-10 was significantly reduced, and the seeds of cr-mkk6-9 and cr-mkk6-10 showed malformed development, such as... Figure 8 -A~ Figure 8 -D is shown; while the seed area of ​​OV-MKK6-4 and OV-MKK6-9 is significantly larger than that of Col-0, such as Figure 8 -E~ Figure 8 -F is shown. The seeds were re-sown on 1 / 2 MS medium, and the primary root lengths of the seedlings were measured. The results showed that, compared to Col-0, the primary roots of atmkk6 showed no significant change, the primary roots of cr-mkk6-10 were shorter, the primary roots of OV-MKK6-4 and OV-MKK6-9 were longer than those of Col-0, while the root development of cr-mkk6-9 was abnormal. See details... Figure 8 -G~ Figure 8 -I. The experimental results demonstrate that BnaA03.MKK6 positively regulates seed size and primary root elongation in plants.

[0099] (2) Phenotypic observation at growth stages

[0100] The above-mentioned plants were transplanted into nutrient soil, and their phenotypic characteristics at different growth stages were observed. The results showed that during the vegetative growth stage, compared to Col-0, atmkk6, cr-mkk6-3-9, and cr-mkk6-3-10 seedlings had smaller leaf areas and weaker growth. Figure 9 As shown in -A. OV-MKK6-4 and OV-MKK6-9 have larger leaves and stronger growth compared to Col-0, as shown in... Figure 9 As shown in -B. During the reproductive growth stage, atmkk6 showed no significant change compared to Col-0, as... Figure 9 -B is shown. However, OV-MKK6-4 and OV-MKK6-9 showed faster growth after bolting compared to Col-0, as shown in Figure 1. Figure 9-C is shown. Observation of mature siliques revealed that the length of OV-MKK6-4 siliques was significantly increased compared to Col-0, while the length of OV-MKK6-9 siliques showed no significant change. Figure 9 -D and Figure 9 -E is shown.

[0101] Example 5. Interactions between BnaA03.MKK6 protein and BnaC03.MPK4 and BnaC03.MPK6 proteins

[0102] In Arabidopsis thaliana, MKK6 has been reported to interact with MPK4 and MPK6, and MPK4 has been confirmed to participate in cell cycle regulation, while atmpk4 has a similar phenotype to atmkk6. Therefore, this invention uses the proteins encoded by the rapeseed homologs BnaC03.MPK4, BnaC03.MPK6, and BnaA03.MPK6 as candidate proteins for interaction with BnaA03.MKK6 and performs yeast two-hybrid validation. The yeast two-hybrid results show that the proteins BnaC03.MPK4, BnaC03.MPK6, and BnaA03.MPK6 interact with the protein BnaA03.MKK6. Figure 10 As shown in -A. To further verify the experimental results, this invention conducted a BiFC experiment, co-injecting Ecc-BnaC03.MPK4 and Ecc-BnaC03.MPK6 vectors with Enn-BnaA03.MKK6 into tobacco leaf cells, and observing the fluorescence signal under a laser confocal fluorescence microscope. The results are shown in... Figure 10 As shown in Figure -B, BnaC03.MPK4, BnaC03.MPK6 and BnaA03.MKK6 proteins interact at the cell nucleus and cell membrane, consistent with the results of the yeast two-hybrid assay.

[0103] Example 6. His-BnaA03.MKK6 DD In vitro phosphorylation of GST-BnaCO3.MPK4

[0104] (1) Protein sequence analysis

[0105] The experiments in Example 5 have demonstrated the interaction between BnaC03.MPK4, BnaC03.MPK6, and BnaA03.MKK6 proteins. To further determine how BnaC03.MPK4, BnaC03.MPK6, and BnaA03.MKK6 proteins function, this invention attempts to verify the phosphorylation relationship between BnaA03.MKK6 and BnaC03.MPK4 and BnaC03.MPK6 proteins. Sequence analysis of BnaA03.MKK6 shows that it possesses a highly conserved A-loop of the MKK family and a Ser / Thr structure, as detailed in [link to example]. Figure 11 -A. Structural analysis of BnaC03.MPK4 and BnaC03.MPK6 proteins revealed the presence of a TEY domain. (See details...) Figure 11 -B, phosphorylation of this domain can be detected by the Anti-Phospho-44 / 42 antibody.

[0106] (2) In vitro phosphorylation experiment

[0107] To prepare for subsequent in vitro phosphorylation experiments, this invention uses a site-directed mutagenesis kit provided by Beyotime to mutate the Ser and Thr residues in the Ser / Thr structure of the A-loop of the BnaA03.MKK6 protein to Asp, as shown below. Figure 12 As shown in -A. BnaA03.MKK6 was amplified using ZS11 cDNA and a site-directed mutagenesis vector as templates. DD BnaC03.MPK4, BnaC03.MPK6. Also, BnaA03.MKK6. DD BnaC03.MPK4 and BnaC03.MPK6 were recombined into pGET-4T1 vector using pET-28a. His-BnaA03.MKK6 was successfully obtained. DD GST-BnaC03.MPK4 and GST-BnaC03.MPK6 vectors, see details. Figure 12 -B~ Figure 12 -D.

[0108] The obtained His-BnaA03.MKK6 DD The GST-BnaC03.MPK4 and GST-BnaC03.MPK6 vector plasmids were retransformed into BL21 E. coli competent cells. The bacterial cells were collected to purify the protein, obtaining purified His-BnaA03.MKK6. DD GST-BnaC03.MPK4 and GST-BnaC03.MPK6 proteins were detected by SDS-PAGE. Figure 12As shown in Figure -E, this invention yielded a His-BnaA03.MKK6 with a size of 50 kDa. DD The GST-BnaC03.MPK4 protein (70 kDa) and GST-BnaC03.MPK6 protein (65 kDa) were purified, with single and clearly visible protein bands, indicating successful purification.

[0109] The purified His-BnaA03.MKK6 DD The proteins were incubated in an in vitro phosphorylation system with GST-BnaC03.MPK4 and GST-BnaC03.MPK6, respectively. Western blotting analysis revealed that GST-BnaC03.MPK4 was phosphorylated by His-BnaA03.MKK6. DD In vitro phosphorylation was observed, with the phosphorylation site located in the TEY domain, while GST-BnaC03.MPK6 was not observed to be phosphorylated by His-BnaA03.MKK6. DD In vitro phosphorylation, see details. Figure 12 -F.

Claims

1. The application of the BnaA03.MKK6 gene in promoting plant seed growth and primary root growth, characterized by, The nucleotide sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO:1; the plant is Brassica napus or Arabidopsis thaliana.

2. The application of an overexpression vector containing the BnaA03.MKK6 gene in promoting seed growth and primary root growth in plants, characterized in that, The nucleotide sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO:1; the plant is Brassica napus or Arabidopsis thaliana.

3. The application of Agrobacterium containing the BnaA03.MKK6 gene overexpression vector in promoting plant seed growth and primary root growth, characterized in that, The nucleotide sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO:1; the plant is Brassica napus or Arabidopsis thaliana.

4. A method for increasing seed size and primary root length in Brassica napus, characterized in that, Includes the following steps: (1) Construct an overexpression vector containing the BnaA03.MKK6 gene of Brassica napus; the nucleotide sequence of the BnaA03.MKK6 gene is shown in SEQ ID NO:1; (2) The overexpression vector obtained in step (1) was transformed by Agrobacterium, and transgenic plants were screened to obtain rapeseed of the Brassica napus type.

5. The method according to claim 4, characterized in that, Step (1) specifically includes: PCR amplification using rapeseed cDNA as a template; recombining the amplified BnaA03.MKK6 gene cDNA into the pENTR / D-TOPO entry vector and transforming it into competent DH5α cells in the large intestine; identifying positive clones and sequencing to verify and obtain positive plasmids; performing an LR reaction between the obtained positive plasmids and the pEarleyGate101 expression vector to obtain an overexpression vector for the BnaA03.MKK6 gene.

6. The method according to claim 5, characterized in that, The PCR amplification primers were p101-BnaA03.MKK6-F with nucleotide sequences as shown in SEQ ID NO:4 and p101-BnaA03.MKK6-R with nucleotide sequences as shown in SEQ ID NO:5.

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