CN112011547B - Major gene for controlling rape leaf shape and application thereof - Google Patents

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a major gene for regulating and controlling rape leaf shape and application thereof. The major gene for controlling the leaf shape of the rape provided by the invention is BnaA10.RCO gene or allele thereof, wherein the nucleotide sequence of the BnaA10.RCO gene is shown as SEQ ID NO: 1 is shown. The major gene for controlling the rape leaf shape is a gene positioned on a rape A10 chromosome and encodes an HD-ZIPI transcription factor related to plant leaf shape development, and the gene is separated and cloned by utilizing a method combining map-based cloned gene positioning and genetic transformation, so that a theoretical basis is provided for the improvement and breeding of the rape leaf shape; meanwhile, the method provides reference for the leaf shape regulation and control research of cross mosaic plants such as cabbage type rape, mustard type rape and the like.

Description

A major gene for controlling leaf shape of rapeseed and its application

technical field

The invention relates to the technical field of plant genetic engineering, in particular to a major gene for regulating the leaf shape of rape and its application.

Background technique

Leaf is one of the most important organs of plants, with functions such as photosynthesis, transpiration, nutrient supply and water transport, which affect the physiological characteristics and even yield of crops. The change of leaf shape affects leaf temperature, water use efficiency and photosynthesis intensity of plants. It is an adaptive performance of plants to the environment and is formed through long-term natural selection. There are abundant natural variations in the shape and size of leaves, and leaves can be divided into whole, lobed and deep-lobed types according to the shape of the leaf margin. In addition, some studies have also found that mosaic traits are also related to plant stress resistance. So far, research on leaf shape has mainly focused on model species such as Arabidopsis, Camelina and tomato, while the localization and molecular regulation mechanism of leaf shape genes in rapeseed are rarely studied.

The mosaic phenotype in rapeseed can be manifested in the early stage of plant development, and the phenotype is stable and not easily affected by environmental conditions. Therefore, mosaic has also become an ideal morphological marker for the production of rapeseed hybrids. At present, the existing application of rape mosaic phenotype is to use the dominant traits of mosaic and leaves as markers to select restorer lines and sterile lines to ensure the purity of hybrid rapeseed. However, there are few studies on the localization and molecular regulation mechanism of leaf shape genes in rapeseed. Therefore, how to better understand the formation mechanism of mosaic leaf shape is of great significance for better genetic improvement of rapeseed by using leaf shape traits.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a major gene for controlling rape leaf shape and its application. The main effect gene provided by the invention can realize the regulation of rape leaf shape, and provides a theoretical basis for the improvement and breeding of rape leaf shape.

The major gene for controlling the leaf shape of rapeseed provided by the present invention is the BnaA10.RCO gene or its allele, wherein the nucleotide sequence of the BnaA10.RCO gene is shown in SEQ ID NO: 1.

The present invention also provides a protein that regulates the leaf shape of rapeseed, and the protein is encoded by the above-mentioned major gene.

The present invention also provides an expression vector containing a major gene for controlling the leaf shape of rapeseed, wherein the expression vector is a PMDC32 vector containing the above major gene.

The invention also provides the main gene for controlling the leaf shape of rapeseed, the protein encoded by the gene and the application of the expression vector containing the gene in regulating the leaf shape of rapeseed.

The present invention also provides a method for constructing an expression vector containing a major gene for regulating the leaf shape of rapeseed, characterized in that it comprises the following steps:

S1-1, Amplify BnaA10.RCO genomic DNA sequence from two materials of HY cleavage leaves and Z9 lobed leaves, respectively;

S1-2, the amplified sequence in step S1 is ligated into the PMDC32 vector with CaMV35S as the promoter by the method of enzyme digestion and ligation.

Wherein, the primers used for amplifying the BnaA10.RCO genomic DNA sequence in step S1-1 are:

A10-117F GGGCttaattaaCTACCATGGGTAAGGCTGTTGC

A10-118R GACAggtaccATGGAATGGTCAACGACGAGC

The present invention also provides a method for cloning a major gene for controlling the leaf shape of rapeseed, comprising the following steps:

S2-1, align the genes annotated with the BnLLA10 segment in the Brassica napus reference genome to the Arabidopsis genome, and perform functional annotation to determine candidate genes;

S2-2, design primers to clone the candidate genes of the two materials of HY incised leaves and Z9 lobed leaves, and compare the cDNA and DNA sequencing results to determine the differential positions of the candidate genes in the two materials. ;

S2-3, analyze the specific expression of candidate genes in the near-isogenic line Z9-NIL of HY and Z9, and finally determine the major gene BnaA10.RCO through overexpression experiments and gene knockout experiments;

S2-4, connect the main gene BnaA10.RCO to the expression vector and transfer it into the recipient cell through the Agrobacterium-mediated hypocotyl transformation method.

Wherein, the candidate genes determined in step S2-1 are BnaA10.RCO and BnaA10.LMI1.

Wherein, the nucleotide sequence of the primer used when the BnaA10.RCO gene is cloned in step S2-2 is:

A10_101F: TTCCCAAGATCCGAAACACCT

A10_101R: ATGGAATGGTCAACGACGAGC.

The invention discloses the isolated and cloned main gene BnaA10.RCO for regulating the leaf shape of rapeseed, and the nucleotide sequence of its allele (HY) as shown in SEQ ID NO: 1 and the amino acid sequence as shown in SEQ ID NO: 2 , in the allelic comparison sequencing, it was found that the allelic differences between the two parental materials HY and Z9 were mainly located in the 5' regulatory region of the gene, and the expression levels of HY and its near-isogenic line Z9-NIL were analyzed by qRT-PCR technology. , found that the expression of BnaA10.RCO gene in different tissues was significantly higher in HY than in near-isogenic lines. Both overexpression and gene knockout transgenic experiments proved that BnaA10.RCO gene positively regulates rape leaf shape.

The invention clones the main gene that regulates the leaf shape of rapeseed in rapeseed, provides a theoretical basis for the improvement and breeding of the leaf shape of rapeseed, and also provides the research on the leaf shape control of cruciferous leaf plants such as cabbage-type rape, mustard-type rape and the like. refer to.

Description of drawings

Figure 1 is the gene structure diagram of BnaA10.RCO; the positions in S1 and S2 indicated by the arrows are the target sequence positions;

Figure 2 is an analysis diagram of the expression level of BnaA10.RCO gene; wherein A is the expression level in leaves of HY and Z9-NIL seedlings at different days, and B is the expression level in different tissues of HY and Z9-NIL seedlings at 10 days;

Figure 3 shows the phenotype and expression levels of overexpressed BnaA10.RCOHY (HY OE) and BnaA10.RCOZ9 (Z9OE) under the background of J9707 and HY; among them, the 15d leaf-aged seedlings: (A) J9707; (B) J9707 HY OE-16, HYOE-55, HY OE-63, HY OE-141 and HY OE-30 transgenic lines in the background; (C) HY; (D) Z9 OE-7, Z9 OE-17 in the HY background , Z9 OE-23; (E) Z9 OE-119 and Z9 OE-122 transgenic lines in HY background; (F) Z9 OE-20 and Z9 OE-25 transgenic lines in HY background; (G-K) correspond to Seedlings in A (G), B (H), C (I), D (J) and F (K) grew to mature leaves at 50 d; L represents the expression levels of BnaRCO and BnaLMI1 in leaves at 10 d, M represents the expression levels of BnaRCO and BnaLMI1 in shoot apex at 10 days.

Figure 4 is a BnaRCO mutant induced by the CRISPR/Cas9 system in the mosaic parent HY. (A) Schematic representation of the Cas9P35s-BnaRCO (SRCO) vector. (B) Target sequence designed for RCO, where the PAM sequence is underlined. (C) Genotype and phenotype of T ₀ and T ₁ generations of BnaRCO edited individual plants. (D) BnaRCO edits sequences near the sgRNA target site of an individual. The PAM sequence is marked with an underline, and the specific mutation sequence information is shown in the annotation on the right; "A" or "C" represents the wild-type allele, and "a" or "c" represents the mutant allele. The ruler is 1cm.

Figure 5 is the detection of the BnaA10.RCO mutant phenotype and the expression level of BnaLMI1 in the mutant. Comparison of phenotypes (A) (two-leaf stage) and leaf shape index (B) of edited individual plants with different genotypes; AaCc represents two RCO gene copies heterozygous mutants, aacc represents two RCO gene copies represents homozygous Mutants or biallelic mutants; (C) BnaLMI1 expression levels in shoot tips of 7d seedlings in BnaA10.RCO edited transgenic lines were examined by normalization of total RNA by qPCR with BnOTC. The ruler is 1cm.

Detailed ways

The principles and features of the present invention will be described below in conjunction with specific embodiments, and the examples are only used to explain the present invention, but not to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the technical field of the present invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

Test material:

HY comes from "Tongling Mosaic" in Anhui Province. It is a local variety. The leaf phenotype is deep-lobed leaves, also called Mosaic. It is provided by the Planting Resource Bank of Jiangxi Academy of Agricultural Sciences.

Z9 is "Zhongshuang No. 9" and abbreviated as Z9. It is a popularized variety, and its leaf phenotype is lobed.

Brassica napus J9707, a lobed leaf shape, was used as the transformation receptor in this study, provided by the Rapeseed Engineering Center of Huazhong Agricultural University.

The expression vector PMDC32 was purchased from the addgene website at https://www.addgene.org .

The PYLCRISPR/Cas935S-H editing vector was provided by the laboratory of Mr. Liu Yaoguang, South China Agricultural University. For the specific sequence of the vector, please refer to Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y .Arobust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015, 8:1274-1284.

Z9-NIL: Two parent materials, HY and Z9, were obtained by reversing crosses to obtain F1 hybrids, wherein the F1 hybrids of HY×Z9 were derived from F2, F3, BC1F2, BC2F2 and BC3F2 through selfing and backcrossing with the HY parent. Segregated groups of different generations. In the BC3F2 generation, the individual plants with the homozygous Z9 allele at the leaf-shaped major locus (BnLLA10 locus) and the leaf shape identical to the donor parent Z9 were screened by molecular marker-assisted selection, and were used as the HY material. The near-isogenic line is called Z9-NIL (Near-Isogenic Line ofHY).

The present invention provides a major gene for controlling the leaf shape of rape, wherein the major gene is the BnaA10.RCO gene or its allele, wherein the nucleotide sequence of the BnaA10.RCO gene is shown in SEQ ID NO: 1. The main gene BnaA10.RCO involved in the present invention is a gene located on the A10 chromosome of rapeseed and encodes an HD-ZIPI transcription factor related to plant leaf shape development. The gene was isolated and cloned by the combined method. The present invention utilizes the fine mapping result of BnLLA10, the locus that controls leaf shape on the A10 chromosome published by our research group, and narrows the mapping segment to the 41.0kb segment corresponding to A10_87 and A10_88 on the A10 chromosome. The BnaA10.RCO gene was identified as a candidate gene by the function prediction of 5 genes in this interval. The candidate gene has 2 introns and 3 exons, encoding 223 amino acid sequences. The genotype of the candidate gene in the two parents, HY and Z9, only has a C-T base mutation in the coding region of the gene 35bp away from ATG, resulting in the mutation of HY from Met12 to Thr12 relative to Z9. However, the mutation was not in the conserved motif segment of the RCO gene, and Met12 also appeared in Yuye 87, which indicated that Thr12 or Met12 were not the key amino acids to determine leaf shape. The 5'-UTR region of BnaA10.RCO has many differences between parents, including 79 SNPs and 21 INDELs, while only 13 SNP differences in the 3'-UTR. The expression level of BnaA10.RCO gene was significantly up-regulated in the stem top tissue of 9d seedlings and the leaves of 10d and 15d seedlings, compared with Z9-NIL. Using overexpression technology and genetic transformation mediated by Agrobacterium, it was found that the expression level of this gene was positively correlated with leaf shape complexity. Using CRISPR/Cas9 gene editing technology, through Agrobacterium-mediated genetic transformation, it was found that knocking out this gene in HY mutated the mosaic leaf shape to normal leaves. There was no significant difference in BnaLMI1 expression levels in the overexpressing transgenic line and the knockout mutant compared to its wild type. Therefore, the BnaA10.RCO gene was finally determined to be the main gene that controls the leaf edge incision of rapeseed.

The present invention also provides a protein that regulates the leaf shape of rapeseed, and the protein is encoded by the above-mentioned major gene.

The present invention also provides an expression vector containing a major gene for controlling the leaf shape of rapeseed, wherein the expression vector is a PMDC32 vector containing the above major gene.

The invention also provides the main gene for controlling the leaf shape of rapeseed, the protein encoded by the gene and the application of the expression vector containing the gene in regulating the leaf shape of rapeseed.

The present invention also provides a method for constructing an expression vector containing a major gene for regulating the leaf shape of rapeseed, characterized in that it comprises the following steps:

S1-1, Amplify BnaA10.RCO genomic DNA sequence from two materials of HY cleavage leaves and Z9 lobed leaves, respectively;

S1-2, the amplified sequence in step S1 is ligated into the PMDC32 vector with CaMV35S as the promoter by the method of enzyme digestion and ligation.

Wherein, the primers used for amplifying the BnaA10.RCO genomic DNA sequence in step S1-1 are:

A10-117F GGGCttaattaaCTACCATGGGTAAGGCTGTTGC

A10-118R GACAggtaccATGGAATGGTCAACGACGAGC.

The present invention also provides a method for cloning a major gene for controlling the leaf shape of rapeseed, comprising the following steps:

S2-1, align the genes annotated with the BnLLA10 segment in the Brassica napus reference genome to the Arabidopsis genome, and perform functional annotation to determine candidate genes;

S2-2, design primers to sequence the candidate genes of the two materials of HY cut leaf and Z9 lobed leaf, and compare the sequencing results of their cDNA and DNA to determine the positions where the candidate genes of the two are significantly different;

S2-3, analyze the specific expression of candidate genes in the near-isogenic line Z9-NIL of HY and Z9, and finally determine the major gene BnaA10.RCO through overexpression experiments and gene knockout experiments;

S2-4, connect the main gene BnaA10.RCO to the expression vector and transfer it into the recipient cell through the Agrobacterium-mediated hypocotyl transformation method.

Wherein, the candidate genes determined in step S2-1 are BnaA10.RCO and BnaA10.LMI1.

Wherein, the nucleotide sequences of the primers used in gene cloning and sequencing in step S2-2 are:

A10_101F: TTCCCAAGATCCGAAACACCT

A10_101R: ATGGAATGGTCAACGACGAGC.

Example 1 Location and cloning of BnaA10.RCO gene

After extensive research by the applicant, the applicant believes that there is a leaf shape gene with a large effect in the A10 chromosome positioning segment, and the regulatory effect of this site on leaf shape is relatively conservative in Brassica.

In the Brassica napus reference genome (http://www.genoscope.cns.fr/brassicanapus/), there are 5 annotated genes within 41.0 kb of the mapped segment, namely BnaA10g26310D to BnaA10g26350D (Table 1). We aligned genes annotated within the BnLLA10 segment to the Arabidopsis genome (http://www.arabidopsis.org/) and performed functional annotation. Among them, BnaA10g26320D (BnaA10.RCO) and BnaA10g26330D (BnaA10.LMI1) are Arabidopsis LMI1-like paralogous genes, which encode HD-Zip I transcription factors. Leaf shape regulation (Vlad et al 2014, Sicard et al 2014). Among the other three genes, BnaA10g26310D is an exocrine family gene; BnaA10g26340D encodes a 3-deoxy-D-mannose octacaprylate transferase; BnaA10g26350D encodes a β-mannan synthase synthase. The annotation information of these three genes has nothing to do with leaf shape regulation. Therefore, the tandem homologous genes BnaA10.RCO and BnaA10.LMI1 are the most likely candidate genes to control rape mosaic.

Table 1 Annotated genes within the mapping segment in the reference genome of Brassica napus

Based on this, according to the resequencing sequences of HY (cut leaf) and Z9 (lobed leaf), we designed the primer pair A10-101F/R to amplify the BnaA10.RCO gene sequence, where the primer sequence is:

A10_101F: TTCCCAAGATCCGAAACACCT

A10_101R: ATGGAATGGTCAACGACGAGC.

At the same time, this pair of primers was used to amplify the cDNA and genome of the gene and carry out TA cloning, wherein the PCR reaction system was: 2ul DNA template, 25ul Phanta Max Buffer (Vazyme, P505-d1), 2ul of primer working solution, 1ul of 10mM dNTP, Phanta Max Super-Fidelity DNA polymerase 1ul and double distilled water were mixed into a 50ul system. The reaction program was: 1: 95°C for 4 minutes; 2: 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute and 30 seconds; 3: Repeat 2 steps 33 times; 4: 72°C for 10 minutes, 25°C for 5 minutes.

Comparison of cDNA sequencing results and DNA sequencing results showed that BnaA10.RCO has 2 introns and 3 exons, encoding 223 amino acid sequences. Compared with BnaA10.LMI1, the protein similarity is 61%, and it is a homologous gene. This type of gene contains a conserved homeobox (Homeobox domain) and leucine zipper domain (Leucine zipper domain). In the allele comparison sequencing results, we found that the BnaA10.RCO gene in the two parents had a C-T base mutation 35bp away from ATG in the gene coding region, resulting in the mutation of HY relative to Z9 from Met12 to Thr12. However, the mutation was not in the conserved motif segment of the RCO gene, and Met12 also appeared in the cut leaf material Yuye 87, indicating that Thr12 or Met12 are not the key amino acids that determine leaf shape. The 5'-gene regulatory region of BnaA10.RCO showed many differences between parents, including 79 SNPs and 21 INDELs, while the 3'-gene regulatory region had only 13 SNP differences (Fig. 1).

Cloning primers for the 5' gene regulatory region of the BnaA10.RCO gene:

A10_111F: GAGCGAGTTGGATGAGTTAG

A10_112F: CTTTAGTCTTGCTGTTCTTGTG

A10_115R: CGGCGATGATGTAGAGATAATA

A10_145F: TTACCTTCGGGTTCTTAGCGT

A10_148F: GTAGAGAGGGAAGTCATGTTGTC

A10_150R: CGATGTCTTGCGGTACTTTAT

3' gene regulatory region cloning primer of BnaA10.RCO gene:

A10_106F: TTCTTCGCAGGAGGTGTA

A10_107F: CAGGTTACGTTCCTCCTTTC

A10_108F: CGGTCTTGCTCCAACATT

A10_109R: TATCATCGCAACAGCCTTAC

A10_110R: GAGGCAAATGAATCTGAACAAC

Example 2 Expression analysis of BnaA10.RCO

The gene expression of BnaRCO was detected in HY and Z9-NIL. The total RNA at the stem tip of the parent HY and Z9-NIL was extracted, and the cDNA obtained by reverse transcription was used as a template, and the expression detection primer of the gene was used for PCR amplification, and the primer sequence was:

RCOF: GGTAGCTGTTTGGTTCCAGAA

RCOR: CCCTCCGGCTATTTGATTGT

The rape gene ORNITHINE TRANSCARBAMYLASE (OTC) gene (Cnops et al 2004) was used as the internal reference gene, and the primer sequences were as follows:

BnOTC-F: CATAACCACCCTTGCCAAATC

BnOTC-R: TTGTTCCCGTCTCCAACATAG

The reaction system was: 2×super mix 7.5ul, left and right primers (2.5uM) 2ul each, cDNA: 5.5ul, and the reaction program was 1: 95°C for 30 seconds; 2: 95°C for 15 seconds, 56°C for 15 seconds, 72 15 seconds at °C, fluorescence collection; 3: Repeat step 2 45 times; 4: Melt curve generation, 10 minutes at 4 °C.

The results of qRT-PCR showed that in the shoot top tissue of 9d seedlings and the leaves of 10d and 15d seedlings, the expression of BnaRCO gene was significantly up-regulated in HY compared with Z9-NIL. In addition, compared with the leaves of 10d and 15d seedlings, the expression levels of candidate genes in the stem apical tissues of 9d seedlings were significantly increased (Fig. 2A); In leaves and cotyledons of 10-day seedlings, the expression level of BnaRCO gene was the highest in the top of the stem (Fig. 2B), indicating that BnaRCO may act in the early stage of leaf development and participate in the regulation of leaf shape. In Figure 2, A is the expression level of BnaRCO gene in HY and Z9-NIL at the stem tip of 9d seedlings, the leaves of 10d seedlings and the leaves of 15d seedlings after germination; B is the expression of BnaRCO gene in different tissues of HY seedlings qRT-PCR uses BnOTC as the internal reference gene, and the expression is expressed as the mean ± SD of 3 biological replicates; Z9-NIL is the HY background in the BC3F2 population, homozygous at the BnLLA10 locus Individuals with Z9 genotype; **, significant level at P<0.01, *, significant level at P<0.05.

Example 3 Overexpression test of BnaA10.RCO

We amplified the BnaA10.RCO genomic DNA sequences from the HY and Z9 genomes, respectively. The above primer information is:

A10-117F: GGGCttaattaaCTACCATGGGTAAGGCTGTTGC

A10-118R: GACAggtaccATGGAATGGTCAACGACGAGC

It was linked into the expression vector PMDC32 with CaMV35S as the promoter to construct an overexpression vector.

The vector simultaneously transformed two acceptor materials of lobed leaf J9707 and mosaic HY. Among them, BnaA10.RCO ^HY is the overexpression vector of BnaA10.RCO allele in HY, referred to as HY OE; BnaA10.RCO ^Z9 is the overexpression vector of BnaA10.RCO allele in Z9, referred to as Z9 OE.

Through the Agrobacterium-mediated hypocotyl transformation method, in the offspring of the HY OE transgenic line with J9707 (lobed leaf shape, excellent transformation acceptor material) as the acceptor material, most of the transgene-positive individual plants were inscribed on the leaf margins. The number of lobes was significantly increased, and some individuals produced a deep-lobed leaf phenotype, similar to HY (Fig. 3B-H). Under the background of HY transformation, in the Z9 OE transgenic line-positive line, the degree of leaf shape incision was more serious than that of HY, only a small amount of mesophyll tissue was left, the growth and cell proliferation between the split leaves were inhibited, and the leaf area was significantly reduced. In the Z9 OE under the HY transgenic background, the two transgenic lines Z9_OE-20 and Z9_OE-119 showed smooth leaf margins, showing the phenomenon of co-expression inhibition (Fig. 3E, F, K). Expression detection by qRT-PCR showed that the expression of BnaA10.RCO was greatly reduced, indicating that there was co-suppression in the two lines (Fig. 3M). Among them, Figure 3 shows the phenotype and expression levels of overexpressed BnaA10.RCOHY (HY OE) and BnaA10.RCOz9 (Z9 OE) in the background of J9707 and HY. 15d leaf-aged seedlings: (A) J9707; (B) HY OE-16, HY OE-55, HY OE-63, HY OE-141 and HY OE-30 transgenic lines in J9707 background; (C) HY; (D) Z9 OE-7, Z9 OE-17, Z9 OE-23 in HY background; (E) Z9 OE-119 and Z9 OE-122 transgenic lines in HY background; (F) Z9 OE in HY background -20 and Z9 OE-25 transgenic lines. (G-K) correspond to the mature leaves when the seedlings in A (G), B (H), C (I), D (J) and F (K) grow to 50 d plants, respectively. BnOTC was normalized by qRT-PCR, and the expression levels of BnaRCO and BnaLMI1 in the leaves (L) and shoot tips (M) of the 10d transgenic lines and their leaves were detected by qRT-PCR; the expression values represent 3 biological replicates Mean ± SD, scale bar, 1 cm.

The above results indicated that increasing the expression of BnaA10.RCO allele in HY or Z9 could increase the complexity of leaf shape; while decreasing the expression of BnaA10.RCO in HY made the leaf margins smoother. Therefore, BnaA10.RCO expression level was positively correlated with leaf shape.

The specific steps of transgenic operation are as follows:

Construction of overexpression vector: 1) Construction of p35S:BnaRCO vector: The BnaA10.RCO gene sequence was amplified from HY and Z9 with primers A10_117F(PacI)/A10_118R(KpnI), and then connected to pMDC32 vector by enzyme ligation . After the constructed vector was verified to be correct by sequencing, the plasmid was extracted and transformed into Agrobacterium GV3101.

Wherein the reaction system of PCR amplification is: 2ul DNA template, 25ul Phanta Max Buffer (Vazyme, P505-d1), each 2ul of primer working solution, 1ul of 10mM dNTP, 1ul of Phanta Max Super-Fidelity DNApolymerase and double distilled water are mixed into a system of 50ul . The reaction program was 1: 95°C for 4 minutes; 2: 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute and 30 seconds; 3: Repeat 2 steps 33 times; 4: 72°C for 10 minutes, 25°C for 5 minutes. The reaction system for enzyme digestion of the expression vector is 15ul of plasmid, 5ul of FastDigest buffer, 1.25ul of PacI and 1.25ul of KpnI, and finally double distilled water is added to mix into a 40ul system; the reaction conditions are 37°C for 15 minutes, and the reaction system during ligation is 4ul of plasmid, T4 DNA 2ul of ligasebuffer, 0.5ul of T4 DNA ligese and double distilled water were mixed into a 10ul system, and the reaction conditions were 20-25°C for 10 minutes.

The main steps of genetic transformation, the culture medium and the preparation method thereof are as follows:

1) Sterilization: The seeds are placed in a 2ml centrifuge tube in a small half tube, and 75% alcohol is added to soak the seeds for 1-3 minutes. Pay attention that the time should not be too long, otherwise the germination will be affected. Remove the alcohol, add 84 disinfectant (diluted twice with distilled water) and soak the seeds for 5-8 minutes; remove the 84 disinfectant and rinse with sterile water 3-5 times.

2) Sowing: Use sterile tweezers to sow sterilized seeds on M0 medium, 20-25 seeds per dish; then, put the petri dish in a sterile incubator, and cultivate at 24°C in dark light for 6 days.

3) Shake bacteria: 4 days after sowing, the target Agrobacterium strains were cultured with LB. Add 4ml of LB culture solution to the sterilized PU bottle, inhale 10μl of the activated target strain, and add antibiotics, that is, 4ml of LB solution plus 4μL of kana and 4μL of Gent and 10μL of bacterial solution; Shake the bacteria in a shaker at 180-220r/min for 12 -15h.

4) Preparation and infection of explants: measure the OD value of the bacteria (OD about 0.4 is better), suck 2ml of the cultured strains into a 2ml sterile centrifuge tube, centrifuge at 6000r/min for 3 minutes, and discard the supernatant ; Resuspend once with 2ml DM, centrifuge at 6000r/min for 3 minutes, discard the supernatant, add 2ml DM to resuspend, and put the suspended bacterial solution at 4°C for later use. Sterile scissors cut the hypocotyls of the seedlings cultured in dark for 6 days and put them in a sterile plate, and add 18mL M1 liquid medium to the plate first; the optimal length of the cut explants is 0.8-1.0cm, and the explants are cut. Try to cut vertically at one time. When there are 150-200 explants per dish, add 2ml of the DM suspension bacteria prepared in the first step, start timed for 10-15 minutes, and shake every 2 minutes during this period. Then quickly aspirate the bacterial liquid, transfer the explants to sterile filter paper, and suck up a large amount of bacterial liquid attached to the explants.

5) Transfer to M1 medium, 30-50 explants per dish, and place at 24°C under dark light.

6) After 24-48h, transfer to M2 medium and cultivate under light conditions at 24°C (16h during the day/8h at night).

7) Transfer to M3 medium after 15-20 days, and subculture every 2-3 weeks until green shoots appear.

8) The green shoots larger than 2cm are transferred to M4 rooting medium for rooting, and the rooting time takes 2-4 weeks. Once robust intact seedlings have grown, the seedlings can be transplanted to the field or greenhouse to continue growing until firm.

Example 4 Gene knockout test of BnaA10.RCO

To further prove that BnaA10.RCO is involved in the regulation of leaf shape, two sgRNA cassettes were designed for the coding region of the BnaA10.RCO gene, and the vector was named Cas9P35S-RCO, or SRCO for short (Figure 4A). The primers for sgRNA construction are:

BnRCOT1-F: gtcACGGGCGTAGACGAATTTCC

BnRCOT1-R: aaacggaaattcgtctacgcccg

BnRCOT2-F: attGCTTCACCCTCCACCGTGCA

BnRCOT2-R: aaactgcacggtggagggtgaag

The two sgRNAs were expressed using the Arabidopsis promoters pU3d and pU6-1, respectively, and simultaneously targeted the two homologous copies of RCO, BnaA10.RCO (BnaA10g26320D) and BnaC04.RCO (BnaC04g00850D) (Figure 4B). The constructed SRCO vector was transformed into HY material by Agrobacterium hypocotyl transformation method. Use carrier-specific primers for positive identification of transgenes, and the above-mentioned specific primer sequences are:

PB-L: GCGCGCGGTCTCGCTCGACTAGTATGG

PB-R: GCGCGCGGTCTCTACCGACGCGTATCC

The amplification system was as follows: 2ul DAN template, 1ul buffer3, 0.16ul 10mM dNTP, 0.1ul Taq DNA polymerase, 0.5ul each of the left and right primers, and double distilled water mixed into a 10ul system. The amplification program was: 95°C for 4 minutes; 2: 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute; 3: repeat step 2 32 times; 4: 72°C for 10 minutes, 25°C for 5 minutes.

As a result, a total of 38 T ₀ generation transgene-positive individual plants were obtained. These individual plants were further identified by high-throughput sequencing (Liu et al 2017), and the primer sequences for gene editing identification were:

C04-1: ggagtgagtacggtgtgcCCTACTTCCCGTTCCCTCAA

A10_266: gagttggatgctggatggATGGAATGGTCAACGACGAG

A10_267: ggagtgagtacggtgtgcGCGTGATAATTCCAAGATTTTTAGAA

A10_269: gagttggatgctggatggGATTCAGACAGGAAGGTGAAG

A10_271: ggagtgagtacggtgtgcACCTCATCGTGCGTTGTC

The results of high-throughput sequencing showed that 21 T ₀ generation individual plants had gene editing events, and the editing efficiency was 55.3%. Among the 21 edited individuals, 12 were homozygous or biallelic mutations in both copies of BnaA10.RCO and BnaC04.RCO, and the mutants showed a lobed leaf shape. To further confirm whether the mutant phenotype can be inherited, we proceeded to breed and genotype five edited T ₀ families. The results showed that in all tested individual plants, when both BnaA10.RCO alleles had frameshift mutations (homozygous or biallelic mutations, aacc genotype) or only BnaA10.RCO had homozygous mutations or bi-isotypes. When both genes were mutated, the mutants all showed the same lobed leaf shape; while when the two genes were heterozygous genotypes (AaCc), the mutants showed an intermediate leaf shape (Fig. 4C-D, Fig. 5A-B). Therefore, we infer that BnaA10.RCO has a decisive effect on leaf shape, and the expression analysis of the two copies of RCO gene shows that BnaC04.RCO is almost not expressed, indicating that BnaC04.RCO has nothing to do with leaf shape regulation. The gene expression level of BnaLMI1 was detected in rco mutant lines. The detection results showed that the gene expression level of BnaLMI1 in the transgenic line material did not change significantly compared with the expression level of the gene in the recipient material (Fig. 5C). Among them, Figure 4 is the BnaRCO mutant induced by the CRISPR/Cas9 system in the mosaic parent HY. (A) Schematic representation of the Cas9P35s-BnaRCO (SRCO) vector. (B) Target sequence designed for RCO, where the PAM sequence is underlined. (C) Genotype and phenotype of T ₀ and T ₁ generations of BnaRCO edited individual plants. (D) BnaRCO edits sequences near the sgRNA target site of an individual. The PAM sequence is marked with an underline, and the specific mutation sequence information is shown in the annotation on the right; "A" or "C" represents the wild-type allele, and "a" or "c" represents the mutant allele. The ruler is 1cm.

Figure 5 is the detection of the BnaA10.RCO mutant phenotype and the expression level of BnaLMI1 in the mutant. Comparison of phenotypes (A) (two-leaf stage) and leaf shape index (B) of edited individual plants with different genotypes; AaCc represents two RCO gene copies heterozygous mutants, aacc represents two RCO gene copies represents homozygous Mutants or biallelic mutants; (C) BnaLMI1 expression levels in shoot tips of 7d seedlings in BnaA10.RCO edited transgenic lines were examined by normalization of total RNA by qPCR with BnOTC. The ruler is 1cm.

This means that the regulation of leaf shape by BnaRCO does not depend on regulating the expression level of BnaLMI1.

CRISPR/Cas9 editing vector construction: Use CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR) online tool to select gene target sites, and then construct according to the method described by Ma et al. (2015) CRISPR/Cas9 editing vector was used, and the backbone vector used was PYLCRISPR/Cas935S-H. The main steps include:

1) The backbone vector is digested with Bsa I, wherein the enzyme digestion reaction system is: take each plasmid such as pU3d, etc.

1μg, 10U Bsa I for reaction in 25μl, cryopreserved, the reaction conditions are: 37°C for 20 minutes, and the backbone fragment is recovered;

2) sgRNA cassette construction, do 2 rounds of nested PCR, do 2 reactions in the first round of PCR, respectively use UF linker reverse primer, and use linker forward primer gR-R; the second round is Overlapping PCR, using position specific The primers amplify the expression cassette product. The UF linker reverse primer is: CTCCGTTTTACCTGTGGAATCG, the linker forward primer gR-R is CGGAGGAAAATTCCATCCAC, and the reaction system is: expression cassette ligation product 0.5ul, primers 0.3ul each, 2mM dNTP 1.5ul, 1.5ul 10×buffer (KOD-Plus , TOYOBO), 5mM MgSO ₄ 0.6ul, KOD plus 0.3ul, add double distilled water and mix into a 15ul system, the reaction conditions are: 94℃ for 2 minutes; 2: 98℃ for 10 seconds, 58℃ for 10 seconds, 68℃ for 10 seconds; 3: Repeat step 2 25 times; 4: 4 minutes at 68°C and 4 minutes at 25°C.

3) Enzymatic cleavage-ligation of binary vector and sgRNA expression cassette. Enzymatic ligation was performed using a variable temperature cycle for approximately 10-15 cycles: 5 minutes at 37°C; 5 minutes at 10°C, 5 minutes at 20°C; and a final 5 minutes at 37°C.

4) After the constructed vector is verified to be correct by sequencing, the plasmid is extracted, and then transformed into Agrobacterium GV3101. For the method of Agrobacterium-mediated hypocotyl transformation, refer to the introduction in Example 3.

The genotype detection method for gene editing is as follows: the present invention uses a high-throughput sequencing method to detect mutant genotypes, and the laboratory mainly adopts the Hi-TOM sequencing method developed by the research group of Mr. Wang Kejian, and performs two rounds of overlapping PCR: first. One round of PCR uses target primers to amplify the target fragments; the second round of PCR uses universal primers to amplify the first round of PCR products, and the universal primers add next-generation sequencing elements and barcode sequences to both ends of the target fragments; Generation of high-throughput sequencing; finally, after simple data extraction, the online website Hi-Tom (http://www.hi-tom.net/hi-tom/) was used to analyze the specific mutant form of each individual plant.

Media used for genetic transformation:

M0 medium: 1/2MS (2.2g/L) + sucrose (30g/L), adjust the pH value to about 5.8, add agar 8g/L, and then sterilize at high temperature;

DM suspension: MS (4.4g/L) + sucrose (30g/L), adjust pH to about 5.8, add agar 8g/L, then sterilize at high temperature, add acetosyringone to final concentration of 100μM;

M1 medium: MS (4.4g/L) + sucrose (30g/L) + mannitol (18g/L) + 2,4-D (1 mg/L) + KT (0.3 mg/L), adjust the pH to about 5.8, add agar 8g/L, then sterilize at high temperature, and add acetosyringone to the final concentration of 100μM;

M2 medium: MS (4.4g/L)+glucose (10g/L)+mannitol (18g/L)+2,4-D (1mg/L)+KT (0.3mg/L), adjust pH to about 5.8 , add agar 8g/L, then sterilize at high temperature, add silver thiosulfate (final concentration 30μM), and antibiotics (such as hygromycin, timementin) when using;

M3 medium: MS (4.4g/L) + sucrose (30g/L) + xylose (0.25g/L) + MES (0.6 g/L), adjust pH to about 5.8, add agar 8g/L, then sterilize at high temperature bacteria. Add hormones ZT (final concentration 2mg/L), IAA (final concentration 0.1mg/L) and AgNO3 (final concentration 3mg/L), and antibiotics (such as hygromycin, Timentin) during use;

M4 medium: MS (4.4g/L) + sucrose (10g/L), adjust pH to about 5.8, add agar 8g/L, and then sterilize at high temperature.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Huazhong Agricultural University

<120> A major gene for controlling rape leaf shape and its application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1769

<212> DNA

<213> Rape (Brassica campestris L)

<400> 1

atggaatggt caacgacgag caatgttgaa aacacgagag ttgcttttat gccacttcag 60

tggctggagt ctaactcatc caactcgctc caaaacttca gctatgatcc ttatgcaggt 120

atattcattc acatgcatct ttatatccat ctttttgtgg atttgttgta attccggttg 180

agttttaaaa tttccaccaa aatatagcta ggtttgcatt aaattttgaa aaggtagcac 240

cattaatggc gagagattaa aaagaaaata ttagtatttt atttttatga ttttttttaaa 300

gaaaattaaa gaaatatgaa tggtcagttg aatgacactt gtataataga attctcaaaa 360

tttgtgcaaa atgtttaaaa ttgaaaccat ttttcctctt ttattatttt tctttaattt 420

taattatgag agactcagag aaatacgcct actatttcca tttttggaaa agattcgatt 480

ttatacagta ctccagtata tgactttttt gagggaacgg gaaaaacgtt ttgcaattct 540

aaaaatcttg gaattatcac gcttttctta tgagaagtag gatatagacc accgatattc 600

ttatgacttt cttggaacca tgctcagtgt ttcaaaagta gaaacgtcgc ttttcaaatg 660

ttttgagatt cgtttttctcg ctcaagaaaa actggaaagt ttttttttttg tattgtattt 720

gaaattatta ttgttgagcg tttgtgtcgt tctctttgtt tttgttgtta agttttacca 780

ggaaattcgt ctacgcccgt ccttacgcaa accggaccgg ttatttctgt accggaatca 840

tcagaaaaga tcaccaatgc gtgcccatat ccaagtaacg acgacgagat gataaagaag 900

aagcagaaac taacgactga acaattagct tcacttgaac agagttttca agaagatatc 960

aaacttgatt cagacaggaa ggtgaagctg tcgaaggagc ttcgtctgca gccacgtcag 1020

gtagctgttt ggttccagaa ccgccgtgca cggtggaggg tgaagcatct cgaggagtcg 1080

tacaactcgc taaggaaaga gtacgacgtg gtttcaagac agaatcaaat gctacacgat 1140

gaggtatata tatatata tattttcttt ttttgacaac gcacgatgag gtatatatat 1200

gcaccattta aaaaaaaaaa tcatgggatc ttagagcatc cgtctccctt tagatattca 1260

cctaggtaat tgatagataa aatattacta gtagttattt aatatagttt atttaatgat 1320

tacattagaa atcatactaa aagtaagcaa atcatagatt gccatataac tttaagagta 1380

tctccagtttt tttttttttt ttttttaagt ttctcaaact ccgtcattta tctctctact 1440

ttttgagttc ttttttttttc ttcactacat tttcaaaatt tcttatttta taatgatcag 1500

atattcggtt gagatacacc aatgtgattt ctaaactatt catcctttgt tgttttagcc 1560

ctttagtttt tgattttgtg acgcaggtga tgaatctgag aggtgtaata ctaaaagacc 1620

atttgatgaa gaggcaaatg aatctgaaca acaatcaaat agccggaggg agtcaaattt 1680

acggtactgc agatcaatat aataatccaa tgtgtgttgc ttcaacttgt tggcccccgt 1740

tatcatcgca acagccttac ccatggtag 1769

<210> 3

<211> 223

<212> PRT

<213> Rape (Brassica campestris L)

<400> 3

Met Glu Trp Ser Thr Thr Ser Asn Val Glu Asn Thr Arg Val Ala Phe

1 5 10 15

Met Pro Leu Gln Trp Leu Glu Ser Asn Ser Ser Asn Ser Leu Gln Asn

20 25 30

Phe Ser Tyr Asp Pro Tyr Ala Val Leu Pro Gly Asn Ser Ser Thr Pro

35 40 45

Val Leu Thr Gln Thr Gly Pro Val Ile Ser Val Pro Glu Ser Ser Glu

50 55 60

Lys Ile Thr Asn Ala Cys Gln Tyr Pro Ser Asn Asp Asp Glu Met Ile

65 70 75 80

Lys Lys Lys Gln Lys Leu Thr Glu Gln Leu Ala Ser Leu Glu Gln

85 90 95

Ser Phe Gln Glu Asp Ile Lys Leu Asp Ser Asp Arg Lys Val Lys Leu

100 105 110

Ser Lys Glu Leu Arg Leu Gln Pro Arg Gln Val Ala Val Trp Phe Gln

115 120 125

Asn Arg Arg Ala Arg Trp Arg Val Lys His Leu Glu Glu Ser Tyr Asn

130 135 140

Ser Leu Arg Lys Glu Tyr Asp Val Val Ser Arg Gln Asn Gln Met Leu

145 150 155 160

His Asp Glu Val Met Asn Leu Arg Gly Val Ile Leu Lys Asp His Leu

165 170 175

Met Lys Arg Gln Met Asn Leu Asn Asn Asn Gln Ile Ala Gly Gly Ser

180 185 190

Gln Ile Tyr Gly Thr Ala Asp Gln Tyr Asn Asn Pro Met Cys Val Ala

195 200 205

Ser Thr Cys Trp Pro Pro Leu Ser Ser Gln Gln Pro Tyr Pro Trp

210 215 220

Claims (9)

1. The application of overexpressing a major gene for controlling the leaf shape of rape in the production of leaf margin cut rape, wherein the nucleotide sequence of the major gene is as shown in SEQ ID NO: 1. 2 . The application of overexpressing a protein that controls the leaf shape of rapeseed in the production of leaf margin cut rape, wherein the protein is encoded by the nucleotide sequence of SEQ ID NO: 1 according to claim 1 . 3 . The application of overexpressing an expression vector containing the control of the leaf shape of rape in the production of leaf edge cut rape, wherein the expression vector contains the nucleotide sequence of claim 1 . 4 . The application of the overexpression-containing expression vector for controlling the leaf shape of rapeseed according to claim 3 in the production of leaf edge cut rape, wherein the expression vector is a PMDC32 vector. 5 . 5. overexpression according to claim 4 contains the application of the expression vector of control rape leaf shape in the production of leaf edge cut rape, it is characterized in that, the construction method of described expression vector comprises the following steps: S1-1, the genomic DNA sequence of BnaA10.RCO was amplified from two materials of "Tongling Mosaic" HY-cut leaves and "Zhongshuang No. 9" Z9 lobed leaves; S1-2, the amplified sequence in step S1 is ligated into the PMDC32 vector with CaMV35S as the promoter by the method of enzyme cleavage and ligation. 6. the application of the expression vector that overexpression contains control rape leaf shape according to claim 5 in the production of leaf edge incised rape, it is characterized in that, in step S1-1, the primer used for amplifying the BnaA10.RCO genomic DNA sequence Are: A10-117F: GGGCttaattaaCTACCATGGGTAAGGCTGTTGC; A10-118R: GACAggtaccATGGAATGGTCAACGACGAGC. 7. the cloning method of the main gene that regulates and controls the leaf edge of rape, is characterized in that, comprises the following steps: S2-1, align the genes annotated with the BnLLA10 segment in the Brassica napus reference genome to the Arabidopsis genome, and perform functional annotation to determine candidate genes; S2-2, design primers to clone the candidate genes of two materials from "Tongling Mosaic" HY-cut leaves and "Zhongshuang No. 9" Z9 lobed leaves, and compare the cDNA and DNA sequencing results to determine the candidate genes The differential position of the allele of the gene in the two materials; S2-3, analyze the specific expression amount of the candidate gene in the near-isogenic line Z9-NIL of HY and Z9, and finally determine the main gene BnaA10.RCO through overexpression experiment and gene knockout experiment; S2-4, connect the main gene BnaA10.RCO to the expression vector and transfer it into the recipient material through the Agrobacterium-mediated hypocotyl transformation method; The construction method of the near-isogenic line Z9-NIL is as follows: two parent materials HY and Z9 are respectively obtained by reciprocal cross to obtain F1 hybrid, wherein the F1 hybrid of HY × Z9 is derived from self-crossing and backcrossing with HY parent. F2, F3, BC1F2, BC2F2 and BC3F2 segregated populations of different generations; in the BC3F2 generation, the molecular marker-assisted selection method was used to screen out the homozygous Z9 allele at the major leaf-shaped locus and the leaf-shaped and donor parent The same single plant of Z9 is obtained. 8 . The method for cloning a major gene for regulating leaf edge incision of rape according to claim 7 , wherein the candidate genes determined in step S2-1 are BnaA10.RCO and BnaA10.LMI1 . 9 . 9. the cloning method of the major gene of regulation rape leaf edge notch according to claim 8 is characterized in that, the nucleotide sequence of the primer used during BnaA10.RCO cloning in step S2-2 is: A10_101F: TTCTCAAGATCCGAAACACCT ; A10_101R: ATGGAATGGTCAACGACGAGC.

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