A rapeseed RNA processing factor NCBP gene and its application

Abstract

The present invention discloses a rape RNA processing factor NCBP gene and its application, and relates to the field of gene engineering technology. The gene is located on the A10 chromosome of rape and has a nucleotide sequence as shown in SEQ ID NO.1, or has a nucleotide sequence complementary to the sequence shown in SEQ ID NO.1. The present invention discloses for the first time the use of the NCBP gene in regulating the flowering period of plants. The rape NCBP gene family is gene-edited by genetically transforming the Brassica napus variety Westar using CRISPR‑Cas9 technology, and further screening is performed to obtain homozygous mutant materials containing target gene mutations. The mutant materials show late flowering, and the overexpression of NCBP.A10 in Arabidopsis shows early flowering, which confirms the use of the NCBP gene in regulating the flowering period of rape. The present invention provides new gene resources and new ideas for cultivating Brassica napus with an appropriate growth period.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a rapeseed RNA processing factor NCBP gene and application thereof. Background Art

[0002] Rapeseed (Brassica napus L., 2n = 38, AACC) is one of the most important edible vegetable oil resources in the world, and rapeseed meal is also an important source of feed protein, which is closely related to the development of animal husbandry. Therefore, vigorously developing the rapeseed industry is of great significance to ensure the effective supply of high-quality edible vegetable oil and feed protein in my country and promote the development of related industries.

[0003] Flowering is the process of transition from the vegetative stage to the reproductive stage of plants. This process is regulated by internal factors (age and gibberellins) and external environmental conditions (photoperiod, vernalization and ambient temperature). The growth period of rapeseed has a significant impact on yield, and flowering time is an important factor affecting the growth period. Different ecological regions have different flowering period requirements for rapeseed varieties. The length of the rapeseed flowering period also determines its planting area. Timely flowering of plants can avoid unsuitable living environments, which is of great significance to increasing plant yields. Exploring rapeseed flowering period regulatory genes has important strategic significance and practical application value for maintaining the country's edible oil supply and ensuring national grain and oil security.

[0004] In view of this, the present invention uses GWAS technology to isolate a QTL located on chromosome 10 of Brassica napus that controls the initial flowering period, final flowering period and maturity period, S10_12378911, and further discovers the candidate gene NCBP.A10 in this interval, which encodes an RNA processing factor. By verifying the candidate gene through CRISPR / Cas9 gene editing and heterologously expressing its overexpression vector in Arabidopsis, the biological function of the gene in controlling flowering time is confirmed, which can provide a theoretical basis for using the gene for genetic improvement of flowering period. . Summary of the invention

[0005] The purpose of the present invention is to provide a method for gene editing of the rapeseed NCBP gene family by CRISPR-Cas9 technology, so as to provide a gene that can regulate the flowering time of plants and provide a basis for cultivating rapeseed varieties with appropriate growth periods.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The invention provides a rapeseed RNA processing factor NCBP gene, which is located on the A10 chromosome of rapeseed and has a nucleotide sequence as shown in SEQ ID NO.1, or has a nucleotide sequence complementary to the sequence shown in SEQ ID NO.1.

[0008] The present invention also provides an application of the rapeseed RNA processing factor NCBP gene in improving the flowering period trait of rapeseed.

[0009] A further improvement is that the rapeseed is Brassica napus.

[0010] A further improvement is that homozygous mutant plants knocking out the NCBP gene in Brassica napus exhibit late flowering.

[0011] A further improvement is that homozygous plants overexpressing the NCBP gene in Brassica napus exhibit early flowering.

[0012] The present invention also provides a method for improving rapeseed flowering traits, comprising the following steps:

[0013] (1) Designing target sequences in the conserved region of the BnaNCBP gene family coding region of rapeseed;

[0014] (2) Synthesizing adapter primers based on the target sequence and constructing a CRISPR / Cas9 recombinant vector for BnaNCBP gene targeting;

[0015] (3) The recombinant vector was transferred into Agrobacterium tumefaciens GV3101, and the Brassica napus spring variety Westar was transformed by Agrobacterium-mediated method to obtain transgenic rapeseed plants;

[0016] (4) Extracting genomic DNA from transgenic positive plants, using gene-specific primers covering the target site for PCR amplification and sequence sequencing, and genotyping the T0 generation transgenic plants;

[0017] (5) The gene-edited mutants were planted in the T1 generation, and the HPT and Cas9 transgenes in the T1 generation plants were amplified by PCR using primers to obtain homozygous BnaNCBP gene-edited mutant plants.

[0018] A further improvement is that the target sequence includes the following three types:

[0019] sgRNA1: 5'-TGCAGTACAAGTTTGCGATTTGG-3';

[0020] sgRNA2: 5'-TCGCAATGCTTCTGATCATCAGG-3';

[0021] sgRNA3: 5'-TACTCTCGCGACCTCAAAGCTGG-3'.

[0022] A further improvement is that in step (5), the primer sequences used for HPT and Cas9 transgenic amplification are:

[0023] HPT-F: 5'-ACACTACATGGCGTGATTTCAT-3';

[0024] HPT-R: 5'-TCCACTATCGGCGAGTACTTCT-3';

[0025] Cas9-F: 5'-GGCCTCGATATTGGGACTAACT-3';

[0026] Cas9-R: 5'-GTCCGCCTTATCTGTGGAGT-3'.

[0027] The present invention also provides a method for obtaining the rapeseed RNA processing factor NCBP gene, which uses the nucleotide sequence of the NCBP gene as a template to design specific amplification primers, uses the wild-type Westar rapeseed variety as a material, extracts RNA and reversely transcribes it into cDNA, and obtains the rapeseed NCBP gene through PCR amplification technology.

[0028] A further improvement is that the specific amplification primers for the PCR amplification are:

[0029] BnaNCBP.A10-F: 5'-GGATCCATGGAGATTACGGAGAGGAGGC-3';

[0030] BnaNCBP.A10-R: 5'-GTCGACTCCTCTCAGCCAAGTGTTCC-3'.

[0031] The present invention provides the following beneficial effects:

[0032] 1. The present invention uses a natural population of Brassica napus to locate a stable major effect QTL—S10_12378911—which controls multiple growth period traits on chromosome A10 based on the results of GWAS analysis. Gene annotation and candidate gene analysis are performed on the located interval to screen out a candidate gene NCBP, which encodes an RNA processing factor.

[0033] 2. The present invention uses CRISPR-Cas9 technology to perform gene editing on the BnaNCBP gene family, and a total of 4 homozygous mutant genotypes are obtained in the T2 generation. Potted experiments show that the flowering time of homozygous mutants is significantly delayed compared with the wild type. The BnaNCBP.A10 overexpression vector was further transformed into Arabidopsis, and the transgenic homozygous plants showed early flowering, confirming that the NCBP gene can control the flowering period. Therefore, the NCBP gene is conducive to the genetic improvement and molecular breeding of the flowering period traits of Brassica napus. This method can be used to quickly and directionally improve existing rapeseed varieties, greatly shorten the breeding cycle, and save time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the GWAS positioning result of the natural population growth period traits of the present invention;

[0035] Figure 2 To perform gene editing of NCBP gene using CRISPR / Cas9 system;

[0036] Figure 3 Sequencing comparison results for wild type and gene-edited mutants;

[0037] Figure 4 The flowering phenotypes of wild-type Westar and T2 generation BnaNCBP gene knockout homozygous mutant rapeseed.

[0038] Figure 5 Flowering phenotypes of wild-type Col-0 and T2 generation BnaNCBP.A10 gene overexpression homozygous Arabidopsis thaliana. DETAILED DESCRIPTION

[0039] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0040] 1. Materials

[0041] Unless otherwise specified, the methods used in this example are conventional methods known to those skilled in the art, and the reagents used are commercially available products unless otherwise specified.

[0042] The Brassica napus varieties used in the present invention: the GWAS positioning material is 588 natural populations collected by the Rapeseed Engineering Research Center of Southwest University; the gene editing material is the conventional spring variety Westar; and the overexpression material is the Arabidopsis thaliana variety Col-0.

[0043] 2. Methods

[0044] 2.1 Identification of candidate genes for flowering time in Brassica napus

[0045] TASSEL software was used to conduct a three-year survey and statistics on five growth-stage-related traits (initial flowering period DIF, final flowering period DFF, flowering duration FP, maturity period MT and full growth period GP) of 588 natural populations of Brassica napus collected at home and abroad, and GWAS analysis was performed based on the resequencing data.

[0046] GWAS analysis found that the SNP marker S10_12378911 located on chromosome A10 was significantly associated with the initial flowering period, final flowering period and maturity period. Figure 1 The 300 kb sequence intervals on both sides of the SNP marker were used as LD intervals, and a candidate gene NCBP was detected in the intervals. This gene is homologous to the Arabidopsis eIF4E gene and encodes an RNA processing factor. It is speculated that it is a candidate gene controlling the flowering period of Brassica napus.

[0047] 2.2 Construction of BnaNCBP gene editing vector containing specific sgRNA

[0048] like Figure 2 As shown, the CRISPR / Cas9 system was used to edit the BnaNCBP gene family. The steps are as follows:

[0049] (1) Design of sgRNA

[0050] The BnaNCBP gene family was found on the BnTIR website (http: / / yanglab.hzau.edu.cn / BnTIR). There are five members of the NCB P gene in Brassica napus, named BnaA10.NCBP, BnaA02.NCBP, BnaA03.NCBP, BnC02.NCBP and BnaC03.NCBP, and their full-length DNA sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0051] Based on the CRISPR-GE online website (http: / / skl.scau.edu.cn / primerdesign / ), the target site sequence for gene editing was designed for the conserved region of the coding region of the NCBP gene family to construct the sgRNA sequence;

[0052] The nucleotide sequence of the sgRNA action site is:

[0053] sgRNA1: 5'-TGCAGTACAAGTTTGCGATT TGG -3';

[0054] sgRNA2: 5'-TCGCAATGCTTCTGATCATC AGG -3';

[0055] sgRNA3: 5'-TACTCTCGCGACCTCAAAGC TGG -3';

[0056] The underlined part is the PAM sequence.

[0057] (2) Construction of CRISPR editing vector

[0058] The target site PCR amplification primers were designed according to the target site. The primers were synthesized by Tianyi Huiyuan Biotechnology Co., Ltd. The target fragment was amplified by PCR using the PGTR plasmid as a template.

[0059] The primer sequences are as follows:

[0060]

[0061]

[0062] The PCR product was constructed into the CRISPR expression vector PKSE401-TRNA containing the Cas9 expression cassette through homologous recombination. The recombinant expression vector was transferred into the competent Escherichia coli DH5α. Single colonies were picked and positive clones were screened and sequenced by bacterial liquid PCR. The correctly sequenced recombinant plasmid was transferred into Agrobacterium GV3101 and then transferred into the Brassica napus variety Westar through the Agrobacterium-mediated rapeseed transformation system. The Agrobacterium transformation and positive seedling screening experiments were commissioned to Wuhan Tianwen Biotechnology Co., Ltd.

[0063] 2.3 Obtaining and identifying BnaNCBP gene knockout rapeseed

[0064] (1) Positive seedling screening

[0065] The transgenic seedlings were selected by hygromycin in the culture medium and then transferred to soil. One week later, the young leaves of individual plants were sampled and the rapeseed leaf DNA was extracted by CTAB method. The positive plants were identified by PCR using primers HPT-F / R and the amplified products were detected by 1% agarose gel electrophoresis.

[0066] The HPT primer sequences are as follows:

[0067] HPT-F: 5'-ACACTACATGGCGTGATTTCAT-3'

[0068] HPT-R: 5'-TCCACTATCGGCGAGTACTTCT-3'

[0069] (2) Edit type detection

[0070] Gene-specific primers were designed near each sgRNA target site for PCR amplification, and the PCR products with the correct amplification band size were sent to the sequencing group of the Rice Research Institute of the Chinese Academy of Agricultural Sciences for Hi-TOM sequencing to obtain the variant sites.

[0071] The gene-specific primer sequences are as follows:

[0072]

[0073]

[0074] Among the transgenic T0 plants, 5 transgenic plants carried the target mutation, including 1 homozygous mutant and 4 heterozygous mutants. Figure 3 , select gene-edited plants for self-pollination and obtain T1 generation seeds.

[0075] 2.4 Phenotypic observation of homozygous plants edited with NCBP

[0076] The genomic DNA of the T1 transgenic plants was extracted, and the editing status of the T1 plants was detected using the above method. The T2 gene-edited homozygous mutant plants with stable inheritance were obtained by self-pollination ( Figure 3 ). A total of 5 homozygous mutant genotypes were obtained in the T2 generation. BnC02.NCBP and BnaA03.NCBP were edited in T2-4-9, BnaA10.NCBP was edited in T2-12-7, BnaA03.NCBP was edited in T2-12-9, and BnaA03.NCBP was edited in T2-51-7. All 5 mutant genotypes resulted in frameshift mutations, which caused premature termination of codons and inactivated protein function.

[0077] The homozygous mutants of the T2 generation knockout and the wild-type Westar were planted together in the greenhouse of Anhui Agricultural University. The wild-type flowering time was 65.5±1.2 days. Compared with the wild-type, the homozygous edited plants all showed late flowering in the T2 generation, and the difference was significant. The flowering time of T2-4-9 was 69.4±2.2 days, the flowering time of T2-12-7 was 68.6±1.6 days, the flowering time of T2-12-9 was 68.4±2.5 days, and the flowering time of T2-51-7 was 68.6±1.8 days. The results are shown in Figure 4 The above results indicate that the BnaNCBP gene controls the flowering time of Brassica napus.

[0078] 2.5 Construction of NCBP.A10 overexpression vector and phenotype observation of transgenic Arabidopsis

[0079] (1) Construction of BnaNCBP.A10 overexpression vector

[0080] Using wild-type Westar rapeseed as material, RNA was extracted and reverse transcribed to generate the first strand of cDNA as a template for PCR amplification. Based on the CDS sequence of the coding region of the BnaNCBP.A10 gene, specific primers were designed to construct an overexpression vector (the horizontal line is the restriction site):

[0081] BnaNCBP.A10-F:5'- GGATCC ATGGAGATTACGGAGAGGAGGC-3′;

[0082] BnaNCBP.A10-R:5'- GTCGAC TCCTCTCAGCCAAGTGTTCC-3',

[0083] The BnaNCBP.A10 coding region fragment with restriction enzyme cutting sites was obtained.

[0084] The BnaNCBP.A10 coding region fragment with restriction sites and the overexpression vector pCambia1300modified (PCMM) were double-digested with BamH I and Sal I, respectively, and the target fragment and vector were recovered by gel cutting. Using T4 ligase, ligation was carried out overnight at 22°C, and the heat shock method was used to transfer into the competent Escherichia coli cell DH5α, cultured at 37°C overnight, and single colonies were picked to screen positive clones by bacterial liquid PCR method and sequence analysis, and finally the plant overexpression recombinant vector PCMM-NCBP.A10 was obtained. The PCMM-NCBP.A10 recombinant plasmid was transferred into the competent Agrobacterium GV3101, and the positive clones were identified by bacterial liquid PCR method.

[0085] (2) Transformation of Arabidopsis thaliana and phenotypic observation of homozygous transgenic plants

[0086] Select the positive bacterial liquid, add 5 mL of LB liquid culture medium containing kanamycin Kan and rifampicin Rif resistance, culture at 28°C 200 rpm for two days, then inoculate it into 100 mL of LB liquid culture medium containing kanamycin Kan and rifampicin Rif resistance at a volume ratio of 1:100, and culture at 28°C 200 rpm until the OD600 value is between 0.6 and 0.8.

[0087] The cells were collected and washed by centrifugation and transferred into wild-type Arabidopsis Col-0 using the pollen tube-mediated method. After the seeds matured (T0), the seeds were screened for hygromycin resistance. The positive seedlings (T1) were cultured in an artificial climate chamber until the seeds matured. The T2 generation homozygous transgenic seeds were screened and the phenotypes were identified after sowing. The results are as follows: Figure 5 shown. Figure 5 It can be seen that the flowering time of the overexpressing transgenic line is significantly advanced compared with the wild type Col-0.

[0088] In summary, the present invention uses GWAS analysis to identify the rapeseed flowering period candidate gene NCBP.A10. According to the NCBP gene sequence information of rapeseed, the BnTIR database is searched by bioinformatics methods, and the homologous genes of NCBP are identified in rapeseed, which are named BnaA10.NCBP, BnaA02.NCBP, BnaA03.NCBP, BnC02.NCBP and BnaC03.NCBP respectively. Using the CRISPR-GE online analysis tool, three gene editing target site sequences are designed and connected to the PKSE401-TRNA vector with a Cas9 expression cassette. Agrobacterium-mediated method is used to transfer into Brassica napus Westar. The mutation type of transgenic plants is analyzed by gene-specific primer PCR detection and sequencing, and 4 homozygous BnaNCBP gene editing mutants are screened. The BnaNCBP.A10 overexpression vector is transformed into Arabidopsis, and the transgenic homozygous plants show early flowering. The present invention finds that the BnaNCBP gene has an important influence on the flowering period of rapeseed and can be applied to improving the flowering period of rapeseed.

[0089] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A rapeseed RNA processing factor NCBP gene, characterized in that: The gene is located on the A10 chromosome of rapeseed, and the nucleotide sequence of the NCBP gene is shown in SEQ ID NO.

1.

2. Use of the rapeseed RNA processing factor NCBP gene as claimed in claim 1 in improving the flowering period traits of rapeseed, characterized in that: The rapeseed is Brassica napus.

3. The use according to claim 2, characterized in that: Homozygous mutant plants knocking out the NCBP gene in Brassica napus exhibited late flowering.

4. A method for improving the flowering period traits of rapeseed, characterized in that: The following steps are involved: (1) Designing target sequences in the conserved region of the coding region of the rapeseed BnaNCBP gene family; the target sequences include the following three types: sgRNA1: 5'-TGCAGTACAAGTTTGCGATTTGG-3'; sgRNA2: 5'-TCGCAATGCTTCTGATCATCAGG-3'; sgRNA3: 5'-TACTCTCGCGACCTCAAAGCTGG-3'; (2) Synthesizing adapter primers based on the target sequence and constructing a CRISPR / Cas9 recombinant vector for BnaNCBP gene targeting; (3) The recombinant vector was transferred into Agrobacterium tumefaciens GV3101, and the Brassica napus spring variety Westar was transformed by Agrobacterium-mediated method to obtain transgenic rapeseed plants; (4) Extracting genomic DNA from transgenic positive plants, using gene-specific primers covering the target site for PCR amplification and sequence sequencing, and genotyping the T0 generation transgenic plants; (5) The gene-edited mutants were planted in the T1 generation, and the HPT and Cas9 transgenes in the T1 generation plants were amplified by PCR using primers to obtain homozygous BnaNCBP gene-edited mutant plants.

5. The method for improving flowering traits of rapeseed according to claim 4, characterized in that: In step (5), the primer sequences used for HPT and Cas9 transgenic amplification are: HPT-F: 5'-ACACTACATGGCGTGATTTCAT-3'; HPT-R: 5'-TCCACTATCGGCGAGTACTTCT-3'; Cas9-F: 5'-GGCCTCGATATTGGGACTAACT-3'; Cas9-R: 5'-GTCCGCCTTATCTGTGGAGT-3'.

6. A method for obtaining the rapeseed RNA processing factor NCBP gene as claimed in claim 1, characterized in that: The specific primers were designed with the nucleotide sequence of NCBP gene as template, and the wild type Westar rapeseed variety was used as material. RNA was extracted and reverse transcribed into cDNA, and the rapeseed NCBP gene was obtained by PCR amplification technology.

7. The method for obtaining the rapeseed RNA processing factor NCBP gene according to claim 6, characterized in that: The specific amplification primers for the PCR amplification are: BnaNCBP.A10-F: 5'-GGATCCATGGAGATTACGGAGAGGAGGC-3'; BnaNCBP.A10-R: 5'-GTCGACTCCTCTCAGCCAAGTGTTCC-3'.

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