CN115927381A - Rape RNA processing factor NCBP gene and application thereof - Google Patents

Abstract

The invention discloses a rape RNA processing factor NCBP gene and application thereof, relating to the technical field of genetic engineering, wherein the gene is positioned on an A10 chromosome of rape and has a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence complementary with the sequence shown as SEQ ID NO. 1. The invention discloses the application of NCBP gene in regulation and control of flowering phase of plants for the first time, wherein a cabbage type rape variety Westar is genetically transformed through a CRISPR-Cas9 technology to carry out gene editing on a rape NCBP gene family, and a homozygous mutant material containing target gene mutation is further obtained by screening, wherein the mutant material shows late flowering, and the overexpression of NCBP.A10 in Arabidopsis shows early flowering, so that the application of the NCBP gene in regulation and control of flowering phase of rape is verified. The invention provides a new gene resource and a new idea for culturing the cabbage type rape with a proper growth period.

Description

Rape RNA processing factor NCBP gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a rape RNA processing factor NCBP gene and application thereof.

Background

Cabbage type rape (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 and is closely related to the development of animal husbandry. Therefore, the method has great significance for ensuring the effective supply of high-quality edible vegetable oil and feed protein in China and promoting the development of related industries when the rape industry is vigorously developed.

Flowering is a process by which plants transition from the vegetative to the reproductive phase, and is regulated by a combination of internal factors (age and gibberellins) and external environmental conditions (photoperiod, vernalization, and ambient temperature). The growth period of rape has obvious influence on yield, and the flowering time is an important factor influencing the growth period. Different ecological regions have different requirements on the flowering phase of rape varieties, the length of the flowering phase of the rape also determines the planting area of the rape, and the plants bloom at proper time to avoid unsuitable living environments, so that the method has important significance for improving the plant yield. Digging rape flowering phase regulation genes has important strategic significance and practical application value for maintaining national edible oil supply and guaranteeing national grain and oil safety.

In view of the above, the present invention utilizes GWAS technology to isolate a QTL, S10_12378911, located on Brassica napus chromosome 10 and controlling the early flowering phase, the final flowering phase and the mature phase, and further finds a candidate gene NCBP.A10 in this interval, which encodes an RNA processing factor. Through carrying out CRISPR/Cas9 gene editing verification on the candidate gene and carrying out heterologous expression on an overexpression vector in Arabidopsis, the biological function of the gene in controlling flowering time is verified, and a theoretical basis can be provided for genetic improvement of flowering phase by using the gene. .

Disclosure of Invention

The invention aims to provide a method for carrying out gene editing on a rape NCBP gene family through a CRISPR-Cas9 technology, so as to provide a gene capable of adjusting plant flowering time and provide a basis for cultivating a rape variety with a proper growth period.

The invention realizes the purpose through the following technical scheme:

the invention provides a rape RNA processing factor NCBP gene, which is positioned on an A10 chromosome of rape and has a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence complementary with the sequence shown as SEQ ID NO. 1.

The invention also provides an application of the rape RNA processing factor NCBP gene in improving the flowering phase characters of rape.

The further improvement is that the rape is cabbage type rape.

A further improvement is that the homozygous mutant plants that knock out the NCBP gene in brassica napus show late flowers.

A further improvement is that homozygous plants overexpressing the NCBP gene in brassica napus show early flowering.

The invention also provides a method for improving the flowering phase character of rape, which comprises the following steps:

(1) Designing a target sequence in a conserved region of a rape BnanCBP gene family coding region;

(2) Synthesizing a linker primer according to a target sequence, and constructing a CRISPR/Cas9 recombinant vector for BnaNCBP gene targeting;

(3) Transferring the recombinant vector into agrobacterium tumefaciens GV3101, and transforming a cabbage type rape spring variety Westar by using an agrobacterium-mediated method to obtain a transgenic rape plant;

(4) Extracting the genome DNA of the transgenic positive plant, performing PCR amplification and sequence sequencing by using a gene specific primer covering a target locus, and performing genotype identification on the T0 generation transgenic plant;

(5) The mutant with gene editing is planted in the T1 generation, and the PCR amplification detection is carried out on HPT and Cas9 transgenes in the T1 generation plant by using the primer, so as to obtain the homozygous BnancBP gene editing mutant plant.

In a further improvement, the target sequence comprises three of:

sgRNA1：5‘-TGCAGTACAAGTTTGCGATTTGG-3’；

sgRNA2：5‘-TCGCAATGCTTCTGATCATCAGG-3’；

sgRNA3：5‘-TACTCTCGCGACCTCAAAGCTGG-3’。

in a further improvement, in the step (5), the primer sequences for amplification of the HPT and Cas9 transgenes are:

HPT-F：5'-ACACTACATGGCGTGATTTCAT-3'；

HPT-R：5'-TCCACTATCGGCGAGTACTTCT-3'；

Cas9-F：5'-GGCCTCGATATTGGGACTAACT-3'；

Cas9-R：5'-GTCCGCCTTATCTGTGGAGT-3'。

the invention also provides a method for obtaining the NCBP gene of the rape RNA processing factor, which takes the nucleotide sequence of the NCBP gene as a template to design a specific amplification primer, takes a wild Westar rape variety as a material, extracts RNA and carries out reverse transcription to form cDNA, and obtains the NCBP gene of the rape by a PCR amplification technology.

The further improvement is that the specific amplification primers for PCR amplification are as follows:

BnaNCBP.A10-F：5'-GGATCCATGGAGATTACGGAGAGGAGGC-3’；

BnaNCBP.A10-R：5'-GTCGACTCCTCTCAGCCAAGTGTTCC-3’。

the invention has the following beneficial effects:

1. according to GWAS analysis results, a cabbage type rape natural population is utilized, a stable main effect QTL-S10-12378911 for simultaneously controlling multiple growth period traits is positioned on an A10 chromosome, gene annotation and candidate gene analysis are carried out on a positioning interval, and a candidate gene NCBP is screened, wherein the NCBP gene codes an RNA processing factor.

2. The invention edits genes of BnanNCBP gene families through a CRISPR-Cas9 technology, and obtains 4 homozygous mutant genotypes in a T2 generation. Pot experiments showed that the homozygous mutant flowering time was significantly delayed compared to the wild type. Further, arabidopsis thaliana is transformed by the BnancBP. A10 overexpression vector, and the transgenic homozygous plant shows early blossoming, so that the NCBP gene can control the flowering phase. Therefore, the NCBP gene is beneficial to genetic improvement and molecular breeding of the flowering phase character of the cabbage type rape. The method can be used for quickly and directionally improving the existing rape varieties, greatly shortening the breeding period and saving the time and the cost.

Drawings

FIG. 1 shows the positioning result of natural population growth period trait GWAS according to the present invention;

FIG. 2 is a gene editing of the NCBP gene using CRISPR/Cas9 system;

FIG. 3 shows the sequencing alignment of wild-type and gene-editing mutants;

FIG. 4 shows the flowering phenotype of wild type Westar and T2 generation BnanCBP gene knockout homozygous mutant rape.

FIG. 5 homozygous Arabidopsis floral phenotype over-expressed by wild type Col-0 and T2 generation BnancBP. A10 genes.

Detailed Description

The present application will now be described in further detail with reference to the drawings, and it should be noted that the following detailed description is given for purposes of illustration only and should not be construed as limiting the scope of the present application, as these numerous insubstantial modifications and variations can be made by those skilled in the art based on the teachings of the present application.

1. Material

The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents used, etc., are commercially available products unless otherwise specified.

The cabbage type rape variety used in the invention is as follows: the GWAS positioning material is 588 natural populations collected by the rape engineering research center of southwest university; the gene editing material is a conventional spring variety Westar; the over-expression material is Arabidopsis thaliana variety Col-0.

2. Method of producing a composite material

2.1 identification of candidate genes for Brassica napus in flowering phase

The TASSEL software is adopted to carry out survey statistics on 588 brassica napus natural population related traits (initial flowering phase DIF, final flowering phase DFF, flowering phase duration FP, maturation phase MT and full growth phase GP) at home and abroad in 5 growth phases continuously for 3 years, and GWAS analysis is carried out on the traits by combining with resequencing data.

GWAS analysis found that the SNP marker S10_12378911 located on the A10 chromosome was significantly correlated with the early flowering stage, the final flowering stage and the mature stage at the same time, as shown in FIG. 1. A300 kb sequence interval at the upstream and downstream of the SNP marker is used as an LD interval, a candidate gene NCBP is detected in the interval, the gene is homologous with an Arabidopsis eIF4E gene, an RNA processing factor is encoded, and the candidate gene is presumed to be a candidate gene for controlling the flowering phase character of the cabbage type rape.

2.2 construction of BnancBP Gene editing vector containing specific sgRNA

As shown in fig. 2, the BnaNCBP gene family is subjected to gene editing by using CRISPR/Cas9 system, and the following steps are performed:

(1) Design of sgrnas

The BnaNCBP gene family is found on a BnTIR website (http:// yanglab. Hzau. Edu. Cn/BnTIR), the NCB P gene has 5 members in the Brassica napus and is respectively named as BnaA10.NCBP, bnaA02.NCBP, bnaA03.NCBP, bnC02.NCBP and BnaC03.NCBP, and the full-length DNA sequence thereof is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

Designing a target site sequence of gene editing aiming at a coding region conserved region of an NCBP gene family based on a CRISPR-GE online website (http:// skl.scau.edu.cn/primedesign /);

the nucleotide sequence of the sgRNA action site is as follows:

sgRNA1：5‘-TGCAGTACAAGTTTGCGATTTGG-3’；

sgRNA2：5‘-TCGCAATGCTTCTGATCATCAGG-3’；

sgRNA3：5‘-TACTCTCGCGACCTCAAAGCTGG-3’；

wherein the underlined part is the PAM sequence.

(2) CRISPR editing vector construction

And designing a target site PCR amplification primer according to the target site. The primers were synthesized by Tianyihui Biotech, inc. The target fragment was amplified by PCR using the PGTR plasmid as a template.

The primer sequences are as follows:

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the PCR product is constructed to a CRISPR expression vector PKSE401-TRNA containing a Cas9 expression cassette in a homologous recombination mode, the recombinant expression vector is transferred into escherichia coli competence DH5 alpha, a single colony is selected, positive clone is screened by adopting a bacterial liquid PCR method and sequenced, a recombinant plasmid with correct sequencing is transferred into agrobacterium GV3101, the brassica napus strain Westar is transferred through an agrobacterium-mediated rape transformation system, and agrobacterium transformation and positive seedling screening experiments are entrusted to be completed by Wuhan Tian Biotech limited company.

2.3 obtaining and identifying BnanCBP Gene knockout rape

(1) Screening of positive seedlings

The transgenic seedlings are screened by hygromycin in a culture medium, then transferred to soil, after one week, the young leaves of a single plant are sampled, DNA of rape leaves is extracted by a CTAB method, a positive plant is identified by PCR through a primer HPT-F/R, and an amplification product is detected by 1% agarose gel electrophoresis.

The HPT primer sequence is as follows:

HPT-F：5'-ACACTACATGGCGTGATTTCAT-3’

HPT-R：5'-TCCACTATCGGCGAGTACTTCT-3’

(2) Edit type detection

Respectively designing gene specific primers near each sgRNA target site to carry out PCR amplification, and sending the PCR product with the correct amplification product strip size to a rice sequencing group of Chinese academy of agricultural sciences to carry out Hi-TOM sequencing to obtain a mutation site.

The gene-specific primer sequences are as follows:

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in transgenic T0 generation plants, 5 transgenic plants have target mutation, wherein homozygous mutant 1 and heterozygous mutant 4 are shown in figure 3, and plants subjected to gene editing are selected for self-breeding to obtain T1 generation seeds.

2.4 phenotypic Observation of plants homozygous for the NCBP Gene editing

Extracting T1 generation transgenic plant genome DNA, detecting the editing condition of T1 generation plant by the method, and selfing to obtain stably inherited T2 generation gene editing homozygous mutant plant (figure 3). 5 homozygous mutant genotypes were obtained in the T2 generation, both 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. These 5 mutant genotypes all resulted in frame shift mutations, leading to premature termination of codons and inactivation of protein function.

Homozygous mutants with the T2 knockout and wild-type wistar were planted together in the greenhouse at the university of agriculture, anhui. The flowering time of the wild type is 65.5 +/-1.2 days, and compared with the wild type, the homozygous edited plants show late flowers in the T2 generation and have obvious difference. The flowering time of T2-4-9 is 69.4 + -2.2 days, the flowering time of T2-12-7 is 68.6 + -1.6 days, the flowering time of T2-12-9 is 68.4 + -2.5 days, the flowering time of T2-51-7 is 68.6 + -1.8 days, and the results are shown in FIG. 4. The results show that the BnanCBP gene controls the flowering time of the cabbage type rape.

2.5NCBP.A10 overexpression vector construction and transgenic arabidopsis phenotype observation

(1) Construction of BnancBP. A10 overexpression vector

The wild Westar rape variety is used as a material, RNA is extracted, and the extracted RNA is subjected to reverse transcription to generate a cDNA first chain which is used as a template for PCR amplification. Designing a specific primer to construct an over-expression vector (the transverse line is an enzyme cutting site) according to a coding region CDS sequence of the BnancBP.A10 gene:

BnaNCBP.A10-F：5'-GGATCCATGGAGATTACGGAGAGGAGGC-3’；

BnaNCBP.A10-R：5'-GTCGACTCCTCTCAGCCAAGTGTTCC-3’，

obtaining the BnancBP.A10 coding region segment with the enzyme cutting site.

The BnancBP. A10 coding region fragment with the correct enzyme cutting site and the over-expression vector pCambia1300modified (PCMM) are subjected to double enzyme cutting by BamH I and Sal I respectively, and the target fragment and the vector are recovered by gel cutting. T4 ligase is used for overnight connection at 22 ℃, escherichia coli competent cells DH5 alpha are transferred by a heat shock method, overnight culture is carried out at 37 ℃, single colonies are selected, positive clones are screened by a bacterial liquid PCR method and are subjected to sequencing analysis, and finally the plant overexpression recombinant vector PCMM-NCBP.A10 is obtained. The PCMM-NCBP. A10 recombinant plasmid is transferred into agrobacterium-sensitive GV3101, and positive clone is identified by a bacterial liquid PCR method.

(2) Transformation of Arabidopsis thaliana and observation of phenotype of transgenic homozygous plant

Selecting positive bacteria liquid, adding 5mL LB liquid culture medium containing kanamycin Kan and rifampicin Rif resistance, culturing at 28 ℃ and 200rpm for two days, and then mixing the materials according to a volume ratio of 1:100 was inoculated into 100mL LB liquid medium containing kanamycin Kan and rifampicin Rif resistance, and cultured at 28 ℃ and 200rpm until OD600 was 0.6-0.8.

Collecting and cleaning thalli by a centrifugal method, transferring the thalli into wild type arabidopsis thaliana Col-0 by using a pollen tube mediated method, after the seeds are mature (T0), carrying out hygromycin resistance screening on the seeds, and culturing a positive seedling (T1) in an artificial climate chamber until the seeds are mature. T2 generation homozygous transgenic seeds were screened and phenotypes identified after sowing, the results are shown in FIG. 5. As can be seen in FIG. 5, the flowering time of the over-expressed transgenic line was significantly advanced compared to the wild type Col-0.

In summary, the present invention utilizes GWAS analysis to identify rape flowering phase candidate gene NCBP.A10, searches BnTIR database by bioinformatics method according to rape NCBP gene sequence information, identifies homologous genes of NCBP in rape, named as BnaA10.NCBP, bnaA02.NCBP, bnaA03.NCBP, bnC02.NCBP and BnaC03.NCBP respectively. 3 target site sequences edited by genes are designed by using a CRISPR-GE online analysis tool and are connected to a PKSE401-TRNA vector with a Cas9 expression cassette. The agrobacterium-mediated transformation method is adopted to transfer the brassica napus Westar. And performing transgenic plant mutation type analysis by gene specific primer PCR detection and sequencing to screen 4 homozygous BnanNCBP gene editing mutants. The BnancBP. A10 overexpression vector is used for transforming Arabidopsis, and a transgenic homozygous plant shows early blossoming. The invention discovers that the BnanNCBP gene has important influence on the flowering phase of rape and can be applied to the flowering phase improvement of the rape.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims (10)

1. A rape RNA processing factor NCBP gene is characterized in that the gene is positioned on the A10 chromosome of rape and has a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence which is complementary with the sequence shown as SEQ ID NO. 1.

2. Use of the RNA processing factor NCBP gene of canola as claimed in claim 1 for improving flowering characteristics of canola.

3. Use according to claim 2, wherein the rape is brassica napus.

4. Use according to claim 3, wherein the mutant plant homozygous for the knockout of the NCBP gene in Brassica napus exhibits late flowering.

5. Use according to claim 3, characterized in that the overexpression of the NCBP gene causes the transgenic Arabidopsis thaliana to show early flowering.

6. A method for improving flowering phase characters of rape is characterized by comprising the following steps:

(1) Designing a target sequence in a conserved region of a rape BnanNCBP gene family coding region;

(2) Synthesizing a linker primer according to a target sequence, and constructing a CRISPR/Cas9 recombinant vector for BnaNCBP gene targeting;

(3) Transferring the recombinant vector into agrobacterium tumefaciens GV3101, and transforming a cabbage type rape spring variety Westar by using an agrobacterium-mediated method to obtain a transgenic rape plant;

(4) Extracting genome DNA of the transgenic positive plant, performing PCR amplification and sequence sequencing by using a gene specific primer covering a target site, and performing genotype identification on the T0 generation transgenic plant;

(5) The mutant with gene editing is planted in the T1 generation, and the PCR amplification detection is carried out on HPT and Cas9 transgenes in the T1 generation plant by using the primer, so as to obtain the homozygous BnancBP gene editing mutant plant.

7. The method for improving flowering traits in oilseed rape of claim 6, wherein the target sequence comprises three of:

sgRNA1：5‘-TGCAGTACAAGTTTGCGATTTGG-3’；

sgRNA2：5‘-TCGCAATGCTTCTGATCATCAGG-3’；

sgRNA3：5‘-TACTCTCGCGACCTCAAAGCTGG-3’。

8. the method for improving flowering characteristics of rape according to claim 6, wherein in the step (5), the primer sequences for HPT and Cas9 transgenic amplification are respectively:

HPT-F：5'-ACACTACATGGCGTGATTTCAT-3'；

HPT-R：5'-TCCACTATCGGCGAGTACTTCT-3'；

Cas9-F：5'-GGCCTCGATATTGGGACTAACT-3'；

Cas9-R：5'-GTCCGCCTTATCTGTGGAGT-3'。

9. a method for obtaining the NCBP gene of RNA processing factor of rape features that the nucleotide sequence of NCBP gene is used as template to design specific amplification primer, the wild Westar rape variety is used as raw material, RNA is extracted and reverse transcribed to become cDNA, and the NCBP gene of rape is obtained by PCR amplification technology.

10. The method for obtaining the NCBP gene of the RNA processing factor of Brassica napus according to claim 9, wherein the specific amplification primers for PCR amplification are:

BnaNCBP.A10-F：5'-GGATCCATGGAGATTACGGAGAGGAGGC-3’；

BnaNCBP.A10-R：5'-GTCGACTCCTCTCAGCCAAGTGTTCC-3’。

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