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CN-122012575-A - CRISPR/dCAS9 vector for improving BnNAC022 gene expression and construction method and application thereof

CN122012575ACN 122012575 ACN122012575 ACN 122012575ACN-122012575-A

Abstract

The invention relates to a CRISPR/dCAS9 vector for improving BnNAC022 gene expression and a construction method and application thereof, wherein the CRISPR/dCAS9 vector is a CRISPR-SL-U6-26-TG1-TG2 vector, the nucleotide sequence of the CRISPR/dCAS9 vector is shown as SEQ ID NO.7, and the CRISPR/dCAS9 vector comprises two core expression cassettes, wherein the expression cassette 1 sequentially comprises an Arabidopsis thaliana U6-26 promoter, a target point 1, an sgRNA2.0 bracket sequence, an At-tRNAGly coding sequence, a target point 2 and an sgRNA2.0 bracket sequence from the 5'-3' end, and the expression cassette 2 sequentially comprises a constitutive promoter, a 3×flag tag coding sequence, a first SV40 NLS, a dCAS9 coding gene, an NLS nuclear positioning signal, a VP64 coding gene, a self-cleaving polypeptide T2A coding gene, an MS2 coding gene, a second SV40 NLS, a P65 coding gene, an HSF1 coding gene and a transcription terminator. The recombinant vector CRISPR-SL-U6-26-TG1-TG2 obtained by construction is an activation vector of a targeted BnNAC022 gene promoter, can obviously improve the BnNAC022 gene expression level in the brassica napus, and enhances the aphid resistance of the brassica napus.

Inventors

  • SHENG LEI
  • ZHAO MANLI
  • HOU SHUMIN

Assignees

  • 安徽省农业科学院

Dates

Publication Date
20260512
Application Date
20260108

Claims (10)

  1. 1. A CRISPR/dCas9 vector for improving BnNAC022 gene expression, which is characterized in that the CRISPR/dCas9 vector is a CRISPR-SL-U6-26-TG1-TG2 vector and comprises two core expression cassettes; The expression cassette 1 is an sgRNA expression cassette, which sequentially comprises an Arabidopsis U6-26 promoter, a target point 1, an sgRNA2.0 bracket sequence, an Arabidopsis At-tRNAGly coding sequence, a target point 2 and an sgRNA2.0 bracket sequence from the 5 'end to the 3' end; the expression cassette 2 is a functional protein expression cassette, and comprises a constitutive promoter, a 3 xFlag tag coding sequence, a first SV40 NLS, dCS 9 coding gene, NLS nuclear localization signal, VP64 coding gene, self-cleaving polypeptide T2A coding gene, MS2 coding gene, a second SV40 NLS, P65 coding gene, HSF1 coding gene and NOS transcription terminator from the 5 'end to the 3' end in sequence.
  2. 2. The CRISPR/dCas9 vector for increasing expression of a BnNAC022 gene according to claim 1, wherein the constitutive promoter is a 35S promoter and/or a UBI promoter.
  3. 3. The CRISPR/dCas9 vector for improving BnNAC022 gene expression according to claim 1, wherein the nucleotide sequence of the CRISPR-SL-U6-26-TG1-TG2 vector is shown in SEQ ID No.7, the nucleotide sequence of the target 1 is shown in SEQ ID No.4, the nucleotide sequence of the target 2 is shown in SEQ ID No.5, and the nucleotide sequence of the arabidopsis U6-26 promoter is shown in SEQ ID No. 6.
  4. 4. The CRISPR/dCas9 vector for increasing expression of BnNAC022 gene according to claim 1, wherein CDS sequence of BnNAC022 gene is shown as SEQ ID No.2, and amino acid sequence of encoded protein is shown as SEQ ID No. 3.
  5. 5. A method of constructing a CRISPR/dCas9 vector for increasing BnNAC022 gene expression as set forth in any one of claims 1 to 4, wherein the CRISPR/dCas9 vector is a recombinant vector CRISPR-SL-U6-26-TG1-TG2, the method comprising the steps of: (1) Constructing a basic gene activation vector CRISPR-SL; (2) Designing and synthesizing two specific sgRNA targets aiming at the upstream region of the BnNAC022 gene translation initiation site, and respectively named as target 1 and target 2; (3) And (3) performing enzyme digestion on a basic gene activation vector CRISPR-SL by using SamI restriction enzyme, and inserting an sgRNA expression unit which is driven by an Arabidopsis thaliana U6-26 promoter, comprises the two specific sgRNA target sequences and is connected with a sgRNA2.0 bracket into the basic gene activation vector CRISPR-SL by using T4 DNA ligase, thereby constructing and obtaining a recombinant vector CRISPR-SL-U6-26-TG1-TG2.
  6. 6. The method of claim 5, wherein in the step (1), the base gene activation vector CRISPR-SL is constructed by double-digestion of pCAMBIA3301 vector with NcoI and PmI restriction enzymes, excision of the original GUS fragment, and insertion of CRISPR-SAM system with dCS 9-VP64 and MS2-P65-HSF1 fusion expression cassette as core by seamless cloning technique.
  7. 7. The method of claim 5, wherein in step (3), the sgRNA expression unit has the structure of an Arabidopsis thaliana U6-26 promoter-target 1-sgRNA2.0 scaffold-At-tRNAGly-target 2-sgRNA2.0 scaffold.
  8. 8. Use of a CRISPR/dCas9 vector according to any one of claims 1-4 for increasing BnNAC022 gene expression for targeted activation of BnNAC022 gene for enhancing aphid resistance of brassica napus.
  9. 9. The use according to claim 8, characterized in that the method for obtaining cabbage type rape with enhanced aphid resistance is that recombinant vector CRISPR-SL-U6-26-TG1-TG2 is transformed into wild cabbage type rape ZS11 by agrobacterium mediation method to obtain T 0 generation transgenic plant, positive transgenic plant is screened by molecular detection and cultivated to T 1 generation to obtain genetically stable strain, and the transgenic homozygous strains JH21 and JH40 with significantly improved BnNAC022 gene expression level are obtained by screening by using real-time fluorescence quantitative PCR technology.
  10. 10. Use according to claim 9, characterized in that the expression level of BnNAC022 gene in the transgenic homozygous lines JH21 and JH40 is 2-4 times that of wild type brassica napus ZS 11.

Description

CRISPR/dCAS9 vector for improving BnNAC022 gene expression and construction method and application thereof Technical Field The invention belongs to the technical field of plant genetic engineering and molecular breeding, and particularly relates to a CRISPR/dCAS9 vector for improving BnNAC022 gene expression, a construction method and application thereof. Background Cabbage type rape is an important oil crop, but is often affected by pests such as aphids in the growth process, so that the yield and quality are seriously reduced, at present, aphids are mainly prevented and treated by chemical pesticides, but the problems of environmental pollution, pesticide residues, enhanced pest resistance and the like are easily caused by long-term use, so that the cultivation of new varieties of insect-resistant rape is a fundamental way for solving the problems. The plant insect resistance is a complex character controlled by multiple genes, NAC transcription factor family plays an important regulation role in plant response biological and abiotic stress processes, and early researches show that BnNAC022 genes are up-regulated in the rape response aphid stress process, and presumably involved in aphid resistance reaction, however, how to utilize the genes to cultivate aphid resistance rape germplasm resources is not reported yet. The traditional transgenic technology realizes function enhancement by introducing exogenous genes, but has the problems of integration randomness, unstable expression, biosafety disputes and the like of the exogenous genes, the CRISPR gene activation technology carries out fusion expression on dCS 9 (D10A, H840A) and transcriptional activation structural proteins, the transcriptional activation proteins are targeted to target gene promoter regions by utilizing the accurate targeting function of the dCS 9, and then the transcription factors and RNA polymerase are recruited to carry out activation expression on the target genes, so that the CRISPR gene has the characteristics of accuracy, safety and high efficiency, shows great potential in crop genetic improvement, and does not have the related research of creating novel aphid resistance varieties of rape by utilizing the CRISPR gene activation technology at present. Disclosure of Invention The invention aims to solve the problems and provide a CRISPR/dCAS9 vector for improving BnNAC022 gene expression, a construction method and application thereof. The invention realizes the above purpose through the following technical scheme: The invention provides a CRISPR/dCAS9 vector for improving BnNAC022 gene expression, wherein the CRISPR/dCAS9 vector is a CRISPR-SL-U6-26-TG1-TG2 vector and comprises two expression cassettes of cores; The expression cassette 1 is an sgRNA expression cassette, which sequentially comprises an Arabidopsis U6-26 promoter, a target point 1, an sgRNA2.0 bracket sequence, an Arabidopsis At-tRNAGly coding sequence, a target point 2 and an sgRNA2.0 bracket sequence from the 5 'end to the 3' end; the expression cassette 2 is a functional protein expression cassette, and comprises a constitutive promoter, a 3 xFlag tag coding sequence, a first SV40 NLS, dCS 9 coding gene, NLS nuclear localization signal, VP64 coding gene, self-cleaving polypeptide T2A coding gene, MS2 coding gene, a second SV40 NLS, P65 coding gene, HSF1 coding gene and NOS transcription terminator from the 5 'end to the 3' end in sequence. As a further optimization of the above invention, the constitutive promoter is a 35S promoter and/or a UBI promoter. As a further optimization scheme of the invention, the nucleotide sequence of the CRISPR-SL-U6-26-TG1-TG2 vector is shown as SEQ ID NO.7, the nucleotide sequence of the target spot 1 is shown as SEQ ID NO.4, the nucleotide sequence of the target spot 2 is shown as SEQ ID NO.5, and the nucleotide sequence of the Arabidopsis thaliana U6-26 promoter is shown as SEQ ID NO. 6. As a further optimization scheme of the invention, the CDS sequence of the BnNAC022 gene is shown as SEQ ID NO.2, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 3. The invention also provides a construction method of the CRISPR/dCAS9 vector for improving BnNAC022 gene expression, wherein the CRISPR/dCAS9 vector is a recombinant vector CRISPR-SL-U6-26-TG1-TG2, and the construction method comprises the following steps: (1) Constructing a basic gene activation vector CRISPR-SL; (2) Designing and synthesizing two specific sgRNA targets aiming at the upstream region of the BnNAC022 gene translation initiation site, and respectively named as target 1 and target 2; (3) And (3) performing enzyme digestion on a basic gene activation vector CRISPR-SL by using SamI restriction endonuclease, and inserting an sgRNA expression unit which is driven by an Arabidopsis thaliana U6-26 promoter, comprises the two specific sgRNA target sequences and is connected with a sgRNA2.0 bracket into the basic gene activation vector CRISPR-SL by usi