Turnip yellow mosaic virus (TYMV)-induced cruciferous endogenous gene silencing method and application thereof

A technology of cruciferous and endogenous genes, which is applied in the field of plant bioengineering, can solve the problems of low infection efficiency and difficulty in constructing TYMV vectors, and achieve the effects of improving infection efficiency, improving silencing efficiency, and reducing plant damage

Inactive Publication Date: 2018-01-09
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

Provides a TYMV virus-mediated gene silencing system, and aims at the difficulty of constructing TYMV vectors and low infection efficiency in the past, and uses infusion enzymes instead of traditional T4 ligases to construct vectors through homologous recombination, and uses The gene gun method replaces the original friction inoculation method, optimizes the method of target gene carrier construction and gene infection, and causes the silencing effect of different organs. The specific technical scheme is as follows:

Method used

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  • Turnip yellow mosaic virus (TYMV)-induced cruciferous endogenous gene silencing method and application thereof
  • Turnip yellow mosaic virus (TYMV)-induced cruciferous endogenous gene silencing method and application thereof
  • Turnip yellow mosaic virus (TYMV)-induced cruciferous endogenous gene silencing method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1 A kind of TYMV virus-induced cruciferous endogenous gene silencing method

[0061] (1) Cloning and sequencing verification of PDS gene

[0062] The PDS gene sequences of two cabbage crops, non-heading cabbage and Chinese cabbage, were searched through the NCBI database. The cDNA was used as the template for PCR amplification, and the target band was cut under UV light to recover the target fragment, connected to the blunt-zero vector (purchased from TransGenBiotech), and chemically transformed into Trans-T1 Escherichia coli, LB plate containing ampicillin 37 Cultivate overnight at ℃, pick a single colony for PCR identification, extract and sequence the positive plasmid, and obtain the full-length sequence of the PDS gene.

[0063] (2) Vector construction and mass extraction of plasmids

[0064] Select the appropriate 40bp sequence and its reverse complement, and the selection principle is as follows image 3 and Figure 4 Shown: the 2-4 bases are TGA, T...

Embodiment 2

[0069] Example 2 Phenotype and PCR detection after silencing

[0070] 15-17 days after the bombardment of the gene gun, the seedlings appeared photobleaching, such as Image 6 (A. Infection with non-heading Cruciferae pTY-s; B. Infection with non-heading Brassicaceae pTY-PDS; C. Infection with non-heading Cruciferae pTY-s; D. Infection with Brassicaceae pTY-s -PDS infestation) and Figure 7 (Nonheading Cruciferae causes silent phenotypes in different organs, 1, Aheading Cruciferae uninfected 2 Aheading Cruciferae pTY-PDS infection 3 Aheading Cruciferae pTY-s infection ), the results showed that the PDS gene was effectively silenced in multiple organs of leaves, stems, roots, pedicels, and fruit pods. To further verify the effect of gene silencing at the molecular level, PCR detection was performed. The total RNA of the seedlings was extracted and reverse transcribed to obtain cDNA, which was first identified by PCR with CP-F / R, and detected by 2% agarose gel electrophoresis...

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Abstract

The invention discloses a turnip yellow mosaic virus (TYMV)-induced cruciferous endogenous gene silencing method and application thereof. The method comprises the steps of taking target gene complementary deoxyribonucleic acid (cDNA) as a template for carrying out polymerase chain reaction (PCR) amplification, purifying a connecting vector, converting escherichia coli, and carrying out positive clone sequencing to obtain a target coding region full-length CDS sequence; according to the sequencing result, selecting a (T / A / G / C) TGA, (T / A / C) TAG or (T / A / C) TAA backwards selection 40bp sequence; adding a reverse complementary sequence into the 3' end, adding sequences, which have the homologous sequence length of 15bp and correspond to the two ends of a linearized vector, into the two ends tosynthesize double-stranded DNA fragments, and enabling the synthesized double-stranded DNA fragments to be in fusion connection with a vector pTY-s, converting the escherichia coli by a product of thefusion connection, culturing and extracting plasmids, carrying out gene gun bombardment when first true leaves of seedlings come up, and rapidly transferring the seedlings into cultivation soil for culturing. The TYMV-induced cruciferous endogenous gene silencing method is easy in vector construction, high in transfection efficiency and obvious in gene silencing effect, and maintains characters to be enduring.

Description

technical field [0001] The invention belongs to the technical field of plant bioengineering, and relates to a gene silencing technology induced by turnip yellow mosaic virus and its application. Background technique [0002] In plants, virus-induced gene silencing (VIGS) belongs to post-transcriptional gene silencing (PTGS) 1 , the phenotypic mutation that occurs after the recombinant virus carrying the cDNA fragment of the target gene infects the plant and induces the silencing of the target gene. It is a simple, fast and reliable tool for studying plant gene function. After the recombinant viral vector infects plants, RNA is first transcribed, and then replicated by endogenous RNA polymerase (RDRP) to generate double-stranded RNA (dsRNA) molecules, which are the key elicitors for triggering post-transcriptional gene silencing. dsRNA is recognized by Dicer enzyme analogs and cleaved to form 21-24nt small interfering RNA (siRNA) 2 . siRNAs are further amplified by RNA-dep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/83A01H6/20
Inventor 侯喜林张昌伟高立伟俞静刘唱刘同坤李英郭明亮肖栋杨洋
Owner NANJING AGRICULTURAL UNIVERSITY
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