Gene silencing method Si-VIGS (Seed imbibition-virus-induced gene silencing) in early stage of cotton

A gene silencing and early gene technology, applied in genetic engineering, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of inability to study genes in the germination stage of seeds, gene silencing has not yet been seen, and seedlings are infected. Effects of long duration of silence, simplified preparation, and damage avoidance

Active Publication Date: 2019-08-27
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the commonly used inoculation method of TRV virus system is the leaf injection method. To infect seedlings at the cotyledon stage, the operation is cumbersome and it is impossible to study the genes in the seed germination stage.
No research report on gene silencing in cotton seed germination stage

Method used

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  • Gene silencing method Si-VIGS (Seed imbibition-virus-induced gene silencing) in early stage of cotton
  • Gene silencing method Si-VIGS (Seed imbibition-virus-induced gene silencing) in early stage of cotton
  • Gene silencing method Si-VIGS (Seed imbibition-virus-induced gene silencing) in early stage of cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The following examples mainly use silent upland cotton GHLS1 As an example, the relevant experimental process is described in detail as follows. It should be noted that the TRV-VIGS system involves pTRV1, positive control pTRV2- GhCLA1 , negative control pTRV2- GFP , experimental group pTRV2- GHLS1 and other vectors, this example uses pTRV2- GhCLA1 Take the carrier as an example (the constructed carrier structure can be partially referred to image 3 As shown), the detailed introduction of its construction process is as follows. Refer to its operation for other carriers.

[0092] (1) Extract RNA and reverse transcribe it into cDNA

[0093] Cotton tissues are rich in polysaccharides and polyphenols, so the Plant Total RNA Isolation Kit Plus from FOREGENE was used to extract the RNA from cotton tissues, and then digested with DNaseI to remove DNA, and then reversed The recording kit was reverse-transcribed into cDNA for later use. The specific operation is des...

Embodiment 2

[0160] On the basis of the bacterial solution for transfection prepared in Example 1, the transfective bacterial solution first infects tobacco and then infects cotton seeds, so as to optimize the operation of related gene silencing. The specific experimental process and experimental results are introduced as follows.

[0161] (1) Agrobacterium-mediated TRV-VIGS infection of tobacco

[0162] First use the needle to lightly touch the back of four-week-old tobacco leaves to cause micro-wounds, and then pTRV2- prepared in Example 1 GhCLA1 , pTRV2- GFP , pTRV2- GHLS1 The Agrobacterium suspension was mixed with the pTRV1 Agrobacterium suspension at a volume ratio of 1:1, and then slowly injected into the tobacco leaves through the wound with a syringe and continued to culture for about 7 days.

[0163] (2) Infection of cotton seeds

[0164] First, weigh 1 g of the Agrobacterium resuspension in step (1) and culture the tobacco leaves containing virus particles for 7 days (the ...

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Abstract

The invention belongs to the technical field of cotton breeding, and specifically relates to a gene silencing method in early germination and seedling stage of cotton. The method comprises the steps of preparing agrobacterium liquid for transfection, performing virus amplification by utilizing tobacco, infecting cotton seeds and the like. According to the invention, a cotton gene silencing technology is optimized and improved by taking a tobacco rattle virus (TRV) as a virus vector. It is proved by a result that a gene silencing phenotype can be obtained through inoculating cotton cotyledon orseeds with TRV homogenate or agrobacteria. Through comparing the similarities and differences of TRV homogenate and agrobacteria inoculation, it is discovered that through applying a seed imbibition(Si) method to inoculation of any infection liquid, the virus inoculation time can all be advanced to the inflorescence primordial formation stage of the seeds; functional genes during germination ofthe seeds can be efficiently silenced within 3 days; virus homogenate-mediated gene silencing can last till the seedling stage of the cotton. Si-VIGS (Seed imbibition-virus-induced gene silencing) involved in the invention can efficiently silence the genes from the early germination stage of the cotton; the operation is simple; the phenotypes are unified; a relatively good application prospect isshown.

Description

technical field [0001] The application belongs to the technical field of cotton breeding, and in particular relates to a gene silencing method for cotton germination and early seedling stages. Background technique [0002] Among the existing genome research methods, virus-induced gene silencing (VIGS), as a relatively mature transgenic technology, is widely used in monocotyledonous and dicotyledonous plants, such as Arabidopsis, tobacco, cotton, soybean, etc. [0003] VIGS was first used to explain the defense mechanism of plants caused by virus infection, and this phenomenon is common in plants. After a plant is infected by a virus, since the viral RNA is inserted into the plant cell, it can induce the plant to produce a sequence-specific genetic immune mechanism. [0004] As a transient gene silencing technology, TRV-VIGS has a principle similar to RNA interference and is mainly used to down-regulate the expression of target genes. The principle of its technical operatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/60
CPCC07K14/415C12N15/8218C12N15/8205
Inventor 师恭曜位芳田保明李晶张斌郝建峰邓凯文彭月
Owner ZHENGZHOU UNIV
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