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VIGS-based Rhododendron pulchrum leaf gene silencing system and construction method thereof

A gene silencing and construction method technology, which is applied in the fields of genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problems of less applied research and achieve the effect of short time, high throughput and fast speed

Pending Publication Date: 2022-07-12
JIYANG COLLEGE OF ZHEJIANG A & F UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although VIGS has been widely used in many plants, the application and related research of this technology on Rhododendron splendens is less

Method used

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  • VIGS-based Rhododendron pulchrum leaf gene silencing system and construction method thereof
  • VIGS-based Rhododendron pulchrum leaf gene silencing system and construction method thereof
  • VIGS-based Rhododendron pulchrum leaf gene silencing system and construction method thereof

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Embodiment 1

[0035] 1. Total RNA extraction and cDNA synthesis of Rhododendron splendid

[0036] The leaves of Rhododendron splendid with good growth and development were sampled, quick-frozen in liquid nitrogen after sampling, and then transferred from liquid nitrogen to -80°C ultra-low temperature refrigerator for preservation for the extraction of total RNA of Rhododendron splendid.

[0037] 1. In this experiment, the RNA kit was used to extract the RNA of Rhododendron splendid leaves. For the specific operation, refer to the instructions of the RNA extraction kit of Tiangen Biochemical Technology Co., Ltd., and finally obtain a high-concentration RNA solution.

[0038] 2. Use the kit of Tiangen Biochemical Technology Co., Ltd. to synthesize cDNA.

[0039] The total RNA of Rhododendron splendor extracted above was subjected to cDNA synthesis, and the specific operations were as follows:

[0040] (1) Prepare the reaction solution in a 1.5mL centrifuge tube

[0041]

[0042] (2) Afte...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a VIGS-based Rhododendron pulchrum leaf gene silencing system and a construction method thereof. The method has the characteristics of short time, high speed, high flux and no dependence on stable genetic transformation, and specifically comprises the following steps: constructing a TRV2 recombinant plasmid containing a specific target gene segment, respectively transforming the TRV2 recombinant plasmid and TRV1 into agrobacterium GV3101 to obtain positive agrobacterium strains, respectively proliferating the positive agrobacterium strains, and mixing to obtain a mixed infection solution; the mixed infection liquid is used for infecting leaves of Rhododendron pulchrum to realize silencing of a specific target gene. The invention explores a method capable of realizing Rhododendron simsii leaf gene silencing, the Rhododendron simsii is infected by using the method disclosed by the invention, the expression level of a target gene can be reduced by 50%, a technical platform is provided for next Rhododendron simsii functional genomics research, and a foundation is laid for next verification of Rhododendron simsii gene functions.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a VIGS-based gene silencing system for Rhododendron splendid leaves and a construction method thereof. Background technique [0002] Virus-induced gene silencing (VIGS) is a mechanism for plants to resist virus infection. By inserting a suitable target gene into the virus genome to construct a recombinant virus vector, it can inhibit the expression of plant endogenous genes. a new genetic engineering technique. VIGS technology does not require the construction of transgenic plants, and has the advantages of short cycle, simple operation and low cost. It is one of the commonly used technical means in the field of functional genomics research. It is widely used in the study of gene function related to plant growth and development, insect resistance and metabolic regulation, including Solanaceae (tobacco, tomato, potato), Legume (soybean, alfalfa, pea,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/12A01H6/36
CPCC07K14/415C12N15/8218Y02A50/30
Inventor 金松恒申建双胡玥
Owner JIYANG COLLEGE OF ZHEJIANG A & F UNIV
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