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Construction and disease-resistant application of a cassava camta gene and its inhibitory expression vector

A cassava, carrier technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem that functional research has not yet been reported, and achieve the effect of fast and efficient silencing

Active Publication Date: 2022-08-02
HAINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, about CAMTA Functional studies of genes in cassava have not been reported yet

Method used

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  • Construction and disease-resistant application of a cassava camta gene and its inhibitory expression vector
  • Construction and disease-resistant application of a cassava camta gene and its inhibitory expression vector
  • Construction and disease-resistant application of a cassava camta gene and its inhibitory expression vector

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Cassava MeCAMTA Construction of gene expression silencing vector

[0025] (1) Cassava MeCAMTA Cloning of specific nucleic acid silencing fragments from 1-600 bp at the 5' end of the gene:

[0026] Take an appropriate amount of cassava leaves of Huanan No. 124 and put them in a mortar cooled by liquid nitrogen, add an appropriate amount of liquid nitrogen, and grind the cassava leaves into a fine powder. Root Biochemical Co., Ltd., the same below), extracted total RNA from cassava. The absorbance value of RNA at 260 nm and 280 nm and the RNA concentration were measured with a protein detector (DM 650 BECKMAN, MSA), and the RNA purity was detected by 1.5% (mass volume ratio) agarose gel electrophoresis. According to the instructions of the reverse transcription kit (purchased from Thermo Fermentas, the same below), the RNA was reverse transcribed into cDNA and stored in a -40°C refrigerator for later use.

[0027] By designing primers, we cloned cassava f...

Embodiment 2

[0032] Example 2: pTRV2- MeCAMTA Application of gene silencing expression vector in cassava disease resistance

[0033] (1) Preparation of cassava materials:

[0034] A. Seed selection: Varieties with good adaptability and high yield should be selected, and Huanan 124 should be selected for planting. Generally, it is better to choose cassava stems with fully mature stems, corked, dense stems, complete epidermis and axillary buds, thick and obvious bud eyes, no pests and diseases, no damage, fresh and firm middle and lower parts of the main stem. Old and young stems should be avoided.

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Abstract

The invention provides a cassava CAMTA gene and the construction and disease-resistant application of its inhibitory expression vector, which is isolated from cassava to obtain a CAMTA Gene. Cassava CAMTA Construction of gene suppression expression vector: RNA was extracted from cassava leaves of Huanan No. 124 and reverse transcribed into cDNA. CAMTA The 1-600 bp fragment at the 5' end of the gene is used as the specific nucleic acid silencing fragment of the virus-induced gene silencing system, and the obtained cDNA is amplified as a template to obtain CAMTA The 1-600 bp fragment at the 5' end of the gene is connected to the TRV virus-inducible gene silencing vector pTRV2 after the gel is recovered to obtain pTRV- MeCAMTA Gene silencing expression vector. By constructing a silent expression vector of this gene and injecting cassava leaves, an effective transient MeCAMTA Gene silencing of cassava provides an effective method for studying gene function.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and technology, and in particular relates to a method for enhancing the resistance of cassava to pathogenic bacteria Xam ( Xanthomonas axonopodis pv. manihotis ) resistant MeCAMTA gene, also involving a cassava CAMTA Gene silencing system and construction method of expression vector, and also relates to a kind of cassava CAMTA Gene silencing expression vectors in enhancing plant resistance to pathogenic bacteria Xam Use in resistance. Background technique [0002] Virus-induced gene silencing (VIGS) is a gene transcription suppression technique used to characterize the function of a gene in plants. In recent years, a variety of VIGS vectors have been widely used in plants. Among them, tobacco rattle virus (TRV) is currently the most widely used vector. It consists of two viral chains, TRV1 and TRV2. TRV1 is used to assist the movement of TRV2 carrying target gene fragments in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/83C12N15/66A01H5/12A01H6/38
CPCC07K14/415C12N15/8218C12N15/8281C12N15/66
Inventor 施海涛昌燕李
Owner HAINAN UNIVERSITY
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