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Method for testing drought tolerances of vigna unguiculata type I and vigna unguiculata type II by fluorogenic quantitative PCR (polymerase chain reaction), diagnostic genes and primers

A fluorescent quantitative and drought-tolerant technology, applied in the field of plant biology, can solve problems that have not yet been reported or developed, and achieve the effect of promoting drought-tolerant breeding of cowpea and improving efficiency

Active Publication Date: 2014-04-02
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no diagnostic gene and its primers that can be used to specifically detect type I and type II drought tolerance have been reported or developed in cowpea

Method used

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  • Method for testing drought tolerances of vigna unguiculata type I and vigna unguiculata type II by fluorogenic quantitative PCR (polymerase chain reaction), diagnostic genes and primers
  • Method for testing drought tolerances of vigna unguiculata type I and vigna unguiculata type II by fluorogenic quantitative PCR (polymerase chain reaction), diagnostic genes and primers
  • Method for testing drought tolerances of vigna unguiculata type I and vigna unguiculata type II by fluorogenic quantitative PCR (polymerase chain reaction), diagnostic genes and primers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The method for screening diagnostic genes and primers for specifically distinguishing cowpea type I and type II drought tolerance comprises the following steps:

[0030] (1) Download and preprocessing of cowpea non-redundant gene sequence: from Harvest cowpea DNA database UP12 (http: / / www.harvest-web.org / hweb / bin / wc.dll?hwebProcess~hmain~&versid=68) Batch download of cowpea unigene sequences, a total of 29,728. Run the VecScreen program (http: / / www.ncbi.nlm.nih.gov / VecScreen / VecScreen.html) to scan and remove vector and adapter sequences contained on the downloaded sequences.

[0031] (2) Cowpea gene chip design: After scanning all 29,728 unigene sequences in the UP12 database, 4 oligonucleotide DNA sequences were designed as probes at different base positions of each sequence. These 4 sequences were in The weighted average of the signal values ​​in the microarray hybridization will define the signal value of a gene. The technical standards for design are: the GC cont...

Embodiment 2

[0037] The differential expression information of the diagnostic gene VuCETS1 in leaf tissue was analyzed by real-time quantitative PCR to identify type I and type II drought-tolerant materials.

[0038] (1) Material preparation: select a plump seed of 1 type I drought-tolerant cowpea variety and 1 type II drought-tolerant cowpea variety seeds and sow them in a nutrient bowl, and the substrate is vermiculite: nutrient soil = 3:1. After the cotyledons were flattened, the water was thoroughly watered, and then the watering was stopped, and the RNA of the leaves under normal growth and under stress conditions was extracted by the conventional Trizol method 14 days after the treatment.

[0039] (2) RAN extraction and reverse transcription: Take 0.1 g of leaf tissue and add liquid nitrogen to grind it into powder, and extract total RNA according to the kit instructions. Dilute the extracted RNA to 0.1 g / L with DEPC-treated water, and add 2 μL of anchor primer (AP) and 2 μL of RNA i...

Embodiment 3

[0042] Differential expression information of the diagnostic gene VuCETS1 in root tissue was analyzed by real-time quantitative PCR to identify type I and type II drought-tolerant materials.

[0043] (1) Material preparation: Select a plump seed of 1 type I drought-tolerant cowpea variety and 1 type II drought-tolerant cowpea variety seeds and sow them in a nutrient bowl, the substrate is vermiculite: nutrient soil = 3:1. After the cotyledons were flattened, the watering was stopped, and the roots of the plants were carefully pulled out 14 days after the treatment, washed with water quickly, and the RNA of the roots under normal growth and stress conditions was extracted by the conventional Trizol method.

[0044] (2) RAN extraction and reverse transcription: Take 0.1 g of root tissue and add liquid nitrogen to grind it into powder, and extract total RNA according to the kit instructions. Dilute the extracted RNA to 0.1 g / L with DEPC-treated water, and add 2 μL of anchor prime...

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Abstract

The invention relates to a method for testing drought tolerances of vigna unguiculata type I and vigna unguiculata type II by fluorescence quantitative PCR, diagnostic genes and primers. According to the method, sequences of the tested diagnostic genes are shown as in SEQIDNO: 1; the fluorescence quantitative PCR primers have the sequences as follows: a forward primer: 5'-TGGATTGTGGTGGACATAC-3' and a downstream primer: 5'-GGTTCCTTCTGAGCATTGA-3'. The method, the diagnostic genes and the primers have the advantages that significant differentially expressed genes in materials of drought tolerances of the vigna unguiculata type I and the vigna unguiculata type II provide a diagnostic gene tool for distinguishing the drought tolerances of different types of vigna unguiculata specifically. In addition, direct technical support is provided for testing drought tolerance types of different breeding parent materials by real-time fluorescence quantitative PCR rapidly and conveniently based on the primer sequences of the tolerance diagnostic genes of the vigna unguiculata type I and the vigna unguiculata type II.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a method for detecting cowpea type I and type II drought tolerance by fluorescent quantitative PCR, diagnostic genes and primers. Background technique [0002] cowpea( Vigna unguiculata L. Walp) originated in the arid region of Africa, and is one of the important grain beans in the world and one of the important vegetable crops in Asian countries. According to the 2006 Statistical Yearbook, the annual output of cowpea in my country is more than 8 million tons, and the output loss due to drought is estimated to be about 20%. The drought tolerance of cowpea was significantly different among different genotypes. Previous studies have shown that there are two different types of drought tolerance responses in cowpea natural resources, namely type I and type II drought tolerance responses (Watanabe et al., 1997; Muchero et al., 2008; Agbicodo et al., 2009) . In the type I drough...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/29C12N15/11G01N21/64
Inventor 徐沛李国景吴晓花汪宝根鲁忠富罗洁王莎刘永华
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES