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Nonsymbiotic hemoglobin gene AhGLB for waterlogging resistance of peanut and its application

A technology for hemoglobin and peanuts, applied in the field of agricultural biology, can solve problems that rarely involve in-depth research on internal mechanisms

Inactive Publication Date: 2013-08-21
SHANDONG PEANUT RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the world's largest peanut production and foreign trade country, and as one of the countries seriously affected by waterlogging, research on peanut waterlogging prevention and control needs to be strengthened urgently. In addition to research, there are few in-depth studies on the internal mechanism. Therefore, the research on the mechanism of peanut waterlogging is urgently needed. At present, the research and management of peanut waterlogging resistance should be strengthened to promote the improvement and innovation of peanut varieties and guide production, so as to ensure the overall stability of the peanut industry. develop

Method used

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  • Nonsymbiotic hemoglobin gene AhGLB for waterlogging resistance of peanut and its application
  • Nonsymbiotic hemoglobin gene AhGLB for waterlogging resistance of peanut and its application
  • Nonsymbiotic hemoglobin gene AhGLB for waterlogging resistance of peanut and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Acquisition of peanut waterlogging tolerance gene AhGLB

[0025] 1. Extraction of total RNA from peanut tissue

[0026] Experimental preparation: All utensils used need special treatment. High-temperature-resistant instruments and utensils were baked at 150°C for 3 hours, and other utensils were soaked in 0.1% DEPC for 12 hours, and then autoclaved to remove DEPC. The solution reagents are treated with 0.1% DEPC (sterilized after 12 hours at 37°C), and the reagents that are not resistant to high temperature are directly treated with DEPC-H 2 O preparation. Total RNA was extracted according to the operation requirements of TIANGEN's Trizol Reagent, the specific steps are as follows:

[0027] 1) Take the young peanut leaves and grind them into powder in liquid nitrogen, then add 1 ml of Trizol solution for every 50 mg of tissue to grind, and note that the total volume of the sample should not exceed 10% of the volume of the Trizol used.

[0028] 2) Centrifu...

Embodiment 2

[0090] Embodiment 2: Utilize fluorescent quantitative PCR technology to detect the expression level of peanut waterlogging tolerance gene AhGLB, detect according to the following steps:

[0091] 2.1 Total RNA extraction and cDNA synthesis

[0092] Extract the total RNA of peanut leaves, refer to the test method in Example 1, reverse transcription process (Table 10):

[0093] Table 10

[0094]

[0095]

[0096] The system was placed at 37°C for 15 minutes, and the cDNA product was stored at -20°C.

[0097] 2.2 Drawing of standard curve

[0098] Design a pair of PCR primers according to the cloned gene fragment: AhGLBRT-S and AhGLB-RT-AS, extract the total RNA of peanut seed coat, reverse transcribe it into cDNA, use it as a template, carry out PCR amplification, recover the amplified product, and The PCR product was ligated with pMD-18, and then transformed into Escherichia coli DH5α. After identification by colony PCR, a large number of positive clones were amplified,...

Embodiment 3

[0110] Example 3: Detecting the Specific Expression of the Peanut Waterlogging Resistance Gene AhGLB Using Fluorescent Quantitative PCR Technology

[0111] In order to detect the specific differential expression of peanut waterlogging tolerance gene AhGLB, three varieties of Xianghua 2008, Yuhua 15 and Zhonghua 8 were selected and carried out in a constant temperature culture room. Take peanut seeds that are plump, undamaged, and relatively uniform in size, soak overnight, and then place them in a petri dish that has been lined with filter paper in advance, and put a small amount of tap water in the petri dish, and cultivate them in an incubator under the condition of light for 13 hours , the light intensity is 230-260μmol.m -2 .s -1 , the temperature is 24-26°C, and the relative humidity is 50-60%, and wait for it to germinate. After the peanuts germinated, they were cultured in hydroponic culture, and when they grew to the eighth true leaf of the seedlings, the peanut seed...

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Abstract

The invention discloses a nonsymbiotic hemoglobin gene AhGLB for waterlogging resistance of peanut and its application. The complete sequence of the gene is shown as SEQ ID NO:5. When peanut is under waterlogging and hypoxia stress caused by long-term rain or flood, the gene can bind to NO to generate NO3-, reduce waterlogging hazards, so as to play an important role when peanut is?suffered from?waterlogging hazard. In addition, the invention also discloses specific expression conditions of the gene when different genotypes of peanut are under different stress treatments, and the expression condition of the gene when transgenic peanut is under waterlogging and hypoxia stress treatment so as to reveal the function of the gene.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a peanut waterlogging tolerance gene AhGLB, and the invention also relates to the use of the peanut waterlogging tolerance gene and the specific expression of the gene when peanuts of different genotypes are subjected to different stress treatments, revealing the gene function. Background technique [0002] Peanut is the main oil crop and economic crop in my country, and it is the most competitive export-earning agricultural product in China. Water is one of the important ecological factors affecting the growth, development, metabolism and ecological geographical distribution of plants. The stress caused by water is generally divided into two types, drought stress and waterlogging and hypoxia stress, both of which cause great harm to plant physiology and ecology. Among them, wetness has a profound impact on the world's peanut production. In Asia (especially Southeast A...

Claims

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Application Information

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IPC IPC(8): C12N15/29A01H5/00
Inventor 单世华李林张廷婷李春娟徐娟闫彩霞周西
Owner SHANDONG PEANUT RES INST
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