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Heat shock transcription factor gene TaHsfA2i of wheat, and encoded protein, and application of heat shock transcription factor gene TaHsfA2i

A technology of heat shock transcription factor and wheat, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as incomplete and redundant sequences, and achieve the effect of improving heat resistance and improving heat resistance

Active Publication Date: 2018-02-09
HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, at least 56 wheat Hsfs have been identified, many of which have redundancy and incomplete sequences

Method used

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  • Heat shock transcription factor gene TaHsfA2i of wheat, and encoded protein, and application of heat shock transcription factor gene TaHsfA2i
  • Heat shock transcription factor gene TaHsfA2i of wheat, and encoded protein, and application of heat shock transcription factor gene TaHsfA2i
  • Heat shock transcription factor gene TaHsfA2i of wheat, and encoded protein, and application of heat shock transcription factor gene TaHsfA2i

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 RNA Extraction

[0023] The roots, stems and leaves of wheat Cang 6005 seedlings with two leaves and one heart were taken respectively, and quickly frozen in liquid nitrogen.

[0024] The frozen plant tissue was quickly ground in a mortar until the plant tissue was powdered, and liquid nitrogen was continuously added during this period.

[0025] Quickly divide the ground plant tissue samples into RNase-free 1.5ml centrifuge tubes, 100mg per tube, add 1ml Redzol (Beijing Saibaisheng) to each centrifuge tube, vortex and let the homogenate stand at 30°C for 5 minutes to fully lyse.

[0026] Phase separation: Add 200 µL of chloroform to each centrifuge tube, vortex and mix well, then let stand at room temperature for 3 minutes.

[0027] Centrifuge at 12,000g for 15 minutes at 4°C. The mixture separated into three phases after centrifugation (lower phenol / chloroform phase, middle white interface, upper colorless aqueous phase). The RNA is all in the colorless a...

Embodiment 2

[0032] Example 2 cDNA first-strand synthesis

[0033]RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo scientific. The reagents in the kit were thawed, mixed gently and placed on an ice box.

[0034] Add 3µg total RNA and 1µL Oligo (dT) to 200µL nuclease-free PCR tubes respectively 18 primer, and make up to 12µL with nuclease-free Water, mix slightly and centrifuge.

[0035] The mixed system was placed in a PCR instrument at 65°C for 5 min and cooled on ice.

[0036] Add 4 µL 5X Reaction Buffer, 1 µL RiboLock RNaseInhibitor (20 U / µL), 2 µL 10 mM dNTP Mix, 1 µL RevertAid M-MuLV RT (200 U / µL) to each PCR tube, mix gently and centrifuge .

[0037] The reaction system was reacted in a PCR machine at 42°C for 60 minutes, denatured at 70°C for 5 minutes, and then stored at -20°C.

Embodiment 3

[0038] Example 3 Gene Cloning

[0039] Front primer (SEQ ID No. 3): ATGGATCCCTTTCACGGCA.

[0040] Back primer (SEQ ID No. 4): TCACTGGTAGCTGTGGGGC.

[0041] Use Takara's high-fidelity enzyme PrimerStar to amplify, and separate the PCR products by agarose gel electrophoresis. figure 1 obtained by cloning wxya Agarose gel electrophoresis image, there are clear bands between 1000bp-2000bp, the target fragments were cut, and TaKaRa MiniBEST Agarose Gel DNA Extraction Kit was used for gel recovery. The concentration of recovered fragments was determined using NanoDrop 2000. Use the p-EASY-Blunt cloning kit of Beijing Quanshijin Biotechnology Co., Ltd. to connect the fragments to the vector, transfer the ligated plasmid into Escherichia coli competent cells, perform resistance screening, and pick a single clone for PCR verification. Sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing.

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Abstract

The invention belongs to the fields of genetic engineering and molecular breeding, and relates to a sequence of a heat shock transcription factor gene TaHsfA2i of wheat and application of the gene sequence. The nucleotide sequence of the heat shock transcription factor gene of the wheat is shown as in SEQ ID No.1. The gene TaHsfA2i is acquired from a heat resistant variety Cang 6005 of the wheat through cloning, the expression is obviously improved because of heat stress, and moreover, the expression of a downstream stress resistance gene is activated. The gene genetically transforms a yeast and arabidopsis, and the heat resistance capability of the yeast and the arabidopsis is obviously improved. A heat shock transcription factor encoding gene of wheat plays an important role in cultivating a crop variety with strong heat resistance.

Description

technical field [0001] The invention belongs to the related technical field of plant molecular biology and transgene. wheat heat shock transcription factor gene wxya High temperature resistance function verification, and the application of this gene in the breeding of new plant varieties resistant to high temperature. Background technique [0002] With the intensification of the global greenhouse effect, global warming has become a severe challenge to the modern agricultural production system. As an important crop in the world, wheat production is increasingly affected by global warming. Temperate wheat in my country often suffers from dry and hot winds during its growth period, which leads to early maturity of wheat. At the same time, high temperature will affect carbon assimilation and Starch synthesis has a serious impact, resulting in a reduction in wheat yield and quality to varying degrees. In normal years, wheat yields are reduced by 5-10%, and in severe years, it ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/82C12N1/19C12N1/21C07K14/415A01H5/00A01H6/20A01H6/46C12R1/01
CPCC07K14/415C12N15/8271
Inventor 段硕楠李国良张华宁刘子会郭秀林
Owner HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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