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Protein GmHAD1 related to plant stress tolerance and encoding gene and application thereof

A stress tolerance, plant technology, applied in the field of genetic engineering, can solve problems such as limiting soybean growth and development, affecting soybean yield and quality, and falling flower pods

Active Publication Date: 2018-05-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, low-phosphorus soil severely limits the growth and development of soybeans, and also causes flower pods to fall off, etc., and ultimately affects the yield and quality of soybeans.

Method used

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  • Protein GmHAD1 related to plant stress tolerance and encoding gene and application thereof
  • Protein GmHAD1 related to plant stress tolerance and encoding gene and application thereof
  • Protein GmHAD1 related to plant stress tolerance and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Acquisition of Soybean Low Phosphorus Tolerance Related Protein GmHAD1 and Its Encoding Gene

[0082] 1. Acquisition of soybean low phosphorus tolerance related protein GmHAD1 and its coding gene

[0083] 1. Acquisition of cDNA

[0084] The plant total RNA extraction kit (TR02, GeneMark) was used to extract the root total RNA of soybean material Huachun No. 2 (low phosphorus tolerance variety), and its integrity was detected by 1% agarose electrophoresis; using PrimeScript TM RT reagent Kit with gDNA Eraser kit reverse transcription to synthesize cDNA.

[0085] 2. PCR amplification

[0086] Using the cDNA obtained in step 1 as a template, PCR amplification was performed using GmHAD1-F and GmHAD1-R primers to obtain a PCR product. The primer sequences are as follows:

[0087] GmHAD1-F: 5'-GGGGAAAACACTTTGGTC-3';

[0088] GmHAD1-R: 5'-AGTTACTGGTTGGCTTTG-3'.

[0089] The PCR reaction system (50μl system) is as follows: 2×Phanta Max Buffer (25μl), ddH 2 O (19...

Embodiment 2

[0110] Example 2, Obtaining GmHAD1 Gene-transferred Hairy Roots of Soybean and Analysis of GmHAD1 Gene Function

[0111] 1. Obtaining hairy roots of transgenic GmHAD1 soybean

[0112] 1. The full-length sequence of the GmHAD1 gene shown in Sequence 1 is forward inserted between the Xba I and Sac I restriction sites of the pTF101.1 vector to construct the recombinant plant expression vector pTF101.1-GmHAD1. The structural diagram of the recombinant plant expression vector pTF101.1-GmHAD1 is shown in figure 2 As shown, it can be seen from the figure that the GmHAD1 gene is located behind the CaMV35S promoter, and the CaMV35S promoter is used to promote the expression of the GmHAD1 gene.

[0113] 2. Transform pTF101.1 and pTF101.1-GmHAD1 into Agrobacterium rhizogenes K599 (purchased from BioVector NTCC Inc.) with a Bio-rad electric shock transformation instrument, and then transform soybeans through the K599 Agrobacterium rhizogenes-mediated transformation method , to obtain t...

Embodiment 3

[0136] Embodiment 3, the acquisition of transgenic Arabidopsis thaliana and the functional analysis of GmHAD1 gene

[0137] 1. Obtaining the transgenic Arabidopsis thaliana

[0138] 1. Transform the recombinant expression vector pTF101.1-GmHAD1 in Example 2 into GV3101 (purchased from Guangzhou Chunni Biotechnology Co., Ltd., item number AC1001S) by freeze-thaw method to obtain the Agrobacterium containing pTF101.1-GmHAD1, and use for infecting Arabidopsis plants.

[0139] 2. Agrobacterium containing pTF101.1-GmHAD1 was used to infect wild-type Arabidopsis thaliana (Columbian ecotype col-0) by flower dipping method. Harvest T 1 The surrogate seeds were planted on nutrient soil and sprayed with 10 mg / L glufosinate-ammonium (purchased from Yisheng Biotechnology Co., Ltd., product number 60221ES03) for screening. The next generation produced by the screened seedlings is screened again, and so on, and finally T 3 The homozygous line of Arabidopsis thaliana was transformed into...

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Abstract

The invention discloses a protein GmHAD1 related to plant stress tolerance and an encoding gene and application thereof. An amino acid sequence of the protein GmHAD1 related to plant stress toleranceis shown in a second sequence, and an encoding gene sequence of the protein GmHAD1 related to plant stress tolerance is shown in a first sequence. It is proved through experiments that the GmHAD1 geneis overexpressed in soybean hairy roots and arabidopsis thaliana, and the low-phosphorus tolerance of transgenic plants can be improved. The gene can be introduced into plants as a target gene to improve the low-phosphorus tolerance of the plants, have important significance to the cultivation of low-phosphorus-tolerant soybean varieties, and lay a foundation for the research on the cultivation of the transgenic plants with high low-phosphorus tolerance.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a protein GmHAD1 related to plant stress tolerance, its coding gene and its application. Background technique [0002] Soybean is an important economic crop, which is widely grown in the world and is the main source of human edible oil and vegetable protein. However, low-phosphorus soil severely limits the growth and development of soybeans, and also causes flower pods to fall off, etc., and ultimately affects the yield and quality of soybeans. During the long-term evolution of plants, a series of low phosphorus tolerance mechanisms have been formed. One of them is that plants secrete acid phosphatase to hydrolyze organic phosphorus in soil into inorganic phosphorus for plant uptake and utilization. Therefore, the research on the regulation of acid phosphatase-related genes in soybean is of great significance to the improvement of soybean resistance to lo...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/82A01H5/00A01H5/06A01H6/54A01H6/20
CPCC12N9/16C12N15/8271C12Y301/03002
Inventor 蔡占东年海程艳波马启彬牟英辉连腾祥
Owner SOUTH CHINA AGRI UNIV
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