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Sodium and hydrogen reverse transport protein PbrNHX2 in birch-leaf pears and application thereof in enhancement of salt resistance of plants

A technology of antiporter and Duli, applied in the field of genetic engineering, can solve the problems of complex genetic background, long childhood, self-incompatibility, etc.

Active Publication Date: 2018-07-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of the existence of pear breeding: complex genetic background, long childhood, self-incompatibility, uncertain breeding goals, etc., the conventional breeding methods have been far from meeting the needs of modern pear varieties.

Method used

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  • Sodium and hydrogen reverse transport protein PbrNHX2 in birch-leaf pears and application thereof in enhancement of salt resistance of plants
  • Sodium and hydrogen reverse transport protein PbrNHX2 in birch-leaf pears and application thereof in enhancement of salt resistance of plants
  • Sodium and hydrogen reverse transport protein PbrNHX2 in birch-leaf pears and application thereof in enhancement of salt resistance of plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Cloning of full-length cDNA of PbNHX2 gene in Du pear

[0049] In the early stage, our research group used a high-efficiency yeast expression system to screen a sodium-hydrogen antiporter PbrNHX2 from Duli pear. According to the sequence of the PbrNHX2 gene and primer premier 5.0, primers were designed, and its full length was amplified from Duli pear by RT-PCR. long. The detailed steps are as follows: take 1 μg of Du pear RNA, treat it with 1U of DNase I at 37°C for 30 minutes, and immediately place it on ice, add 1 μl of 50mM EDTA, treat it at 65°C for 10 minutes, and immediately place it on ice. The synthesis of the first strand of cDNA was carried out according to the operation manual of the TOYOBO reverse transcription kit. The resulting first-strand cDNA was used for amplification of the PbrNHX2 gene. PCR was completed according to the following procedure: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 90 s, extension at...

Embodiment 2

[0053] qRT-PCR analysis of PbrNHX2 gene and subcellular localization of PbrNHX2 gene under different stress conditions

[0054] In order to analyze the response pattern of PbrNHX2 gene to low temperature, high salt and dehydration in Duli pear, the expression pattern of PbrNHX2 gene was analyzed using Real-time PCR technology. The RNA was extracted by the CTAB method, and the synthesis of the first strand of DNA was carried out according to the operation manual of the TOYOBO reverse transcription kit. In the 20 μl reaction system, there are: 10 μl 2×Mix, 0.1 μl cDNA, 5 μl primers (using ubiqutin as internal reference primers (SEQ ID NO.5 and SEQ ID NO.6), the length is 208), 4.9 μl water. The program of quantitative PCR is shown in Table 1:

[0055] Table 1 Quantitative PCR program

[0056]

[0057] The results are shown in Figure 2. Fig. 2 is a schematic representation of expression of a PbNHX2 encoding gene of the present invention under salt, dehydration and low tempe...

Embodiment 3

[0059] Subcellular localization of the gene encoding PbrNHX2

[0060] According to the nucleotide sequence of the coding gene of PbrNHX2 and the pJIT166-GFP vector map, SalI and BamHI restriction sites were added before and after the gene sequence. The target gene extraction plasmid with correct sequencing results was used as a template, and amplified with primers (SEQ ID NO.7 and SEQ ID NO.8) with added restriction sites. The PCR program used was: 94°C pre-denaturation for 3 minutes; 94°C denaturation 30s, 58°C annealing for 1min, 72°C extension for 1min 30s, 35 cycles; 72°C extension for 10min. The stop codon TAG was removed at the 3' of the gene in order to allow the gene to be fused to GFP. After the PCR products were subjected to 1% agarose gel electrophoresis, the target band was recovered using a gel kit. The recovered and purified amplified fragment was cloned into pMD19-T vector, and transformed into Escherichia coli competent DH5α. The transformed bacterial liquid...

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Abstract

The invention provides sodium and hydrogen reverse transport protein PbrNHX2 in birch-leaf pears and application thereof in enhancement of salt resistance of plants, and belongs to the technical fieldof gene engineering. The invention provides sodium and hydrogen reverse transport protein in birch-leaf pears, a coding gene, a primer pair for cloning the cDNA sequence of the coding gene, and the application of the sodium and hydrogen reverse transport protein in birch-leaf pears, the coding gene or the coding gene cloned by the primer pair in enhancement of salt resistance of plants. A tobaccotransformation vector and an ussurian pear transformation vector are constructed, and obtained positive plant seedlings are subjected to salt treatment; and the results show that compared with wild type plant seedlings, the positive plant seedlings have the advantages that the conductivity is obviously reduced, the chlorophyll content is obviously increased, the survival rate is obviously increased, and the anti-ROS capability is better. The over-expressed coding gene can effectively enhance the active oxygen scavenging capability of transgenic plants, thereby enhancing the salt resistance ofthe plants.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the sodium and hydrogen antiporter PbrNHX2 in pear pear and its application in improving the salt tolerance of plants. Background technique [0002] Pear is one of the main fruit tree species planted in the world. The distribution of pear trees in China is very extensive. The layout of the main pear producing areas is "three regions and four points". There are pear trees planted from Northeast to Guangxi, and from Yunnan to Shandong. The layout and planning of the dominant pear producing areas have greatly promoted the development of my country's pear industry, but precisely because of the wide distribution, they are easily affected by various environmental factors, such as drought, waterlogging, salinity and so on. Therefore, whether can breed good stress-resistant new varieties has just become the most crucial factor of pear industry development. Becau...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/82A01H5/00A01H6/82A01H6/74
CPCC07K14/415C12N15/8273
Inventor 黄小三张绍铃邢才华刘月赵梁怡胡轼李凌高俊芝姚征宏
Owner NANJING AGRICULTURAL UNIVERSITY
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