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Pear guard cell potassium ion absorption channel gene PbrKAT1 and application thereof

A technology of guard cells and potassium ions, which is applied in the application field of regulating the potassium ion absorption capacity of plant guard cells, can solve problems such as no reports and no determination of potassium ion effects, and achieves improving agricultural economic benefits, reducing agricultural production costs, and improving Effects of Plant Salt Tolerance

Pending Publication Date: 2020-07-14
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genes related to potassium ion uptake and transport have not been identified in pear and the role of potassium channel genes in pear in plant salt tolerance has not been reported.

Method used

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  • Pear guard cell potassium ion absorption channel gene PbrKAT1 and application thereof
  • Pear guard cell potassium ion absorption channel gene PbrKAT1 and application thereof
  • Pear guard cell potassium ion absorption channel gene PbrKAT1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096]Cloning of embodiment 1 pear PbrKAT1 gene full-length cDNA and promoter

[0097] A pear guard cell potassium uptake channel gene PbrKAT1 was screened by screening the pear whole genome cDNA library, and the 2000 bp upstream of the PbrKAT1 gene was cloned as a promoter. For the sequence, Primer Premier 5.0 was used to design primers, and the full-length PCR amplification was carried out using the cDNA and DNA of the leaves of 'Dangshan Suli' as templates. The detailed steps are as follows:

[0098] The research material Dangshansu pear is planted in the National Pear Engineering Center of Nanjing Agricultural University, and its seedling age is 3 to 5 years. Select young leaves of Dangshansu pears with vigorous growth and no pests and diseases, weigh 500 mg samples at random, and immediately freeze them with liquid nitrogen. Total RNA and DNA were extracted by the CTAB method. RNA-free blue tips, yellow tips, white tips and 1.5ml centrifuge tubes were prepared before th...

Embodiment 2

[0101] Example 2 qRT-PCR analysis of the potassium ion uptake channel gene PbrKAT1 in pear guard cells in different tissues of Dangshansu pear

[0102] In order to analyze the expression pattern of PbrKAT1 gene in different pear tissues in Dangshansu pear, the expression pattern of PbrKAT1 gene was analyzed by Real-time PCR technology. According to the sequence of the coding region of the PbrKAT1 gene, the primer primer 5.0 software was used to design the upstream and downstream PCR primers for amplifying the entire coding region of the gene according to the general principle of primer design. The kit was used to extract RNA, and the synthesis of the first strand of cDNA was carried out according to the operation manual of Thermo Scientific RevertAid FirstStrand cDNA Synthesis Kit. The 20 μL reaction system includes: 10 μL SYBR Green, 5 μL sterilized ultrapure water, 1 μL cDNA, 2 μL forward primer, F3: 5'-GATCTAGCACAACAGCAAGG-3' (SEQ ID No.7), 2 μL reverse primer, R3: 5 '-GAT...

Embodiment 3

[0105] Example 3 Pear regulates the tissue localization of the potassium ion uptake gene PbrKAT1

[0106] 1. Plant transformation vector construction

[0107] According to the characteristics of homologous recombination and the sequence of PbrKAT1 gene promoter (2000bp upstream of PbrKAT1 gene), pear leaf DNA was used as template for cloning. After the PCR product was subjected to 1% agarose gel electrophoresis, the target band was recovered using a gel kit. The recovered and purified amplified fragment was recombined with the expression vector, and transformed into Escherichia coli competent DH5α by heat shock method. The transformed bacterial solution was tested by PCR, and the positive bacterial solution identified by PCR was sent for sequencing. The correct recombinant destination vector was named PbrKAT1-pB35S-GFPXB-4. The recombinant vector PbrKAT1-pB35S-GFPXB-4 was introduced into Agrobacterium GV3101 by freeze-thaw method (refer to Sam Brook, translated by Huang Pei...

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Abstract

The invention discloses a pear guard cell potassium ion absorption channel gene PbrKAT1 and application thereof. The gene is a DNA molecule with a nucleotide sequence shown in SEQ ID No.1, or has homology of 85% or greater with SEQ ID No.1, and is a DNA molecule for encoding related proteins for regulating potassium ion absorption and transferring and resisting salts in plant guard cells. Electrophysiological functions verify that the pear guard cell potassium ion absorption channel gene PbrKAT1 disclosed by the invention has the properties of absorbing and transferring potassium ions and being inhibited by extracellular sodium ions. The PbrKAT1 gene disclosed by the invention aims to regulate and control absorption and transferring of the potassium ions of plants, regulate and control movement of pore guard cells and improve salt resistance of the plants, and is beneficial to research on salt resistance mechanisms of the plants, so that the agricultural production cost can be reduced,and the agricultural economic benefits can be increased.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to the construction of pear guard cell potassium ion absorption channel gene PbrKAT1 and its recombinant expression vector and its application in regulating the potassium ion absorption capacity of plant guard cells. Background technique [0002] Potassium ion is the most abundant cation in plant cells. It is not only an important nutrient for plants, but also participates in the regulation of basic physiological characteristics in the process of plant growth and development, such as regulating the turgor of guard cells, thereby controlling cell expansion and guard cell movement (Szczerba et al. 2009). In the past ten years, the molecular mechanism of potassium ion uptake and transport in plant roots has been intensively studied. Therefore, many potassium ion channel genes have been identified at the molecular level ( et al. 2001). Among them, the Shaker family o...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H5/00A01H6/74
CPCC07K14/415C12N15/8273C12N15/8243
Inventor 吴巨友陈国栋王莉张绍铃齐开杰谢智华殷豪谷超王鹏黄智
Owner NANJING AGRICULTURAL UNIVERSITY
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