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 pea

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

Example Embodiment

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

[0097] By screening the whole genome cDNA library of pear, a gene PbrKAT1, which regulates the potassium ion absorption channel of pear guard cells, and the 2000bp upstream of PbrKAT1 gene were cloned as a promoter. According to the characteristics of homologous recombination expression vector recombination and the PbrKAT1 gene and its promoter For the sequence, primers were designed with Primer premier 5.0, and full-length PCR was carried out using cDNA and DNA from the leaves of'Dangshan Su Pear' as templates. The detailed steps are as follows:

[0098] The research material Dangshan Su pear was planted in the National Pear Engineering Center of Nanjing Agricultural University, and its seedling age was 3 to 5 years. Select the young leaves of Dangshan crisp pear that are vigorously growing and free from diseases and insect pests, weigh 500 mg samples at random, and quickly freeze them with liquid...

Example Embodiment

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

[0102] In order to analyze the expression pattern of PbrKAT1 gene in Dangshan Su pear in different tissues of pear, the expression pattern of PbrKAT1 gene was analyzed using Real-time PCR technology. According to the coding region sequence of the PbrKAT1 gene, the primer primier 5.0 software was used to design the upstream and downstream PCR primers to amplify the entire coding region of the gene according to the general principle of primer design. The RNA was extracted with the kit, 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 20μL reaction system includes: 10μL SYBR Green, 5μL sterile ultrapure water, 1μL cDNA, 2μL forward primer, F3:5'-GATCTAGCACAACAGCAAGG-3'(SEQ ID No.7), 2μL reverse primer, R3:5 '-GATACAGGTGATCACC...

Example Embodiment

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

[0106] 1. Plant transformation vector construction

[0107] According to the characteristics of homologous recombination and the sequence of the promoter of the PbrKAT1 gene (2000bp upstream of the PbrKAT1 gene), the pear leaf DNA was used as a template for cloning. After the PCR product was electrophoresed on a 1% agarose gel, the target band was recovered using a gel kit. The purified amplified fragment was recovered and recombined with the expression vector, and transformed into the competent E. coli DH5α by the heat shock method. The transformed bacterial solution is tested by PCR, and the bacterial solution that is positive by PCR is sent for sequencing. The correct recombinant target 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 Sambrook, translated by Huang Peitang...

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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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