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
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[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...
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[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...
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[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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