Plant salt stress tolerant protein and encoding gene and application thereof
A plant stress tolerance and stress tolerance technology, applied in the direction of plant genetic improvement, application, plant peptides, etc., can solve problems such as limited economic value, and achieve the effect of improving salt tolerance and drought resistance, and high application value.
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Embodiment 1
[0031] Embodiment 1, the acquisition of plant stress tolerance protein and its coding gene
[0032] The ESTs obtained from the cDNA library constructed from the leaves of soybean variety Suinong 14 were analyzed, and an osmotic stress-related EST was found. The tblast program was used to search the GenBank database, and a pair of primers were designed. A cDNA of a plant stress tolerance protein was cloned from leaves of Feng 7 (unified number: ZDD02611; provided by China National Germplasm Bank). The specific method is:
[0033] Soak the roots of soybean variety Wenfeng 7 seedlings grown for 2 weeks after germination with 200mM NaCl solution for 6 hours, harvest the leaves, extract RNA and reverse transcribe it into the first strand of cDNA. Using the cDNA as a template, primer PK1F(5' -TGCTACGCACACACAAAACACA-3′) and PK1R
[0034] (5'-TATGTCCCATGCGGAAAACTT-3') for PCR amplification.
[0035] The PCR reaction system is: 10×Buffer2.5μl, dNTP2.5mM, PK1F5μM, PK1R5μM, cDNA20ng, ...
Embodiment 2
[0038] Example 2, Functional Verification of Plant Stress Tolerance Protein and Its Encoding Gene
[0039] 1. Construction of overexpression vector in Arabidopsis
[0040] Clone the GmPK ORF into the pBI121 vector and place it under the control of the 35S promoter. The specific method is:
[0041] Primers were designed according to the above GmNHX2 cDNA sequence, the sequence is as follows: F1: 5'-CGC GTC GAC ATGGAGAAATACGAGCTCGT-3' (underlined is the Sal I enzyme recognition site); R1: 5'-CTG CTCGA GTTAACTGACATGAAATTCCC-3' (Xho I enzyme recognition site is underlined).
[0042]Soak the roots of soybean variety Wenfeng 7 seedlings grown for 2 weeks after germination in 200mM NaCl solution for 6 hours, extract RNA from the leaves and reverse transcribe to obtain the first-strand cDNA, using the cDNA as a template and using F1 and R1 as primers Perform PCR amplification.
[0043] A fragment of 1077bp was amplified by PCR, and sequencing showed that the fragment had the nuc...
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