Drought-resistant and salt-tolerant gene IpNY-B1 as well as encoding protein and application thereof
An ipny-b1, protein technology, applied in the field of plant genetic engineering and molecular biology, can solve problems to be developed and so on
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experiment example 1
[0023] Experimental Example 1: Cloning of the Austeng IpNY-B1 gene and construction of the pYES2-IpNY-B1 vector.
[0024] Specifically, the method comprises the following steps: three steps of total RNA extraction, cDNA library construction and gene cloning. The main steps are summarized as follows:
[0025] (1) Total RNA extraction
[0026] Take about 2g of the mixed sample of thick rattan root, leaves, rattan and leaf buds, add liquid nitrogen to grind thoroughly, and use the MAGEN RNA extraction kit to extract RNA according to the instructions. The obtained RNA was dissolved with 50 μl RNase-free water. The quality and concentration of RNA were determined with a Nanodrop micro-spectrophotometer.
[0027] (2) Construction of cDNA primary library
[0028] Use PromegaM-MLV Reverse Transcriptase Reverse Transcription Kit to synthesize the first strand of cDNA, take about 2 μg RNA, add 0.5 μg Oligo(dT)Primer and RNase-free H2O to a total volume of 15 μL; incubate at 70°C for...
experiment example 2
[0033] Experimental example 2: Overexpression of IpNY-B1 gene in yeast improves the salt tolerance and oxidative stress tolerance of yeast
[0034] (1) Yeast strain transformed with pYES2-IpNY-B1
[0035] The concentration of pYES2-IpNY-B1 recombinant plasmid was adjusted to 1 μg / μL, and the salt-sensitive yeast mutant strain AXT3 and the corresponding wild-type yeast strain (W303) were respectively transformed by lithium acetate transformation method. In addition, using the same method to convert to H 2 o 2 Sensitive yeast mutants skn7Δ and yap1Δ and corresponding wild-type yeast strains (WT). At the same time, the yeast expression vector empty vector pYES2 was used as a control to transform the above yeast strains respectively.
[0036] (2) Overexpression of IpNY-B1 in yeast salt-sensitive mutant strain AXT3 and wild-type strain W303 increased the resistance of transgenic yeast
[0037] Pick the single clones of yeast strain AXT3 transformed with the IpNY-B1 gene overexp...
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