New anti-drought and cold-resistant gene CaDHN1 in caragana korshinskii
A caragana and gene technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of destroying free radical metabolism of plant cells, increasing active oxygen free radicals, and cell membrane system damage
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Embodiment 1
[0009] Embodiment 1, the construction of twisted bar leaf full-length cDNA library
[0010] The caragana plant was cultivated in a cultivation pot, cultivated in a greenhouse for 60 days, and then subjected to drought treatment until the whole plant wilted. The young leaves were quick-frozen in liquid nitrogen, ground, and total RNA was extracted with TRIzole and dissolved in water. The mRNA was processed by the mRNA purification kit. The mRNA is taken for reverse transcription, and then the second strand is synthesized. Ligate with pDNR-LIB vector after homogenization (see Creator for details TM SMART TM cDNA Library Construction Kit (Clontech Cat. No. 634903) and transform Escherichia coli. Single clones were obtained by smearing the plates and stored separately.
Embodiment 2
[0011] Example 2. Random EST sequencing of cDNA library: 3000 single clones were randomly selected from the library for 5'-end EST sequencing, and the successfully sequenced sequences were subjected to BLAST to select ESTs similar to verified drought-resistance-related genes.
Embodiment 3
[0012] Example 3 Select ESTs similar to dehydrin by BLAST, and pass through their corresponding clones to obtain the full-length DNA sequence of the candidate stress resistance gene. If the full-length sequence cannot be obtained, use the EST to design primers, and obtain the full-length sequence by 5' or 3' RACE.
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