A Tanggut white thorns NTCIPK9 gene and its expression protein and application
A gene and Nitraria technology applied in the field of plant genetic engineering to increase resources and improve salt tolerance
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Embodiment 1
[0027] Using the leaves of Nitraria tangutica as material, total RNA was extracted and reversed into cDNA, and corresponding primers were designed for PCR. After agarose gel electrophoresis, the target band was recovered, connected with pMD19-T vector, and transformed into E. coli. Sequence and analyze. After determining the target sequence, design RACE primers based on the sequence, obtain 5' and 3' sequences by PCR, and after sequencing analysis, splicing to obtain NtCIPK9 full-length sequence. Primers were designed based on the full-length sequence, the full-length fragment was obtained by PCR, connected to the pMD19-T vector, transformed into E. coli, sequenced and analyzed again, and after the full-length fragment was confirmed, positive clones were picked for plasmid extraction, and enzyme cutting sites were added After spotting, it was simultaneously double-digested with the vector pBI121, ligated under the action of T4 ligase, and then transformed into Agrobacterium E...
Embodiment 2
[0063] Example 2 Gene function verification
[0064] Build 35S: CIPK9 Expression vector, transformed into wild-type Arabidopsis thaliana, to observe Nitraria tangutica NtCIPK9 Whether the gene can enhance the salt tolerance of Arabidopsis, compare the phenotypic differences between transgenic Arabidopsis and wild-type Arabidopsis, and speculate that Nitraria tangutata CIPK9 The function of the gene.
[0065] 1) Construction of the carrier
[0066] The Escherichia coli strain used in the present invention is E. coli JM109 (purchased from Biovector (Dalian) Co., Ltd.); the expression vector is pBI 121 (purchased from Biovector Co., LTD).
[0067] The specific process is as follows:
[0068] 1. By PCR to CIPK9 BamH I and SmaI restriction sites are added to the upstream and downstream of the gene fragment respectively. The PCR system and reaction conditions are the same as the full-length amplification. The primers are:
[0069] NtCIPK9F+BamH: 5'-CGCGGATCCATGAATAAGGTACCGG...
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