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A Tanggut white thorns NTCIPK9 gene and its expression protein and application
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A gene and Nitraria technology applied in the field of plant genetic engineering to increase resources and improve salt tolerance
Active Publication Date: 2017-03-01
NANJING FORESTRY UNIV
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In addition, so far, no similar genes have been found in species other than plants, so CIPK Gene is unique to plants for Ca 2+ Ser / Thr-like phosphoprotein kinase genes in signal pathway
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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:
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Abstract
The invention discloses a nitraria tangutorum CBL-interacting protein kinase 9 (NtCIPK9) gene, expressed protein of the NtCIPK9 gene and application of the NtCIPK9 gene. The nucleotide sequence of the NtCIPK9 gene is shown as the SEQ ID NO.1, and the amino acid sequence of the NtCIPK9 gene is shown as the SEQ ID NO.2. According to the NtCIPK9 gene, a full-length gene related to stress resistance of nitraria tangutorum is cloned in a homological mode by using leaf blade tissue of the nitraria tangutorum on the basis of a part of existing transcriptome data according to the stress resistance characteristic of the nitraria tangutorum, and is named NtCIPK9 according to a homologous gene in arabidopsis. The function of plant salt tolerance of the NtCIPK9 gene is proved through analysis of salt tolerance of an NtCIPK9 gene homozygous arabidopsis plant, and resources are increased for a plant stress resistance gene pool.
Description
technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a Nitraria tangutata NtCIPK9 Genes and their expressed proteins and applications. Background technique [0002] Nitraria tangutica ( Nitraria tangutorum ) belongs to the genus Nitraria (Zygophyllaceae) of the family Zygophyllaceae ( Nitraria ), a strong xerophytic deciduous shrub, is a unique Nitraria species in my country, mainly distributed in the northwestern region of China. Nitraria tangut is resistant to drought, salt and alkali, wind and sand, and barrenness. It can improve saline-alkali soil, increase soil fertility, prevent wind and sand, maintain oasis, and protect ecological balance. Its berry-shaped drupe is rich in vitamin C, flavonoids, protein, 19 kinds of amino acids and 21 kinds of trace elements, and has edible value. In addition, its fruit also has medicinal value such as invigorating the spleen and stomach, lowering blood fat, lowering ...
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