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| Cloning and Expression Analysis of DoCDPK6 and Promoter in Dendrobium officinale |
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Abstract Calcium-dependent protein kinase (CDPK) is a kind of protein kinase that can respond to abiotic stresses. Over the past few years, many CDPK genes have been isolated from Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. To study the structure, function and expression patterns of CDPK genes in Dendrobium officinale, DoCDPK6 (GenBank No. KY682702) and its promoter were cloned from D. officinale, sequence characteristics, structure and evolutionary relationships were analysed by bioinformatics, which showed that the length of cDNA sequence of DoCDPK6 were 1 976 bp, with 1 725 bp open reading frame which encoded 574 amino acids. The molecular formula was C2845H4500N766U858S24 and the molecular weight is 63.9 kD. Structural analysis showed that DoCDPK6 gene had 4 typical conserved structure of CDPK genes: variable domain, protein kinase domain, autoinhibitory domain and calmodulin-like region. The phylogenetic analysis showed that it is most relative to Phalaenopsis amabilis, and in accordance with botanical classification status, which illustrated that DoCDPK6 gene was conservative in evolution. The core promoter elements of TAAT-box and CAAT-box were found in the 1 500 bp upstream sequence of DoCDPK6 gene, and adverse signal related response elements like light, temperature, salicylic acid, abscisic acid (ABA), auxin, gibberellic acid (GA) and so on, which was speculated that it could participate in the regulation of DoCDPK6 response to stress. By qRT-PCR the expression of DoCDPK6 could be detected in roots, stems, leaves of D. officinale, the most in leaves, followed by stems and roots, which showed the expression of DoCDPK6 has tissue specificity. It was found that the expression of DoCDPK6 were up to varying degrees, through the low temperature of 4 ℃, ABA and NaCl treatment. Under 4 ℃ treatment the expression of DoCDPK6 increased and reached the peak at 4 h, and had 3.5 times as much as 0 h. Then as time went on it decreased, the expression at 24 h returned to the level as much as 0 h, that low temperature induces the expression of DoCDPK6 can inferred .Under the ABA treatment the expression of DoCDPK6 was up-regulated in all times periods and reached the peak at 12 h and was 4 times as much as the level at 0 h, which showed the expression was regulated by ABA of high concentration. Under NaCl treatment the expression of DoCDPK6 increased to the highest level at 4 h, 4.9 times as much as 0 h, after that the expression decreased but also significantly higher than 0 h, which indicated that the expression was regulated by NaCl of high concentration. CDPK is a kind of Ser/Thr protein kinase response to the second messenger of Ca2+ and plays an important role in plant growth, adversity stresses and cell signaling. The study indeed proves DoCDPK6 can response to stresses such as low temperature, NaCl and ABA, it is speculated that DoCDPK6 plays a role in stresses, which provides information to the regulatory mechanism and pathway of signal transduction of DoCDPK6 gene and its promoter in abiotic stress in the next study.
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Received: 10 November 2016
Published: 31 March 2017
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ZHAO Zhi-Dong, TIAN Hong-Shan, JIANG Yan-Yan, SHI Bin-Gang, LIU Xiu, LI Xu-Peng, WANG Deng-Zhe, CHEN Jin-Lin, HU Jiang. Polymorphisms of ACSL1 Gene Promoter and Their Association Analysis with Milk Quality Traits in Yak (Bos grunniens)[J]. 农业生物技术学报, 2019, 27(9): 1596-1603. |
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