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| Expression Pattern and Stress Response Elements Analysis of CDPK12 Gene Promoter in Rice (Oryza sativa ssp. japonica) |
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Abstract As is known to all, rice (Oryza sativa) is one of the most important food crops to human beings, but a variety of adverse environmental stress severely affect the growth and development and limits the production of rice. Further study of the structure, function and expression pattern of plant promoter have great significance for further exploring the adaptation mechanism of plants to its growing environment and creating stress-resistance improved transgenic rice. Calcium-dependent and calmodulin-independent protein kinases (CDPKs) are a Ser/Thr protein kinase family which exist only in the plant and parts of protista, and play a very important role in mediating the signal transductions of growth and development, and the abiotic stresses. The research mainly analyzed the expression pattern and cis regulatory elements of OsCDPK12 gene promoter which named 12P. Firstly, 12P with 2 109 bp in length of upstream regions was cloned by PCR from the genomic DNA of rice Nipponbare (Oryza sativa ssp. japonica) and transformed into rice zhonghua11 (Oryza sativa ssp. japonica) after fused with the report gene β-glucuronidas (GUS). The histochemical staining results showed that GUS gene driven by the promoter of 12P mainly express in root-stem transition zone, stem node, anther and seed. Subsequently, eight different length of 12P 5' deletions were fused to GUS gene and transformed into rice zhonghua11, respectively. The result of deletion analysis indicated that GUS gene driven by the deletions of 12P1975, 12P1828, 12P1570, 12P1351, 12P1194, 12P966 and 12P472 expressed in root-stem transition zone, tender panicle, branch, stem node and mature seed, respectively, but not in leaf, sheath and root. 12P687 drived GUS gene expressed in all the tissues above, and showed a constitutive expression pattern. The histochemical staining results of different deleting promoters transgenic plants showed that there are several important negatively regulatory elements locate at the fragments from -966 bp to -687 bp, several positive regulatory elements locate at the fragments from -687 bp to -472 bp. Quantitative analysis of GUS activity showed that GUS activity of the 12P687 promoter in the tissue of leaf and leaf sheath was about half of the 35S promoter, and 12P687 GUS activity in the root was about 1/3 of the 35S promoter. Furthermore, the different deleting promoters transgenic plants undertook low temperature, low nitrogen and high salt stress treatment, respectively, the result showed that GUS gene in12P1828 transgenic rice plants was up-regulated expressed in the leaf after low temperature treatment, while, in 12P687 transgenic rice plants it's down-regulated in the root after salt stress treatment. These results identified the expression characteristics of 12P which can drive downstream target genes expressed and some potentially important regulatory element that existing, and also verified the expression of OsCDPK12 gene is regulated by low temperature and high salt stress preliminarily, so it will be conducive to provide reference for further work of cultivating stress resistant transgenic rice and researching the function of gene.
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Received: 21 April 2015
Published: 24 July 2015
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