Promoter Analysis Of Gene OsCDPK12 And Research Of Protein Interactions Between CDPKs And GRXs In Rice

CDPKs(calcium-dependent and calmodulin-independent protein kinases)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. Thirty one CDPK genes have been found in rice so far, however only few of them have been well characterized, the most other genes needs further research.This research aimed at two genes: Os CDPK12 and Os CDPK24. Main research results of this study are as follows:1. Promoter analysis of the gene Os CDPK121.1 The Os CDPK12 promoter which named 12 P with 2109 bp in length of upstream regions was cloned by PCR from the genomic DNA of rice Nipponbare(Oryza sativa L ssp. Japonica) and transformed into rice zhonghua11(Oryza sativa L ssp. Japonica) after fused with the report gene β-glucuronidas(GUS). The histochemical staining results showed that GUS gene driven by the promoter of 12 P mainly express in root-stem transition zone, stem node, anther and seed. Subsequently, eight different length of 12 P 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 express in root-stem transition zone, tender panicle, branch, stem node and mature seed, respectively, but not in leaf, sheath and root. 12P687 drive GUS gene expressed in all the tissues above, and show a constitutive expression pattern.1.2 Analysis the histochemical staining results of different deleting promoters transgenic plants found that there were several important negatively regulatory elements locate at the fragments between-966 bp to-687 bp, several positive regulatory elements locate at the fragments between-687 bp to-472 bp.1.3 Quantitative analysis of GUS activity showed that GUS activity of the 12P687 promoter in the tissue of leaf and leaf sheath is about half of the 35 S promoter, and 12P687 GUS activity in the root is about 1/3 of the 35 S promoter.1.4 The different deleting promoters transgenic plants were analysised also undertook low temperature, low nitrogen and high salt stress treatment, respectively, the results 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 was down-regulated in the root after salt stress treatment.2. Research of protein interactions between CDPKs and GRXs in rice2.1 22 of GRXs gene in rice were cloned, respectively: OsGRX3、OsGRX4、OsGRX5、Os GRX6、Os GRX8、Os GRX9、Os GRX11、Os GRX12、Os GRX14、Os GRX15、Os GRX16、Os GRX17、Os GRX19、Os GRX20、Os GRX21、Os GRX22、Os GRX24、Os GRX25、Os GRX26、Os GRX27、Os GRX28、Os GRX29, and fused with the AD carrier, then obtained 22 recombinant vectors. The self-activation detection found that they all have no potential activation of transcription activity.2.2 Identified the protein interaction between Os CDPK24 and Os GRX family indicated that Os GRX8, Os GRX16, Os GRX26, Os GRX28 protein respectively interact with full length protein Os CDPK24-1, while Os GRX8, Os GRX14, Os GRX16, Os GRX26, Os GRX28 protein respectively interact with Os CDPK24-2 truncated protein.2.3 Full length and truncated sequences of OsCDPK2、OsCDPK20、OsCDPK21、Os CDPK23、Os CDPK25 and Os CDPK29 were cloned, respectively, and fused with the BD carrier, then obtained the recombinant vectors. The self-activation detection found that with the exception of Os CDPK23-1, the others all have no potential activation of transcription activity.2.4 Identified the protein interaction between CDPKs and GRXs in rice indicated that CDPKs proteins(included Os CDPK4, Os CDPK12, Os CDPK20, Os CDPK21, Os CDPK25 and Os CDPK29) all have no interactions with GRXs proteins.2.5 Multiple sequence alignment of Os GRX8, Os GRX14, Os GRX16, Os GRX26 and Os GRX28 amino acid sequences by tools of DNAMAN software, the result showed that these sequences have no obvious conserved sequence; However, multiple sequence alignment of Os GRX8, Os GRX26 and Os GRX28 these three amino acid sequences, found that there exist obvious cconserved sequences.