| Followed by the genome sequence complemented, functions of many genes have been reported one after another in rice. But there are few investigations about the role of rice DHHC-type zinc finger protein genes, and even most of them were not reported as yet. Therefore, a series of bioinformatic analysis and function researches on rice DHHC zinc finger protein gene were conducted in this project. The main results obtained are as follows:(1) In this project, we cloned a DHHC znic finger protein gene OsDHHC1 based on homologous blast. Method using amino acid sequence homology screening, we found that rice DHHC znic finger protein Os02g0819100 with about 800bp CDS shared high homology with At5g04270, which involved in regulating shoot branchs in Arabidopsis. When we cloned Os02g0819100 by RT-PCR, we found two bands in the gel, the long one with about 800 bp and the short one with about 700 bp, importantly, the short band was very bright but the long band was very dim. The sequence result showed that the bright band come from the Os02g0819100 gene which removes 147bp between 112-259 sites of the CDS. At the same time, amino acid sequence deduced from the short band has a complete open reading frame and can be able to fully red. Moreover, it is a new gene which has not been recorded in the NCBI database, and we named it as OsDHHC1. So the result suggested that OsDHHC1 is the alterative-splicing mode of Os02g0819100 in rice.By using the bioinformatics methods the cDNA sequence and amino acid sequence of OsDHHC1 and Os02g0819100 were analysed. By using the method of homology modeling the three-dimensional structure models of OsDHHC1 and Os02g0819100 were built. Bioinformatics analysis showed that the two proteins are membrane proteins. OsDHHC1 had three major transmembrane structures and a secondary transmembrane structure, but Os02g0819100 had four major transmembrane structures and a secondary transmembrane structure. The two proteins contain three sulfur acyltransferase motifs: DPG, DHHC-CRD and TTxE. More significantly, the predicted result showed the two proteins had the existence of active sulfur acyltransferase activity center, which implied that the two proteins should have the sulfur acyltransferase activity.(2) Temporal and spatial expression and subcellular localization of OsDHHC1 gene in rice were clarified, and what role it plays in the resposnses to stresses was also elucidated. The result indicated that OsDHHC1 is the other alterative-splicing mode of Os02g0819100 in rice, but OsDHHC1 plays the key role in rice plants. Bioinformatics analysis showed that the promoter of the gene had many cis acting elements related stress such as light, cold, hot, drought, salt and ABA. Q-PCR analysis revealed that the expression of the gene increased 45 times after ABA treatment compared with no treatment, which indicated that ABA stress could induce OsDHHC1's transcription. GFP fusion protein in onion epidermal cell transient expression analysis showed OsDHHC1 and Os02g0819100 protein localized in the plasmid membrane. The results of pOsDHHC1::GUS showed that the cloned 1639bp sequence in the upstream of OsDHHC1 gene possessed a complete promoter activity and specificity of temporal and spatial gene expression patterns, which controled the high level expression of OsDHHC1 in embryo and veins.(3) The sulfur acyltransferase activity of OsDHHC1 and Os02g0819100 was demonstrated by two ways of recombinant proteins in vitro and yeast morphology. By using of pColdTF vector, the recombinant prokaryotic expression vector pColdTF-OsDHHC1 and pColdTF-Os02g0819100 were successfully constructed and two purified soluble recombinant proteins were got. With biotin (biotin-HPDP) labeling, we confirmed the existence of S-acyltransferase activity in the two proteins. Furthermore, by using PYES263-OsDHHC1 and PYES263-Os02g0819100 to transform the aim gene into sulfur acyltransferase-deleted yeast strain ak1â–³mutant, we improved the S-acyltransferase activity by investigating the phenotype changes of transgenic yeast cell. All these results indicated that OsDHHC1 and Os02g0819100 had sulfur acyltransferase activity.(4) By constructing over-expression vector pCAMBIA1301-OsDHHC1, pCAMBIA1301-Os02g0819100 and the interference vector OsDHHC1-RNAi18 to transform rice Zhonghua 11, stable transgenic lines were generated through Agrobacterium-mediated transformation. Homozygous of overexpressed OsDHHC1 transgenic plants displayed that the number of tillers increased 23%-31% and seed yield increased by 9.6% per acre compared with wild type. However, there's no a noticeable change in the number of tillers and seed yield in overexpressed Os02g0819100 or RNAi transgenic plants. In addition, we constructed PEGAD- OsDHHC1 expression vectors and transformed it into Arabidopsis, and transgenic homozygous Arabidopsis were consistent with the above in the phenotype of tillers and seed yield. So it seemed that OsDHHC1 had the function to augment crop production through increasing tillering (branch) and controlling ideal plant architecture.(5) We have illustrated that the function domain of OsDHHC1 is the motif which contains DHHC zinc finger fragment. In order to confirm its function domain, OsDHHC1 gene was divided into three sections: OsDHHC1A, OsDHHC1B and OsDHHC1C, and then the three fragments accompanied with OsDHHC1 were cloned into the expression vector PEGAD and transformed into Arabidopsis, respectively. The phenotype showed that only OsDHHC1A homozygous transgenic plants had a 3-6 folds increase in the numbers of branches compared with wild-type, which was very similar with the transgenic Arabidopsis overexpressing the full-length CDS of OsDHHC1. This result indicated that the motif which contains DHHC zinc finger fragment was OsDHHC1's function domain.In conclusion, we first cloned a novel of DHHC type zinc finger protein gene OsDHHC1 made a systematic study on its function in this project. Firstly, OsDHHC1 was the other alternative splicing mode of Os02g0819100 gene in rice, importantly, OsDHHC1 played the key role in regulating number of tillers, constructing ideal architecture and increasing seed yield in rice. Secondly, OsDHHC1 had high expression level in the embryo and veins in rice and its transcription was induced by ABA. Thirdly, analysis of recombinant proteins in vitro and yeast morphology indicated OsDHHC1 had sulfur acyltransferase activity. Finally, the different phenotypes of three types of transgenic Arabidopsis which overexpressed three different truncated fragments of OsDHHC1 revealed that the function domain of OsDHHC1 is the motif which contains DHHC zinc finger fragment. Therefore, researches on the function of OsDHHC1 gene in rice may provide the theory basis and guidance for further constructing the ideal architecture to increase crop yields. |
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