【作者】 周波；

【导师】 刘选明；

【作者基本信息】 湖南大学 ， 分析化学， 2011， 博士

【摘要】 随着水稻基因组测序的完成，很多水稻基因的功能已经陆续被报道，但DHHC锌指蛋白基因在水稻中的具体功能还研究甚少，大部分基因功能未见报道。因，本文对水稻中DHHC型锌指蛋白开展了生物信息学分析及基因功能研究，获得如下主要研究结果：(1)采用同源对比的方式克隆了1个新的DHHC型锌指蛋基因OsDHHC1。通同源对比的方式找到了水稻中与调控拟南芥的分枝基因At5g04270编码的氨基序列高度相似的DHHC型锌指蛋白基因Os02g0819100,长度约800bp。采用RT-PCR技术克隆Os02g0819100基因时发现了一条大小约800bp的弱带和一条700bp左右的亮带，测序结果显示这条亮带是从Os02g0819100基因开放阅读框里掉112-259位点之间的147bp后剩余序列的拼接结果。它有完整的开放阅读框且够通读。但是该基因在NCBI数据库中没有收藏，说明这是我们克隆获得的一新基因，将其命名为OsDHHC1，并明确了OsDHHC1是Os02g0819100在水稻内的另一种剪接方式。利用生物信息学方法分析了OsDHHC1和Os02g0819100的核酸及其编码氨酸序列，利用同源建模方法构建了OsDHHC1和Os02g0819100蛋白的三维结构型。研究发现OsDHHC1和Os02g0819100都是膜蛋白，其中OsDHHC1存在3主要的跨膜结构域和一个次要的跨膜结构域，而Os02g0819100有四个主要的跨结构域和一个次要的跨膜结构域。2个蛋白都具有DPG、DHHC-CRD和TTxE 3S-酰基转移酶的motif和S-酰基转移酶的活性中心，预测该蛋白应该具有S-酰转移酶活性。(2)揭示了OsDHHC1基因的时空表达特性及其编码蛋白的亚细胞定位，初步察了OsDHHC1基因在胁迫应答中所发挥的作用。明确了OsDHHC1是Os02g0819100在水稻体内的另一种剪接方式，但主要以OsDHHC1的剪接方式在物体内发挥作用。分析发现该基因的启动子上存在很多与光、冷、热、旱、盐胁迫和ABA响应相关的顺式作用元件。Q-PCR结果显示ABA处理2周龄的幼后，该基因的2种剪接方式mRNA的总表达量相比处理前增加了45倍，说明基因的转录受ABA的诱导。GFP融合蛋白在洋葱表皮细胞中的瞬时表达分析显，OsDHHC1和Os02g0819100这2个蛋白都定位在细胞膜上。OsDHHC1::GUS验结果表明所克隆的OsDHHC1基因上游的1639bp的DNA片段具有完整的OsDHHC1基因的启动子活性，能够调控OsDHHC1基因的时空表达特异性，控制基因在胚和叶脉中高量表达。(3)利用体外重组蛋白和酵母形态观察2种方式证明了OsDHHC1和Os02g0819100蛋白都具有S-酰基转移酶活性。第一种方式是构建了载体pColdTF-OsDHHC1和pColdTF-Os02g0819100，并对这2个基因编码蛋白进行了大肠杆菌表达，纯化得到了大量可溶的重组蛋白，然后采用生物素(biotin-HPDP)标记法证明了2个蛋白具有S-酰基转移酶活性。另一种方式是构建了载体PYES263-OsDHHC1和PYES263-Os02g0819100，并转化S-酰基转移酶缺失的酵母菌株ak1△，通过酵母形态观察确定了这2个基因的S-酰基转移酶的活性。(4)构建了pCAMBIA1301-OsDHHC1，pCAMBIA1301-Os02g0819100过表达载体和OsDHHC1-RNAi18干扰载体，通过农杆菌浸染愈伤组织的方法转化水稻中花11。过表达的转化纯合子表型表明OsDHHC1转基因植株的分蘖数比野生型增加了23%-31%，亩产比野生型增加了9.6%，而Os02g0819100转基因植株没有较野生型在分蘖和产量上都没有明显的变化。干扰OsDHHC1-RNAi18植株的转化纯合子表型显示OsDHHC1表达量降低对分蘖数和产量没有明显的影响。同时我们构建了PEGAD-OsDHHC1的过表达载体转化拟南芥，纯合子表型与前述表型一致。这些结果说明OsDHHC1的主要功能是通过增加分蘖(枝)来构建合理株型从而增加作物的产量。(5)阐明了OsDHHC1的功能域是含有DHHC锌指结构域的motif。将OsDHHC1基因分成OsDHHC1A，OsDHHC1B和OsDHHC1C等3段，分别克隆到PEGAD表达载体上并转化拟南芥，纯合子表型显示包含有DHHC结构域的OsDHHC1A的转基因植株的分支数比野生型增加了3-6倍，与OsDHHC1的全长CDS过表达拟南芥的表型一致，由此可知该基因的功能域应该是含有DHHC结构域的OsDHHC1A片段。综上所述，本研究首次克隆了1个新的DHHC型锌指蛋白基因OsDHHC1，系统地研究了该基因的功能，并发现了它是Os02g0819100基因在水稻体内的另一种剪接方式，但主要以OsDHHC1这种剪接方式在水稻体内发挥促进水稻分蘖、构建合理株型以增加水稻产量的功能。明确了该基因在胚和叶脉里高量表达，且受ABA诱导表达。通过两种方式证明了OsDHHC1具有S-酰基转移酶的活性。在拟南芥中分段表达OsDHHC1基因的结果显示其功能域是含有DHHC锌指结构域的motif。该基因的功能研究为将来利用基因工程构建合理株型从而提高作物产量提供了理论依据，具有一定的理论价值和现实的指导意义。更多还原

【Abstract】 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 ak1△mutant, 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.更多还原