【作者】 陈章权；

【导师】 陈守才；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2002， 博士

【摘要】 本研究以具有明显育性转换特征的水稻温敏核雄性不育系Tb7S为材料，通过抑制性消减杂交(Suppression Subtractive Hybridization，SSH)技术筛选减数分裂期的Tb7S幼穗在高温可育和低温不育条件下的差异表达cDNA片段。经SSH富集cDNA片段构建了两个cDNA质粒文库，即cDNA正向消减和反向消减质粒文库。 分别从正向消减和反向消减质粒文库中随机选取39个和35个克隆进行分析。重组质粒EcoR1酶切鉴定表明随机挑取的74克隆中均载有约200-600bp大小的插入片段，进一步的测序表明cDNA片段大小介于200bp-300bp之间的约占41％，100bp-200bp的约24％，300bp-400bp的约23％，400bp以上的约12％，与理论预期值大小相符。在序列分析过程中发现有部分克隆是重复检出的同一基因，也有些克隆是同一基因的不同片段，但在两个cDNA消减文库中的cDNA未发现有相同的序列，说明所进行的SSH消减杂交效率高，同时SSH消减效率鉴定实验结果也证明了这一点。序列分析表明除去重复克隆，74个cDNA克隆代表了61个不同的基因，说明约82％的克隆代表不同的基因，表明cDNA消减群体内部重复拷贝少，不同基因间丰度的差异得到了很好的均衡。对这61个序列进行BLAST检索查询，结果发现有17个为已知基因，约占26％，有44个为未知功能的基因，约为74％。 从测序的克隆中随机选取10个(每个文库5个克隆)，用RT-PCR技术分析这些克隆所代表的基因在Tb7S幼穗处于高温可育和低温不育条件下的差异表达情况。结果鉴定出2个………………… ．华南热带农业大学博士学位论文…．，……………．·￥cDNA在高温可育幼穗中特异表达，另有2个cDNA在低温不育幼穗中特异表达，提示这4个。DNA代表的基因的表达可能与水稻温敏核不育系Tb7S的育性转换相关。 在RT．PCR的鉴定出差异表达。DNA片段的基础上，选取一个未知功能的SZcDNA片段，用RACE技术克隆其全长CDNA。对扩增的片段进行测序和拼接，结果获得全长为2976hp的 SZ cDNA，其编码区为 2367hp，此外，还含有 75 hp的 5’－UTR 534hp的 3’－UTR。对推导的全长氨基酸序列用 3DPSSM Protein Prediction软件和 PredictProtein软件进行分析，结果提示SZ基因的编码产物可能是一个参与信号转导的调控因子。通过PSORT进行蛋白质定位信号分析，提示该基因的表达产物含有一段线粒体靶肽（mitochondrial枯吧eting peptide），推测该基因编码产物可能在干扰线粒体的功能方面起作用。如果该推测成立，暗示三系和两系水稻之间可能存在某种联系。Southern杂交分析表明在Tb7S基因组中SZ基因为单拷贝基因。 对获得的新基因有必要进行生物学功能的鉴定，而RNAi技术是一种高效的阻抑基因表达的新技术。本研究成功构建了SZ C DNDNA 片段的RN＊1 植物表达载体pCAMBIA130lAP人石，Sallnyll和Pmll／BstEll酶切鉴定表明正向和反向特异片段正确插入表达载体，EcoR I／Sma酶切鉴定表明 ubiquitiin启动子也正确插入。构建的载体含有潮霉素筛选标记基因，利用农杆菌介导法导入Tb7S愈伤组织，获得了抗性愈伤组织，后续工作正在进行。更多还原

【Abstract】 OBJECTIVE: The aim of the research is to isolate the genes associated with fertility transformation of thermo-sensitive genie male sterile rice. METHODS: Two-directional (forward and backward) Suppression Subtractive Hybridization (SSH) was performed on cDNAs of fertile young panicle and sterile young panicle of thermo-sensitive genie male sterile rice (Tb7S), and two Subtracted cDNA libraries were established with the forward and the backward subtracted cDNAs, positive clones from each subtracted cDNA libraries were selected for sequencing and BLAST analysis, the differential expression of 10 clones between fertile young panicle and sterile young panicle of Tb7S were analysed by RT-PCR; RACE technique was used to amplify the 5’ cDNA fragment and 3’ cDNA fragment of S2; The plant expression vector of pCAMBIA1301-P-A-S was constructed. In pCAMBIA1301-P-A-S, gene-specific sequences in the antisense (A) and sense (s) orientations were linked with a 1300-bp of the GUS gene and controlled by ubiquitin promoter, the 400-bp gene-specific sequences in the antisense (A) and sense (s) orientations were inserted in the Sal IIIBgl II and Pml l/Bst Ell sites, the ubiquitin promoter was inserted in the EcoR HSma I site, the callus from tissue cultured young embryo were used as material in agrobacterium-mediated genetic transformation.RESULT: The experiment of subtractive efficiency indicated that the subtractive efficiency is high, and the plasmid digestion by EcoR I indicated that all the positive clones picked randomly contained cDNA fragments of 200-600bp. The sequenceanalysis demonstrated that picked randomly 74 clones represented 61 different genes, which showed the repeat copies of subtracted population of cDNA fragments were few and the abundance between different genes was normalized. Sequencing and BLAST homology search revealed that 17 clones contain sequences of known gene fragments and 44 possibe novel genes show no sequence homologies with any know sequences in the database. 4 clones of 10 clones picked randomly were identified as differentially expressed genes by RT-PCR, among which 2 clones were expressed specifically in the fertile young panicle of Tb7S and the other 2 clones were expressed specifically in the sterile young panicle of Tb7S. A novel full-length of S2 cDNA containing 2976bp was obtained by RACE technique, which contain the ORF of 2367bp, 5’-UTR of 75bp and 3’-UTR of 534bp. The results of 3DPSSM Protein prediction and protein domain prediction indicated that it might be a regulatory factor involved in signal transduction;. The result of PSORT (Prediction of Protein Localization Sites) indicates that the protein encoded by S2 has a mitochondrial targeting peptide. Southern Blotting showed that the S2 gene is a single cope gene.The plasmid digestion showed that the 400-bp gene-specific sequences in the antisense (A) and sense (s) orientations were inserted in the Sal 11/Bgl II and Pml l/Bst Ell sites, the ubiquitiin promoter was inserted in the EcoR l/Sma I site of the improved pCAMBIA1301. CONCLUSION: (1) All results confirmed the effectiveness and sensitivity of SSH;(2) S2 gene is a single copy gene; (3) S2 gene is expressed specifically in the sterile young panicle of Tb7S ,which suggest that S2 gene might be a novel gene associated with fertility transformation of thermo-sensitive genie male sterile rice;(3) The fact that the protein encoded by S2 cDNA has a mitochondrial targeting peptide suggests that there might be relationship between the two-line rice and three-line rice; (4) The RNAi plant expression vector(pCAMBIA1301A-P-A-S) was constructed successfully. In the pCAMBIA1301A-P-A-S, the gene-specific sequences in the antisense (A) and sense (s) orientations were linked with a fragment of the GUS gene and controlled by the ubiquitin promoter.更多还原