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以盐胁迫下苹果的耐盐砧木珠眉海棠为材料,用SMARTTM方法构建了cDNA文库。随机挑取5 000个cDNA克隆制备芯片,得到差异表达的基因388个,其中249个表达上调、139个表达下调。分析其中变化量在8倍以上的42个基因,并对部分基因进行RT-PCR验证,结果与芯片杂交结果一致。根据功能把这些基因分为5类:光合作用相关基因、物质转运相关基因、基础代谢相关基因、逆境相关基因以及其他基因。其中直接参与盐应答的基因占34%,包括上游调控蛋白、合成植体内小分子渗透调节物的限速酶、胁迫产生的活性氧清除剂、直接调节离子运输和储存的蛋白等。用此cDNA微列阵体系,将基因芯片技术与cDNA文库相结合,成为全面研究盐胁迫下基因表达的有效途径。为进一步研究盐应答基因功能及盐胁迫机制提供参考。
The cDNA library was constructed by using SMARTTM method under salt stress, the salt-tolerant rootstock, Begonia maculata. A total of 388 cDNAs were randomly selected from 5,000 cDNA clones. Among them, 249 genes were up-regulated and 139 genes were down-regulated. 42 genes with more than 8-fold variation were analyzed, and some genes were verified by RT-PCR. The results were consistent with the results of hybridization. According to the function of these genes are divided into five categories: photosynthesis related genes, substance transport related genes, basic metabolism related genes, stress related genes and other genes. Which directly involved in salt response genes accounted for 34%, including the upstream regulatory proteins, synthesis of small molecule osmotica agents in the rate-limiting enzyme, stress generated reactive oxygen species scavenger, direct regulation of ion transport and storage of protein. Using this cDNA microarray system, the combination of gene chip technology and cDNA library has become an effective way to comprehensively study gene expression under salt stress. This study provides a reference for further study on the function of salt-responsive genes and the mechanism of salt stress.