香蕉幼果SSH文库构建及初步分析

Construction and primary analysis of a SSH cDNA library of the young fruit of banana

【目的】构建香蕉果实发育抑制差减文库,筛选果实发育相关基因。【方法】以皇帝蕉幼果为实验组,幼叶为对照组,构建差减文库,并用Blast2GO软件进行生物信息学分析。【结果】挑选982个重组子菌液PCR筛选cDNA文库,得到750个候选克隆。对其中200个克隆进行测序、去载体序列、拼接后,获得92个unigenes。在GenBank中进行同源性比较,共有81个找到同源序列,11个cDNA未找到同源序列。75个unigenes在GO数据库中有功能注释,涉及到的细胞组分、分子功能和生物过程分别为53个、72个和60个,显示果实发育差异主要与在细胞内部组分进行的代谢过程的强度,以及其他一些因子和激酶的影响有关。【结论】成功构建皇帝蕉果实的差减cDNA文库,为进一步开展香蕉果实发育相关基因如tubulin、P450等基因的克隆及功能鉴定奠定了重要基础。

[Objective]The aim of this experiment is to construct suppression subtractive cDNA Library of fruit-development, and then to isolate related differentially expressed genes. [Method]The young fruits of Musa AA were treated as tester and young leaves as driver. Suppression subtractive hybridization （SSH） was utilized for the isolation of cDNA fragments for differentially expressed genes. Blast2GO software was used for the bioinformatic analysis. [Result]982 recombinants of Subtractive cDNA Library were different- ly screened through bacterial PCR, and 750 cDNA clones were identified as candidate cloning. After se- quencing of 200 cDNA clones, removing redundant cDNAs and assembling, 92 unigenes were obtained. The results showed that 81 of these unigenes were homologous to known genes, and 11 unigenes are ex- pected to be novel genes, because no sequence homology with any known sequences was found in Gen- Bank databases. In Gene Ontology database, 75 unigenes were assigned functional description, and cell component, molecular function and biological process were involved 53,72 and 60 unigenes. The differ- ence in fruit development almost existed in intracellular region after function annotation and category for expressed sequence tags in SSH cDNA library, most of the differentially expressed genes were involved in basic biological processes and the process may be regulated by some factors and kinases. [ConclusionlThe SSH cDNA libraries were successfully constructed. The results establish the foundation for cloning the whole length of the homeotic genes and identifying the function such as tubulin,P450 during fruit develop- Bent.