导　　师： 庄伟建;郑奕雄

学科专业： G1010

授予学位： 硕士

作　　者： ;

机构地区： 福建农林大学

摘　　要： 就现在所知而言，大多数植物抗病基因为组成型表达；尽管也有诱导表达的，如xal，但是目前知道的诱导表达的基因仅这一个。有人反辩说即使是组成型表达，在病菌的诱导下，表达量可能会有低水平的增强。但是这无疑也表明，表达量是否增加并不影响基因的检出。否则，就无法判别表达量是否增加或减少。本研究通过直接构建花生各组织混合全长cdna文库及相关生物信息学初步分析，为本实验室以后进行花生功能基因组研究打下坚实的基础。同时通过同源克隆技术克隆抗性基因类似物片段(rga)，为抗性相关基因全长的克隆做好前期准备。具体结果如下： 1成功构建了一个花生各组织混合的全长cdna文库。分别提取花生各个组织的rna，等量混合后分离mrna，然后利用改良的smart技术合成双链cdna。利用限制性内切酶sfiⅠ酶切。将经过分级分离得到的cdna片段连接到质粒载体pdnr-lib，成功构建花生各组织混合全长cdna文库。从所得到的初级文库分析，该初级文库含有1.98×106个克隆，已足够用于某一稀有基因的筛选。pcr和酶切鉴定检测插入片段大小，结果表明该文库重祖率很高，而且插入条带集中分布于1000-2000bp，平均插入大小约1300bp。插入片段较长，可以满足全长基因筛选的要求。随机挑取16个克隆进行全长测序分析，其中有9条序列为全长序列。将所得到的初级文库扩增后进行保存，检测扩增文库的滴度为8.5×109cfu/ml，可以满足后续实验的要求。 2随机挑取克隆进行5端测序，共获得400条质量较高的est序列，并对其进行了初步的生物信息学分析。碱基组成分析和blast比对结果表明，在双子叶植物拟南芥和单子叶植物水稻中，花生更相似于拟南芥，和传统的植物分类结果一致。但blastx结果也表明花生似乎更相似于藤本植物葡萄，而和已知 So far as I know, the majority of plant disease resistance genes are constitutive expression; even though also reported for induced gene, such as xal, which has been the only induced gene reported in the paper. Though someone argued that even if genes of constitutive expression would still enhance low-level in bacteria-induced condition, that also indicated whether the scalar on the expression of genes increased does not affect detection.Otherwise, the expression will not be able to determine whether the volume was increased or decreased. In this study, so a mixed full-length cDNA Library with whole tissues was directly constructed and a study related to a preliminary analysis of biological information was carried in progress; aiming to lay a solid foundation for functional genomics study of peanut for our laboratory. And some fragments for resistance gene analogues /(RGA/) have also been cloned through homology cloning in this study, on purpose that pre-ready work should be done to do a good job for cloning full-length resistance-related genes. Specific results are as follows: RNA was extracted from the various tissues or organs of the peanut, and mRNA was isolated from an equivalent RNA mixtures. Double-stranded cDNA was synthesized by the improved SMART technology. It was then digested by the restriction endonuclease Sfi I. After fractionation the cDNA fragments were connected to plasmid vector and a mixed full-length cDNA library was constructed successfully in the peanut. The obtained primary library contained 1.98×106 clones which have been enough for a rare gene screening. The insert fragments and its size were confirmed by both PCR and restriction enzyme digestion. It showed that the library had a high rate of recombinant with the inserts varied from 1000 to 2000bp and the average size about 1300bp. Sixteen clones were randomly selected for sequencing analysis and nine of them were full-length cDNA. It indicated that this library could be used for full-length genes screening and low abundance genes

关 键 词： 花生 全组织 全长 文库 生物信息学分析 基因克隆

领　　域： [农业科学]