【作者】 黄湘文；

【导师】 庄伟建； 郑奕雄；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 硕士

【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Sfi I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,而和已知的花生序列的相似性只排在14位,这与所获的序列绝大部分为花生未报道的基因有关。在BLASTX基础上,利用BLAST2GO对其进行了GO功能注释,结果表明,各个功能类的基因基本上都含有。这为后续的大规模测序的可行性提供了理论上的证据,也为相关的研究提供了必要的线索。3获得了37条RGA序列,其中登录了18条。根据已登录的花生RGA片段经过多元比对共设计了五组简并引物,利用同源克隆技术对花生品种闽花六号进行了RGA片段的扩增,通过测序BLASTN分析证实,有37条序列为RGA片段序列。其中18条序列已登录,登录号为GenBank EU639668-EU639685。在这五组引物中,扩增所得片段从测序结果看,有一组中的序列90%为RGA片段序列,比例最低的一组序列也有20%多。而通过对已知的别的物种的抗性基因全长进行多元比对后,设计简并引物进行RGA片段扩增的结果表明:从测序来看,没有和已知RGA片段序列相似的序列。通过本物种已知的RGA序列来扩增相应的RGA片段是有效的,这为RGA的后续研究利用提供了一条捷径。花生功能基因组学研究和别的物种相比总体还很薄弱。本研究可丰富花生功能基因组学的研究内容,为最终的花生品种的遗传改良的提供基础。更多还原

【Abstract】 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 cloning. The full-length cDNA library was stocked after amplification, the titer of which was estimated as 8.5×109cfu/ml, meeting the requirements of the following experiments.Clones were randomly selected and then sequenced from 5’-end, obtaining a total of 400 high quality EST sequences. A preliminary study was conducted on the biological information analysis. Bases composition analysis and BLAST comparison results show that between the known genomes of dicotyledon Arabidopsis thaliana and monocot rice, it is more similar to Arabidopsis thaliana for peanut, and it is in accord with traditional plant classification results. However BLASTX results also show that the obtained sequences of peanut seem to be more similar to those of vine grapes than to those of peanut on web; which ranks only in the fourteenth from the point of sequence similarity. At the basis of BLASTX, GO annotation was carried out using BLAST2GO, which showed that they contained various functional gene categories basically. The theoretical evidence has been provided for the feasibility of large-scale subsequent sequencing, as well as the necessary clues for related research.A total of five group degenerate primers were designed according to the result of multiple sequence alignment for entile RGA sequences in peanut. RGA fragments were amplified using homology cloning on the peanut variety Minhua number 6. By BLASTN sequence analysis confirmed that there is 37 RGA sequences sequence, 18 sequences of which have been submitted; GenBank numbers are EU639668-EU639685. In the five pairs of primers, in terms of the sequencing results of the amplified fragments, there is one pair of primer whose amplified products contained 90 percent of sequences similar to RGA sequences, and a pair of primer set with the lowest proportion of RGA still amplified over 20 percent of RGA sequences. It was showed that there was no sequence obtained in the study similar to known sequences of RGA according to the sequencing results. So this study shows that it is more efficiently to amplify corresponding RGA fragments by means of the RGA sequence known to this species, which provides a shortcut for following RGA study and use.In comparison with other species, peanut genomics study is still very weak on the whole. This study can extend the scope of peanut functional genomics research and lay a foundation for the eventual settlement on the issues related to peanut improvement.更多还原