本文選題：獼猴桃 + 生物信息學��； 參考：《浙江大學》2016年碩士論文

【摘要】：獼猴桃病害嚴重影響了獼猴桃的品質與產(chǎn)量,其中由丁香假單胞桿菌獼猴桃致病變種P. Syringae pv. actinidae(PSA)引起的獼猴桃細菌性潰瘍病是最重要的病害之一。本研究從獼猴桃的抗病基因入手,分析植物抗病基因的序列結構與其功能的關系,找到能輔助獼猴桃抗?jié)儾≈仓攴肿佑N的標記,結果如下：1.以核苷酸結合位點(Nucleotide binding site, NBS)類基因保守序列,對‘紅陽’獼猴桃全基因組中所有96條NBS-LRR類基因,利用Kiwifruit Genome Database、P fam和Coils Server網(wǎng)站,和Bioedit、MEGA 5.0、ClustalW軟件,分析確定‘紅陽’獼猴桃的NBS-LRR基因類型、結構和系統(tǒng)發(fā)育學關系。在‘紅陽’獼猴桃全基因組中共有96個NBS-LRR類基因,其中27條NBS-LRR類基因分布在基因簇內,可根據(jù)其結構進一步劃分為20類基因。并對NBS-LRR類基因家族進行了氨基酸多序列比對和系統(tǒng)進化樹分析,發(fā)現(xiàn)整個NBS-LRR基因家族可分為三個亞家族。相對其它物種,紅陽獼猴桃NBS-LRR類基因總數(shù)少,缺少TIR結構,具有LRR結構的基因所占比重低,NBS結構不完整,這些特點可能與其感病性有關聯(lián)。2.以易感獼猴桃潰瘍病品種‘紅陽’獼猴桃全基因組中所有數(shù)據(jù)為基礎,尋找到有可能存在獼猴桃抗?jié)儾』虻膬蓚�抗病基因家族Pto-like基因家族和NBS-LRR基因家族的基因序列,并基于該序列設計引物,以潰瘍病抗病品種‘徐香’獼猴桃的全基因組DNA為模板進行PCR擴增,克隆并測序最終得到41條能夠通讀的不重復序列序列,分析其結構特點并與‘紅陽’獼猴桃的NBS-LRR基因序列結構進行對比分析,發(fā)現(xiàn)兩個品種的NBS-LRR基因中幾乎都沒有Tir結構域,且NBS結構域結構特點有一定差異：在構成NBS結構的8個結構域中,相比‘紅陽’獼猴桃,‘徐香’獼猴桃的NBS-LRR序列都具有更完整的Kinase2結構域,但是缺失RNBS-D結構和MHDVmotif結構比較嚴重；總體上來說,‘徐香’獼猴桃的NBS結構域更為完整。3.在‘紅陽’獼猴桃全基因組序列中國搜集整理得到NBS家族(共96條)和RLK家族(共261條)兩大抗病基因家族的序列,應用利用GRAMENE網(wǎng)站在線工具分析其結構,找到68對SSR位點,以此設計和篩選出最終產(chǎn)物在110-340bp,引物長度為18-22bp的引物,以17個品種獼猴桃全基因組DNA為模板擴增,最終篩選得到的兩對引物基本能夠在17個獼猴桃品種中表現(xiàn)出良好的擴增效果并表現(xiàn)出與潰瘍病抗性有一定的相關性。
[Abstract]:The quality and yield of kiwifruit were seriously affected by kiwifruit disease. The bacterial canker of kiwifruit caused by P. Syringae pv. actinidae, a pathogenic variety of Actinidia, is one of the most important diseases. In this study, we analyzed the relationship between the sequence structure and function of disease resistance genes in kiwifruit, and found the markers that can assist molecular breeding of kiwifruit canker resistant plants. The results are as follows: 1. Using the conserved sequence of nucleotide binding site, NBS) class gene at nucleotide binding site, all 96 NBS-LRR class genes in the whole genome of 'Hongyang' kiwifruit were obtained by using Kiwifruit Genome Database P fam and Coils Server website, and BioeditGA-MEGA 5.0 / ClustalW software. The NBS-LRR gene type, structure and phylogenetic relationship of 'Hongyang' kiwifruit were analyzed. There are 96 NBS-LRR class genes in the whole genome of 'Hongyang' kiwifruit, of which 27 NBS-LRR genes are distributed in the gene cluster, which can be further divided into 20 classes according to their structure. The amino acid sequence alignment and phylogenetic tree analysis of NBS-LRR gene family showed that the whole NBS-LRR gene family could be divided into three subfamilies. Compared with other species, the total number of NBS-LRR genes in Actinidia deliciosa is less, the TIR structure is lacking, and the proportion of genes with LRR structure is low. These characteristics may be related to the susceptibility of Actinidia deliciosa. 2. Based on all the data in the whole genome of susceptible kiwifruit canker variety 'Hongyang', the gene sequences of two resistant gene families, Pto-like gene family and NBS-LRR gene family, which may exist in kiwifruit canker resistance genes were found. Based on this sequence, primers were designed to amplify the whole genomic DNA of the disease resistant cultivar 'Xuxiang' kiwifruit by PCR amplification. Finally, 41 non-repeat sequences were cloned and sequenced. It was found that there was almost no Tir domain in the NBS-LRR gene of the two cultivars by analyzing its structural characteristics and comparing it with the sequence structure of the NBS-LRR gene of 'Hongyang' kiwifruit (Actinidia deliciosa). The structural characteristics of NBS domain were different: the NBS-LRR sequences of 'Hongyang' kiwifruit 'Xuxiang' kiwifruit had more complete Kinase2 domain than 'Hongyang' kiwifruit 'Xuxiang' kiwifruit's NBS-LRR domain. In general, the NBS domain of Actinidia chinensis was more complete. 3. Two major resistant gene families of 'Hongyang' kiwifruit ('Hongyang' kiwifruit) were collected and sequenced in China. The sequences of the NBS family (96 genes) and the RLK family (261 genes) were sequenced. Using the GRAMENE website online tools, 68 pairs of SSR loci were found. The primers with the length of 110-340 BP and primer length of 18-22bp were designed and screened. The genomic DNA of 17 varieties of kiwifruit was used as template. The two pairs of primers obtained from the final screening showed good amplification effect in 17 kiwifruit cultivars and showed a certain correlation with the resistance to canker disease.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S436.634.1

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