象耳豆根结线虫转录组分析及4个候选寄生致病基因的鉴定

发布时间：2018-03-04 20:06

本文选题：象耳豆根结线虫　切入点：转录组　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：象耳豆根结线虫为经济上重要的固着型内寄生植物病原线虫,耐高温,寄主范围广,致病力强,可克服多种作物如番茄的Mi和辣椒的Me等抗线虫基因,给农作物生产造成严重经济损失,国际上已将其作为重要的检疫对象。本研究以象耳豆根结线虫混合龄期RNA为样本,进行454测序。获得408663个reads,read的平均长度为403 bp,经序列拼接后获得8 193个Contigs和31860个singlests,长度中值分别为961 bp和416 bp。其中有8143个Contigs和29043个Singlets具有预测的开放阅读框(ORF),其中有4521个ORFs具有GO功能注释。应用orthAgogue软件对象耳豆根结线虫与秀丽隐杆线虫、松材线虫、马铃薯金线虫、南方根结线虫、北方根结线虫的直系同源家族进行了分析,并将6个不同线虫种中保守的1022个单拷贝直系同源家族进行全基因组发育分析,结果表明象耳豆根结线虫与南方根结线虫亲缘关系最近。进一步分析了5类情况的ortholog family.GO富集后发现9个GO注释在三个根结线虫中都有,2个GO注释存在于象耳豆根结线虫和非根结线虫属,1个只为象耳豆根结线虫和南方根结线虫共有,11个为象耳豆根结线虫和北方根结线虫共有。通过KAAS分析发现8个pathway特异存在于象耳豆根结线虫,涉及到四环素生物合成、β-内酰胺抗性、双酚降解、肽聚糖生物合成、阿特拉津降解、类固醇降解、含铁细胞组非核糖体肽合成和疟疾；5个特异存在于南方根结线虫；1个特异存在于北方根结线虫；3个涉及的KO数在象耳豆根结线虫中有扩增,41个在南方根结线虫中有缺失。通过SignallP和TMHMM预测象耳豆根结线虫有1464个分泌蛋白,利用维恩图比较了分泌蛋白基因Ortholog family在3种线虫中的保守和特异情况,发现701个为象耳豆根结线虫特异的,679个是三种线虫共有的。另外,分析了象耳豆根结线虫的免疫反应、RNAi现象、神经递质、神经肽、滞育和致死表型,为寻找防治象耳豆根结线虫的新靶标并制定新的防治策略提供理论依据。通过与其他已报道的植物线虫效应因子进行比对,发现有241个象耳豆根结线虫转录本与之匹配,涉及帮助象耳豆根结线虫寄生的细胞壁降解酶、蛋白酶、参与线虫的防卫和对寄主防御的抑制以及调控植物发育的激素信号等生理生化过程。在此基础上,克隆了4个具有信号肽、食道腺分泌的候选新基因,利用根癌土壤杆菌介导的PVX病毒表达系统,在模式植物本氏烟上对这4个候选新基因进行了初步分析,发现5号、10号候选基因可以抑制BAX诱导的PCD,而8号候选基因能引起烟草叶片细胞坏死,7号候选基因既不抑制BAX诱导的PCD也不引起烟草细胞坏死。烟草细胞亚细胞定位结果显示,3个候选效应蛋白主要在细胞核表达,另外一个主要在质膜上表达。以上结果为深入研究新效应子的功能奠定了基础。
[Abstract]:Root-knot nematodes are economically important pathogen nematodes of endoparasitic plants with high temperature tolerance, wide host range and strong pathogenicity, which can overcome nematode resistance genes of many crops, such as tomato Mi and pepper me. It has been regarded as an important quarantine object in the world. In this study, the mixed age RNA of root-knot nematodes was used as a sample. 454 sequencing showed that the average length of 408663 READSMANs was 403bp. after sequence splicing, 8,193 Contigs and 31860 singlests were obtained, with the median length of 961bp and 416bp.Of them, 8143 Contigs and 29043 Singlets had the predicted open reading frame (ORF). There are 4521 ORFs with go function annotation. OrthAgogue software was used to study root-knot nematodes and cryptophagous nematodes. The homologous families of pine wood nematode, potato golden nematode, southern root knot nematode and northern root knot nematode were analyzed, and 1022 single-copy lineal homologous families of 6 different nematode species were analyzed for the whole genome development. The results showed that the relationship between Rhizodendron Elephantus and Rhizodendron elegans was most recent. After further analysis of the enrichment of ortholog family.GO in 5 kinds of cases, it was found that 9 go annotations were found in all three root-knot nematodes, and 2 go annotations existed in the root of Elephantus aurantia. Among them, 1 was found to belong to the genus Elephyllum root-knot nematode and 11 to the southern root knot nematode, and 11 to the root knot nematodes of the north. By KAAS analysis, 8 pathway were found to exist specifically in the root-knot nematodes. Involving tetracycline biosynthesis, 尾 -lactam resistance, bisphenol degradation, peptidoglycan biosynthesis, atrazine degradation, steroid degradation, Non-ribosomal peptide synthesis and malaria in iron-containing cell group; 5 specific in southern root knot nematodes; 1 in northern root knot nematodes; 3 in root-knot nematodes of Elephant bean and 41 in southern root knot nematode. SignallP and TMHMM predicted that there were 1, 464 secretory proteins in root-knot nematodes. The conservation and specificity of secretory protein gene Ortholog family in three species of nematodes were compared by Vine map. It was found that 701 of them were common to three species of nematodes. The immunoreactive RNAi, neurotransmitters, neuropeptides, diapause and lethal phenotypes of root-knot nematodes were analyzed. In order to find a new target for root-knot nematode control and to provide theoretical basis for new control strategy, 241 transcripts of root-knot nematodes were found to match by comparing with other reported plant nematode effect factors. Involved in physiological and biochemical processes such as cell wall degradation enzymes, protease, defense of nematodes and inhibition of host defense, and hormone signals that regulate plant development, which contribute to parasitism of root-knot nematodes. Four novel candidate genes with signal peptide and esophageal gland secretion were cloned. Using Agrobacterium tumefaciens mediated expression system of PVX virus, the four candidate genes were preliminarily analyzed on the model plant Bentworth tobacco. It was found that candidate gene 5 and 10 could inhibit PCDinduced by BAX, while candidate gene 8 could induce necrosis of tobacco leaf cells. Candidate gene 7 could neither inhibit PCD induced by BAX nor induce necrosis of tobacco cells. The localization results showed that three candidate effector proteins were mainly expressed in the nucleus. The other one is mainly expressed on the plasma membrane, which lays a foundation for the further study of the function of the new effector.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.45

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