本文選題：象耳豆根結(jié)線蟲　切入點(diǎn)：轉(zhuǎn)錄組　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：象耳豆根結(jié)線蟲為經(jīng)濟(jì)上重要的固著型內(nèi)寄生植物病原線蟲,耐高溫,寄主范圍廣,致病力強(qiáng),可克服多種作物如番茄的Mi和辣椒的Me等抗線蟲基因,給農(nóng)作物生產(chǎn)造成嚴(yán)重經(jīng)濟(jì)損失,國(guó)際上已將其作為重要的檢疫對(duì)象。本研究以象耳豆根結(jié)線蟲混合齡期RNA為樣本,進(jìn)行454測(cè)序。獲得408663個(gè)reads,read的平均長(zhǎng)度為403 bp,經(jīng)序列拼接后獲得8 193個(gè)Contigs和31860個(gè)singlests,長(zhǎng)度中值分別為961 bp和416 bp。其中有8143個(gè)Contigs和29043個(gè)Singlets具有預(yù)測(cè)的開放閱讀框(ORF),其中有4521個(gè)ORFs具有GO功能注釋。應(yīng)用orthAgogue軟件對(duì)象耳豆根結(jié)線蟲與秀麗隱桿線蟲、松材線蟲、馬鈴薯金線蟲、南方根結(jié)線蟲、北方根結(jié)線蟲的直系同源家族進(jìn)行了分析,并將6個(gè)不同線蟲種中保守的1022個(gè)單拷貝直系同源家族進(jìn)行全基因組發(fā)育分析,結(jié)果表明象耳豆根結(jié)線蟲與南方根結(jié)線蟲親緣關(guān)系最近。進(jìn)一步分析了5類情況的ortholog family.GO富集后發(fā)現(xiàn)9個(gè)GO注釋在三個(gè)根結(jié)線蟲中都有,2個(gè)GO注釋存在于象耳豆根結(jié)線蟲和非根結(jié)線蟲屬,1個(gè)只為象耳豆根結(jié)線蟲和南方根結(jié)線蟲共有,11個(gè)為象耳豆根結(jié)線蟲和北方根結(jié)線蟲共有。通過KAAS分析發(fā)現(xiàn)8個(gè)pathway特異存在于象耳豆根結(jié)線蟲,涉及到四環(huán)素生物合成、β-內(nèi)酰胺抗性、雙酚降解、肽聚糖生物合成、阿特拉津降解、類固醇降解、含鐵細(xì)胞組非核糖體肽合成和瘧疾；5個(gè)特異存在于南方根結(jié)線蟲；1個(gè)特異存在于北方根結(jié)線蟲；3個(gè)涉及的KO數(shù)在象耳豆根結(jié)線蟲中有擴(kuò)增,41個(gè)在南方根結(jié)線蟲中有缺失。通過SignallP和TMHMM預(yù)測(cè)象耳豆根結(jié)線蟲有1464個(gè)分泌蛋白,利用維恩圖比較了分泌蛋白基因Ortholog family在3種線蟲中的保守和特異情況,發(fā)現(xiàn)701個(gè)為象耳豆根結(jié)線蟲特異的,679個(gè)是三種線蟲共有的。另外,分析了象耳豆根結(jié)線蟲的免疫反應(yīng)、RNAi現(xiàn)象、神經(jīng)遞質(zhì)、神經(jīng)肽、滯育和致死表型,為尋找防治象耳豆根結(jié)線蟲的新靶標(biāo)并制定新的防治策略提供理論依據(jù)。通過與其他已報(bào)道的植物線蟲效應(yīng)因子進(jìn)行比對(duì),發(fā)現(xiàn)有241個(gè)象耳豆根結(jié)線蟲轉(zhuǎn)錄本與之匹配,涉及幫助象耳豆根結(jié)線蟲寄生的細(xì)胞壁降解酶、蛋白酶、參與線蟲的防衛(wèi)和對(duì)寄主防御的抑制以及調(diào)控植物發(fā)育的激素信號(hào)等生理生化過程。在此基礎(chǔ)上,克隆了4個(gè)具有信號(hào)肽、食道腺分泌的候選新基因,利用根癌土壤桿菌介導(dǎo)的PVX病毒表達(dá)系統(tǒng),在模式植物本氏煙上對(duì)這4個(gè)候選新基因進(jìn)行了初步分析,發(fā)現(xiàn)5號(hào)、10號(hào)候選基因可以抑制BAX誘導(dǎo)的PCD,而8號(hào)候選基因能引起煙草葉片細(xì)胞壞死,7號(hào)候選基因既不抑制BAX誘導(dǎo)的PCD也不引起煙草細(xì)胞壞死。煙草細(xì)胞亞細(xì)胞定位結(jié)果顯示,3個(gè)候選效應(yīng)蛋白主要在細(xì)胞核表達(dá),另外一個(gè)主要在質(zhì)膜上表達(dá)。以上結(jié)果為深入研究新效應(yīng)子的功能奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S432.45

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