本文選題：小黑豆 + SCN3��； 參考：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：大豆絲氨酸羥基轉(zhuǎn)移酶(serine hydroxymethyltransferase)基因(SHMT)是Rhg4位點(diǎn)的抗胞囊線蟲基因。本研究參考已有的PCR引物對28份小黑豆品種及2份感病品種進(jìn)行了 SHMT基因分子標(biāo)記、克隆編碼區(qū)序列、實(shí)時熒光定量PCR,研究其遺傳多樣性及與大豆對胞囊線蟲抗性的關(guān)系,進(jìn)一步探究小黑豆抗大豆胞囊線蟲的抗病機(jī)理,為大豆資源的合理利用提供依據(jù),取得了如下進(jìn)展。在供試的30份大豆材料中,分子標(biāo)記研究表明均有Rhg4和rhg1位點(diǎn),對SHMT編碼區(qū)進(jìn)行克隆測序,研究共發(fā)現(xiàn)了 6個SNP位點(diǎn)(2個轉(zhuǎn)換,4個顛換)和2個單堿基插入的Indels;多態(tài)性位點(diǎn)中2個為簡約信息位點(diǎn);SHHMT基因序列多樣性π=0.08190和θ=0.10394;30份供試大豆品種分成6個單倍型,多樣性計(jì)算結(jié)果為0.447;Tajima's D和FuandLi's D和Fisher's檢驗(yàn)發(fā)現(xiàn)不同供試大豆SHMT基因在進(jìn)化過程中可能受自然選擇壓力;卡方測驗(yàn)表明SHMT基因在389G/C、1072T/A位點(diǎn)與大豆對SCN3的抗性有直接關(guān)聯(lián);遺傳進(jìn)化樹將供試30份大豆的SHMT基因聚類在3個類群,該基因在品種間的遺傳關(guān)系與生態(tài)區(qū)存在一定聯(lián)系;利用生物信息學(xué)分析軟件預(yù)測8個存在多態(tài)性位點(diǎn)大豆品種的蛋白三級結(jié)構(gòu),SHMT基因單核苷酸的變化會導(dǎo)致編碼氨基酸理化性質(zhì)的改變,使其具有不同的酶切位點(diǎn),從而表現(xiàn)出不同的電泳圖譜結(jié)果。該基因編碼的蛋白質(zhì)均屬于親水性蛋白,定位于細(xì)胞質(zhì)中,不經(jīng)過跨膜直接參與相關(guān)酶促反應(yīng),推測供試大豆根系中SHMT基因編碼的蛋白可能影響一碳單位的合成及數(shù)量,進(jìn)而間接參與大豆對胞囊線蟲3號生理小種的抗病過程。實(shí)時熒光定量PCR表明SHMT基因在抗感品種均有表達(dá),且相對表達(dá)量在SCN3侵染前期(6d)、中期(12d)、后期(35d)差異顯著,抗病品種在SCN3侵染12d后,該基因相對表達(dá)量普遍開始上調(diào),而感病品種均在中期表達(dá)少而在后期表達(dá)較多,推測在抗性品種中該基因可能參與抗SCN3發(fā)育過程。
[Abstract]:Soybean serine hydroxyltransferase (serine hydroxymethyltransferase) gene is a Rhg4 locus resistant to cysts nematode. In order to study the relationship between genetic diversity and resistance of soybean to cyst nematode, the molecular markers of SHMT gene, cloning coding region sequence and real-time quantitative PCR were used to study the genetic diversity and resistance of 28 black bean cultivars and 2 susceptible varieties to soybean cyst nematode. In order to provide basis for rational utilization of soybean resources, the mechanism of resistance to soybean cyst nematode was further studied. In 30 soybean materials tested, molecular marker analysis showed that all of them had Rhg4 and rhg1 loci, and cloned and sequenced the coding region. Six SNP loci (2 transversions, 4 transversions) and 2 single base insertions were found in the study, among which 2 were simple information loci (SHHMT), and 30 soybean cultivars were divided into 6 haplotypes, including 蟺 -0.08190 and 胃 0.10394. The results of diversity analysis showed that the SHMT gene in different soybean samples might be under the pressure of natural selection in the course of evolution, and that SHMT gene was directly related to the resistance of soybean to SCN3 at 389G / C1072T / A locus. The genetic evolution tree clustered the SHMT gene of 30 soybean samples into three groups, and the genetic relationship between the two cultivars was related to the ecological region. Bioinformatics analysis software was used to predict that the change of single nucleotide of SHMT gene in 8 soybean varieties with polymorphic loci would result in the change of physicochemical properties of encoded amino acids and make them have different restriction sites. The results showed different electrophoretic patterns. The proteins encoded by this gene belong to hydrophilic proteins, which are located in the cytoplasm and take part in the related enzymatic reactions without transmembrane. It is speculated that the protein encoded by SHMT gene in soybean root system may affect the synthesis and quantity of one carbon unit. Furthermore, it was indirectly involved in the disease resistance of soybean to Cystic nematode No. 3. Real-time fluorescent quantitative PCR showed that SHMT gene was expressed in all susceptible varieties, and the relative expression level was significantly different in the early (6 d), 12 d (12 d) and 35 d (later stage) of SCN3 infection. The relative expression of SHMT gene began to up-regulate after 12 days of SCN3 infection. The susceptible varieties expressed less in the middle stage and expressed more in the later stage, which suggested that the gene might be involved in the development of SCN3 resistance.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S435.651

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