小黑豆抗胞囊线虫SHMT基因序列多样性及功能分析

发布时间：2018-07-11 16:41

本文选题：小黑豆 + SCN3　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：大豆丝氨酸羟基转移酶(serine hydroxymethyltransferase)基因(SHMT)是Rhg4位点的抗胞囊线虫基因。本研究参考已有的PCR引物对28份小黑豆品种及2份感病品种进行了 SHMT基因分子标记、克隆编码区序列、实时荧光定量PCR,研究其遗传多样性及与大豆对胞囊线虫抗性的关系,进一步探究小黑豆抗大豆胞囊线虫的抗病机理,为大豆资源的合理利用提供依据,取得了如下进展。在供试的30份大豆材料中,分子标记研究表明均有Rhg4和rhg1位点,对SHMT编码区进行克隆测序,研究共发现了 6个SNP位点(2个转换,4个颠换)和2个单碱基插入的Indels;多态性位点中2个为简约信息位点;SHHMT基因序列多样性π=0.08190和θ=0.10394;30份供试大豆品种分成6个单倍型,多样性计算结果为0.447;Tajima's D和FuandLi's D和Fisher's检验发现不同供试大豆SHMT基因在进化过程中可能受自然选择压力;卡方测验表明SHMT基因在389G/C、1072T/A位点与大豆对SCN3的抗性有直接关联;遗传进化树将供试30份大豆的SHMT基因聚类在3个类群,该基因在品种间的遗传关系与生态区存在一定联系;利用生物信息学分析软件预测8个存在多态性位点大豆品种的蛋白三级结构,SHMT基因单核苷酸的变化会导致编码氨基酸理化性质的改变,使其具有不同的酶切位点,从而表现出不同的电泳图谱结果。该基因编码的蛋白质均属于亲水性蛋白,定位于细胞质中,不经过跨膜直接参与相关酶促反应,推测供试大豆根系中SHMT基因编码的蛋白可能影响一碳单位的合成及数量,进而间接参与大豆对胞囊线虫3号生理小种的抗病过程。实时荧光定量PCR表明SHMT基因在抗感品种均有表达,且相对表达量在SCN3侵染前期(6d)、中期(12d)、后期(35d)差异显著,抗病品种在SCN3侵染12d后,该基因相对表达量普遍开始上调,而感病品种均在中期表达少而在后期表达较多,推测在抗性品种中该基因可能参与抗SCN3发育过程。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.651

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