灰皮支黑豆抗胞囊线虫谷胱甘肽合成途径相关基因的克隆与表达分析

发布时间：2018-06-02 20:53

本文选题：灰皮支黑豆 + 大豆胞囊线虫　；参考：《沈阳农业大学》2016年硕士论文

【摘要】：大豆胞囊线虫病(Soybean Cyst Nematode, SCN)是世界大豆生产上最重要的病害之一,其具有分布广、危害重、寄主范围宽等特点,在我国东北和黄淮海地区发生普遍,对我国大豆生产危害日益加重。本文研究了抗性品种灰皮支黑豆和感病品种辽豆15对大豆胞囊线虫的侵染组织抗性,活性氧检测及大豆谷胱甘肽合成相关基因hGSHS、GSHS、γ-ECS差异表达分析和基因克隆,初步揭示了灰皮支黑豆对大豆胞囊线虫3号生理小种(SCN3)的抗性机理。主要研究结果如下：1通过显微观察明确了灰皮支黑豆对SCN3的组织抗性机理。对抗、感品种进行根内线虫侵染数量检测及根染色和冰冻切片观察,明确了灰皮支黑豆对SCN3具有抗线虫发育作用和组织结构抗性。2.揭示了胞囊线虫侵染后抗、感品种根系内活性氧的变化规律。对抗、感品种进行根内活性氧检测,大豆在受SCN3侵染后,抗、感品种根系内均H202升高,灰皮支黑豆在早期第5d增长幅度较大,随后虽有增长但幅度较小；而辽豆15在10、15 d大幅度增加,高峰出现和灰皮支黑豆相比较晚,推测在抗病品种中的根内防御酶系活性较感病品种高,及时清除根内H2O2。表明抗性品种在SCN胁迫后根系H202不同动态表现与抗性相关。3.实时荧光定量PCR检测了抗、感品种内hGSHS、GSHS、γ-ECS基因的差异表达,明确了灰皮支黑豆根内谷胱甘肽代谢与SCN3的发育过程和抗性的相关性。大豆胞囊线虫侵染5、10、15d后,谷氨酰胺半胱氨酸合成酶基因(y-ECS)在抗、感品种的处理组和对照组中,三个时间点的表达差异不显著；而谷胱甘肽合成酶基因(GSHS)在抗、感品种中呈现出表达差异,尤其在第15 d,在抗病品种灰皮支黑豆中表达显著下调,表达量仅为对照组的2%；而在辽豆15中表达上调4.23倍；H型谷胱甘肽合成酶基因(hGSHS)在抗性品种灰皮支黑豆中显著下调,表达量分别是对照的76%、11%、10%；谷胱甘肽相关基因的下调与灰皮支黑豆对SCN3的抗性呈正相关。4.对灰皮支黑豆的谷胱甘肽代谢相关基因hGSHS、GSHS、γECS进行了克隆和生物信息学分析,hGSHS, GSHS、γECS序列全长分别为1781bp、1654 bp、1559 bp,且有完整的开放阅读框(ORF),分别编码498、544、503个氨基酸,利用Clustal、DNAMAN等分析软件以及常用数据库和在线分析工具,对所克隆基因编码的蛋白进行了物理性质、跨膜域、信号肽、亲疏水性等特征的分析。
[Abstract]:Soybean Cyst Nematode (SCN) is one of the most important diseases in soybean production in the world. It has the characteristics of wide distribution, heavy damage and wide host range. It is widespread in the northeast of China and in the Yellow Huai sea area, and is becoming more and more harmful to soybean production in China. The resistance of soybean 15 to the infection tissue of soybean cyst nematode, the detection of reactive oxygen species and the differential expression analysis and gene cloning of hGSHS, GSHS, and gamma -ECS related genes of soybean glutathione synthesis, preliminarily revealed the resistance mechanism of black pea black bean to 3 physiological races of Soybean Cyst Nematode (SCN3). The main results are as follows: 1 is clear by microscopic observation. The mechanism of the resistance of black bean to the tissue of SCN3. Antagonism, the detection of the number of infected roots and the observation of root staining and frozen section, it was clear that the anti nematode development and tissue structure resistance of gray pea black bean to SCN3 revealed the resistance to the infection after the infection of the cyst nematode, and the change of the reactive oxygen in the root of the susceptible varieties. The antagonism and sense of.2. The variety carried out the Rox detection in the root. After SCN3 infection, the H202 increased in the root of the resistant and susceptible varieties. The growth amplitude of the gray pea black bean in the early 5D was larger, and then increased but the amplitude was small; and the liaopea 15 was greatly increased in 10,15 D, and the peak appeared late in the gray pea black bean, presumably in the root defense of the resistant varieties. The activity of the enzyme system was higher than that of the susceptible variety, and it was clear in time that the root H2O2. showed the different dynamic performance of the root H202 after SCN stress and the resistance related.3. real-time quantitative PCR detection of the resistance, the differential expression of hGSHS, GSHS, and gamma -ECS in the susceptible varieties, and the development and resistance of the Uchiya Ka metabolism and SCN3 in the black bean root. When soybean cyst nematode was infected with 5,10,15d, the expression of glutamine cysteine synthetase gene (y-ECS) was not significant at three time points in the treatment group and the control group, while the glutathione synthetase gene (GSHS) showed a difference in the resistant and susceptible varieties, especially at fifteenth D, in the resistant variety of gray skin branch. The expression was only 2% in the control group, and the expression was up to 2% in the control group. The expression of H type glutathione synthetase gene (hGSHS) was down significantly down in the resistant varieties of black bean, and the expression amount was 76%, 11%, 10%, respectively. The down regulation of glutathione related genes and the resistance of black pea black bean to SCN3 was positive. Cloning and bioinformatics analysis of glutathione metabolizing genes related to glutathione metabolism related genes (hGSHS, GSHS, and gamma ECS) of gray pea black beans were cloned and bioinformatics. The whole length of hGSHS, GSHS, and gamma ECS sequences were 1781bp, 1654 BP, 1559 BP, and a complete open reading frame (ORF), encoding 498544503 amino acids respectively, using Clustal, common numbers, and other analysis software. According to the database and online analysis tool, the physical properties, transmembrane domain, signal peptide, hydrophobicity and hydrophobicity of the protein encoded by the cloned gene were analyzed.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.651

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