刺萼龙葵种子萌发相关的甘露聚糖酶基因的克隆及表达研究

发布时间：2019-01-24 12:03

【摘要】：刺萼龙葵是原产北美的有毒有害入侵杂草,由于其适应性广、竞争能力强等特点在我国许多地区扩散定植,严重破坏了入侵地的生物多样性。种子是杂草发生危害和传播扩散的根源,刺萼龙葵种子具有休眠特性,不利于种群的集中防控和彻底根除。为了探究刺萼龙葵种子休眠萌发的分子调控机制,本研究针对种子萌发过程中降解胚乳组织的关键酶——甘露聚糖酶,克隆了甘露聚糖酶家族的2个基因SrMAN1和SrMAN2,在转录水平上检测了外源激素作用下种子在浸种和萌发过程中的基因表达变化,进行了目的基因的原核表达,并采用凝胶扩散法验证了重组蛋白的功能。主要研究结果如下:1.从刺萼龙葵中克隆得到两个β-甘露聚糖酶相关基因,其中SrMAN1基因全长1921 bp,SrMAN2基因全长3075 bp。SrMAN1基因的编码序列为1143 bp,编码380个氨基酸,蛋白分子量为42.9 kD,等电点为5.4。SrMAN2基因的编码序列为1242 bp,编码413个氨基酸,蛋白分子量为46.6 kD,等电点为5.2。2.实时荧光定量PCR分析表明,赤霉素处理下,SrMAN1和SrMAN2基因均在胚根伸长约10 mm,胚轴露出约2 mm时达到表达高峰。随着种子吸水时间的延长,脱落酸作用下两个基因的相对表达量较低;而在赤霉素与清水处理下,两个基因的表达量先升高再降低,并且赤霉素使两个基因的表达高峰提前出现;赤霉素处理下,SrMAN1基因的表达高峰高于清水处理下的表达高峰,SrMAN2基因的表达高峰低于清水处理下的表达高峰。SrMAN1和SrMAN2基因在刺萼龙葵种子萌发前后均有表达,其表达无显著的组织特异性和时间特异性。赤霉素可在一定程度上影响SrMAN1和SrMAN2基因的表达,但相比于SrMAN1基因,赤霉素对SrMAN2基因的作用较弱。3.成功构建了SrMAN1和SrMAN2基因的重组表达载体pET-32a-SrMAN1和pET-32a-SrMAN2,实现了在E.coli BL21(DE3)中的诱导表达。菌液SDS-PAGE表明,SrMAN1、SrMAN2的标签融合蛋白的出现位置与预测的SrMAN1融合蛋白(62.78 kD)、SrMAN2融合蛋白(66.37 kD)大小接近。凝胶扩散检测表明,成功表达的菌株孵育液在甘露聚糖凝胶底物上出现显著增大的水解圈,证实了融合蛋白具有甘露聚糖酶水解活力。
[Abstract]:Solanum nigrum is a poisonous and harmful weed native to North America. Because of its wide adaptability and strong competitive ability, it has spread and settled in many areas of China, which has seriously damaged the biodiversity of the invading land. Seed is the root of weed damage and spread. The seed of Solanum nigrum has dormancy characteristics, which is not conducive to the centralized control and eradication of the population. In order to explore the molecular regulation mechanism of dormancy germination of Solanum nigrum seeds, two genes of mannanase family, SrMAN1 and SrMAN2, were cloned for the key enzyme of degradation of endosperm tissue during seed germination. The changes of gene expression during seed soaking and germination were detected at the transcriptional level, the prokaryotic expression of the target gene was carried out, and the function of the recombinant protein was verified by gel diffusion method. The main results are as follows: 1. Two 尾 -mannanase related genes were cloned from Solanum nigrum. The coding sequence of 3075 bp.SrMAN1 gene of 1921 bp,SrMAN2 gene of SrMAN1 gene was 1143 bp, encoding 380 amino acids and the molecular weight of protein was 42.9 kD,. The coding sequence of 5.4.SrMAN2 gene with isoelectric point is 1242 bp, encoding 413 amino acids and the molecular weight of protein is 46.6 kD, isoelectric point 5.2.2. Real-time fluorescence quantitative PCR analysis showed that the expression of SrMAN1 and SrMAN2 genes reached the peak when the radicle elongation was about 10 mm, and the cotyledon was exposed to about 2 mm under gibberellin treatment. With the prolongation of water absorption time, the relative expression of the two genes was lower under the action of abscisic acid. Under the treatment of gibberellin and water, the expression of the two genes increased first and then decreased, and gibberellin made the expression peak of the two genes appear earlier. Under gibberellin treatment, the peak of SrMAN1 gene expression was higher than that of water treatment, and the peak of SrMAN2 gene expression was lower than that of clear water treatment. Both SrMAN1 and SrMAN2 genes were expressed before and after germination of Solanum nigrum seeds. There was no significant tissue specificity and time specificity. Gibberellin could affect the expression of SrMAN1 and SrMAN2 genes to some extent, but the effect of gibberellin on SrMAN2 gene was weaker than that of SrMAN1 gene. The recombinant expression vectors of SrMAN1 and SrMAN2 genes pET-32a-SrMAN1 and pET-32a-SrMAN2, were successfully constructed to induce expression in E.coli BL21 (DE3). SDS-PAGE showed that the location of the SrMAN1,SrMAN2 fusion protein was similar to that of the predicted SrMAN1 fusion protein (62.78 kD), SrMAN2 fusion protein (66.37 kD). The gel diffusion test showed that the successfully expressed strain incubated on the gel substrate of mannan showed a significantly increased hydrolysis circle, which confirmed that the fusion protein had the activity of hydrolysis of mannanase.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S451

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