高温胁迫对西花蓟马抗氧化酶活性的影响及CAT基因的克隆与表达

发布时间：2019-02-28 08:44

【摘要】：西花蓟马是为害园艺作物和经济作物的世界性害虫。该虫于2003年首次在北京发现,之后迅速蔓延至我国的大部分地区,为害日益严重。西花蓟马在入侵过程中,常常遭受夏季高温胁迫,但这并未能限制其种群的快速扩张。本文采用生理生化和分子生物学的方法,分析抗氧化酶系统在高温胁迫条件下的作用,以了解西花蓟马对高温胁迫的耐受机制,为西花蓟马种群扩散研究提供理论依据。主要研究结果如下：1.研究了高温胁迫对西花蓟马抗氧化酶活性的影响。结果表明,西花蓟马受到0.5、1和2h的高温胁后,超氧化物歧化酶、过氧化物酶和过氧化氢酶的活性显著升高而后降低；谷胱甘肽-S-转移酶的活性则在0.5h高温处理条件下显著升高,而在1和2h高温胁迫下活性变化不显著。说明西花蓟马抗氧化酶系统能够有效抵御高温胁迫对其造成的氧化损伤。2.通过RT-PCR技术克隆了7个西花蓟马内参基因的部分片段,包括18SrRNA,EF-1、 GAPDH、H3、TUB、ACT和RPL32。利用qRT-PCT技术研究了西花蓟马7个内参基因在不同实验条件下的稳定性,运用常用的内参基因筛选程序(NormFinder、geNorm和BestKeeper)以及比较ΔCt法对各内参基因进行了排名,最后使用RefFinder网页程序进行综合评估,得出最佳的内参基因方案。最合适的内参基因组合如下：不同发育阶段为EF-1和RPL32；高温胁迫为RPL32和GAPDH;低温胁迫为18S和EF-1；高温时间梯度为TUB和GAPDH;低温时间梯度为EF-1和TUB。最后,对筛选的内参基因进行验证,表明内参基因的筛选对目的基因定量研究标准化非常必须。3.采用RT-PCR和RACE的方法从西花蓟马中克隆得到过氧化氢酶基因,命名为FoCAT。 FoCAT的cDNA全长为1870bp,开放阅读框为1521bp,编码506个氨基酸,预测分子量为57.11kda。序列分析表明,FoCAT十分保守,与其他昆虫的CAT具有很高的相似性,FoCAT具有两个CAT家族的签名序列,这也表明FoCAT属于CAT基因家族的一员。采用实时定量PCR研究了西花蓟马过氧化氢酶基因在不同发育历期和不同温度条件下的表达模式。结果表明高温胁迫能诱导CAT的表达,而低温胁迫对CAT表达的影响不显著；CAT在西花蓟马二龄若虫阶段表达量显著高于其它龄期的表达量；不论是高温还是低温不同时间梯度处理均导致CAT表达量显著上调,且在处理2h达到最高值。综上所述,西花蓟马能够耐受较强的高温胁迫是其迅速入侵扩散的重要基础,其内在机制可能与抗氧化酶系统抵御高温胁迫免受损伤有关。
[Abstract]:Western flower thrips are worldwide pests that harm horticultural and cash crops. The insect was first discovered in Beijing in 2003 and quickly spread to most parts of China. Western flower thrips are often subjected to high temperature stress in summer during invasion, but this does not limit the rapid expansion of their populations. In this paper, physiological and biochemical methods and molecular biology methods were used to analyze the role of antioxidant enzyme system under high temperature stress, in order to understand the tolerance mechanism of thrips to high temperature stress, and to provide a theoretical basis for the study of population diffusion of thrips westerners. The main findings are as follows: 1. The effects of high temperature stress on antioxidant enzyme activity of thrips were studied. The results showed that the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (catalase) were significantly increased and then decreased after being exposed to 0.5, 1 and 2 h high temperature stress in thrips. The activity of glutathione-S-transferase (GSH-S-transferase) increased significantly under 0.5 h high temperature stress, but not under 1 and 2 h high temperature stress. The results show that the antioxidant enzyme system of thrips can effectively resist oxidative damage caused by high temperature stress. 2. Seven fragments of the internal reference gene of thrips were cloned by RT-PCR technique, including 18s rRNA, EF 1, GAPDH,H3,TUB,ACT and RPL32.. The stability of seven internal reference genes of thrips japonicus under different experimental conditions was studied by means of qRT-PCT technique. The internal reference genes were ranked by the commonly used internal reference gene screening program (NormFinder,geNorm and BestKeeper) and the comparison of 螖 Ct method. Finally, the RefFinder web program was used for comprehensive evaluation, and the best internal reference gene scheme was obtained. The most suitable internal reference gene combinations were as follows: EF-1 and RPL32; high temperature stress was RPL32 and GAPDH; low temperature stress was 18s and EF-1; low temperature time gradient was TUB and GAPDH; low temperature time gradient was EF-1 and TUB.. Finally, the screening of internal reference genes was verified, indicating that the screening of internal reference genes is very necessary for the standardization of quantitative study of target genes. 3. The catalase gene was cloned from thistle by RT-PCR and RACE and named FoCAT.. The full-length cDNA of FoCAT is 1870bp, the open reading frame is 1521bp, encoding 506amino acids, and the predicted molecular weight is 57.11kda. Sequence analysis showed that FoCAT was very conservative and had high similarity to CAT of other insects. FoCAT had two signature sequences of CAT family, which also indicated that FoCAT belonged to one of the CAT gene families. Real-time quantitative PCR was used to study the expression patterns of catalase gene in thistle at different developmental stages and at different temperatures. The results showed that high temperature stress could induce the expression of CAT, but the effect of low temperature stress on the expression of CAT was not significant, and the expression level of CAT in the second instar nymphs of thrips was significantly higher than that of other ages. The expression of CAT was up-regulated at different time gradients both at high temperature and low temperature, and reached the highest value at 2 h after treatment. In conclusion, the ability of thrips to tolerate high temperature stress is an important basis for its rapid invasion and diffusion, and its intrinsic mechanism may be related to the protection of antioxidant system from damage under high temperature stress.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.89

【相似文献】