江苏地区斑潜蝇发生危害及三叶斑潜蝇HSP70家族基因克隆与表达

发布时间：2018-02-16 09:30

本文关键词： 斑潜蝇三叶斑潜蝇发生危害热激蛋白70 内参筛选基因表达模式　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：斑潜蝇属Liriomyza隶属于双翅目Diptera、潜蝇科Agromyzidae、植潜蝇亚科Phytomyzidae。斑潜蝇种类繁多,种间竞争与替代迅速,不断向世界各地蔓延危害。三叶斑潜蝇Liriomyza trifolii,美洲斑潜蝇Liriomyza sativae和南美斑潜蝇Liriomyza huidobrensis是入侵我国危害严重的三种多食性斑潜蝇。近年的调查显示,在江苏地区发生危害的主要是美洲斑潜蝇和三叶斑潜蝇,未发现南美斑潜蝇的危害。此外,物种对温度胁迫的耐受能力与其它因素一起决定它们的生存、分布、竞争和扩散。热激蛋白被认为是与昆虫温度耐受性关系最密切的一类蛋白质,其中热激蛋白70(HSP70)家族与温度胁迫的关系最为密切,而有关三叶斑潜蝇的HSP70研究甚少。本研究于2014-2016年对江苏地区斑潜蝇发生危害情况进行了调查;同时克隆了三叶斑潜蝇的4条HSP70基因,并对其表达模式进行了研究,同时还与其近缘斑潜蝇种类的HSP70基因进行比较,从分子水平上揭示其抗逆境和生态适应能力,为阐明其入侵及成灾机制提供理论依据。主要研究结果如下:1、2014-2016年连续三年对江苏地区斑潜蝇的发生危害进行了调查,结果表明:江苏地区主要发生危害的是美洲斑潜蝇和三叶斑潜蝇,且美洲斑潜蝇主要在苏北地区发生危害,三叶斑潜蝇主要在苏南苏中地区发生危害,两种斑潜蝇共同发生危害的区域主要集中在苏中和沿江的部分地区。此外,不论在寄主种类、危害程度还是危害面积上,三叶斑潜蝇较美洲斑潜蝇具有更大的竞争优势,而且扬州地区三叶斑潜蝇基本取代美洲斑潜蝇,成为该地区蔬菜上的优势种斑潜蝇,同时三叶斑潜蝇在扬州地区全年有3个发生危害高峰,其中8月上中旬的种群数量最大。2、采用 RT-PCR 和 RACE(Rapid-amplification of cDNA ends)技术,从三叶斑潜蝇中克隆得到4条HSP70基因,分别为Lthsc70、Lthsp701、Lthsp702和Lthsp703。4条热激蛋白基因cDNA序列全长分别为2332、2261、2078和2296 bp,开放阅读框长度分别为1956、1938、1899和1923 bp,分别编码651、645、632和640个氨基酸,同时这4个热激蛋白都具有HSP70家族的典型结构域。序列分析发现,这4条Lthsp70s与其它物种的hsp70具有很高的相似性,并在系统进化树上被归为两类。通过比对基因组序列和cDNA序列发现,Lthsp701、Lthsp702和Lthsp703均不含有内含子,而Lthsc70含有1个内含子,长度为1273 bp,内含子的基因组剪切位点符合经典的GT-AG规则。3、通过RT-PCR技术克隆了 9条三叶斑潜蝇内参基因片段,包括ACTIN,18S,TUB,AK,EF-1,CAD,RPL32,GAPDH和H3 利用 qRT-PCR(Quantitative real-time PCR)技术研究了三叶斑潜蝇9条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ACt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ACTIN和18S;性别为TUB和18S,高温胁迫为ACTIN和GAPDH;低温胁迫为ACTIN和18S。最后,利用目的基因Lthsp21.7对筛选的内参基因进行验证,结果表明内参基因的筛选对目的基因定量标准化研究非常必须。4、采用qRT-PCR技术研究了三叶斑潜蝇4条hsp70s在高低温胁迫和不同发育阶段条件下的表达情况。结果表明高低温胁迫都能诱导Lthsp70s的表达,但表达情况各不相同。Lthsc70和Lthsp702对温度胁迫不敏感其表达量最高时的温度(Tmax)的表达倍数与对照组相比显著比Lthsp701和Lthsp703低;4条hsp70s在三叶斑潜蝇各发育阶段中均能表达,Lthsc70和Lthsp702在各发育阶段中表达差异显著,Lthsc70在雌成虫中的表达量最高,Lthsp702在幼虫和预蛹期的表达量最高,而Lthsp701和Lthsp703在各发育阶段表达差异不显著。
[Abstract]:Liriomyza Liriomyza belongs to Diptera Agromyzidae Diptera, Agromyzidae, Phytomyzidae. subfamily of leafminer Liriomyza species, interspecific competition and substitution rapidly, spreading all over the world. The harm to Liriomyza trifolii Liriomyza trifolii, Liriomyza huidobrensis and Liriomyza sativae Liriomyza huidobrensis is my intrusion in three kinds of hazards of polyphagous leafminer seriously. Recent surveys show that in the Jiangsu area is mainly the occurrence of Liriomyza sativae Blanchard and Liriomyza trifolii, not of L.huidobrensis found. In addition, species on temperature stress tolerance and other factors together determine their survival and distribution. Competition and diffusion. Heat shock protein is considered to be a kind of insect protein most closely with the temperature tolerance relation, in which heat shock protein 70 (HSP70) family and the relationship between the temperature stress is most closely related, and leaf spot HSP70 study of miner understood. In this study, 2014-2016 years of Jiangsu Blanchard hazards were investigated; meanwhile we cloned 4 HSP70 genes of Liriomyza trifolii, and their expression patterns were studied, and its relative species of Liriomyza HSP70 gene were compared, revealing the anti adversity and ecological adaptation at the molecular level, and provide a theoretical basis for clarifying the intrusion and disaster mechanism. The main results are as follows: 12014-2016 for three consecutive years occurred in Jiangsu area of leafminer damage were investigated, the results show that the Jiangsu area is mainly the occurrence of liriomyzasativae and clover leaf miner flies, and leafminer mainly in the northern Jiangsu area occurrence. Liriomyza trifolii mainly in South of Jiangsu Jiangsu occurrence, two leafminers common occurrence areas are mainly concentrated in the Soviet Union and the Ministry of land along the Yangtze River No matter in the area. In addition, the host species, the degree of harm or damage area, Liriomyza trifolii is liriomyzasativae have a greater competitive advantage, and Yangzhou area of Liriomyza trifolii replaced the leafminer, become the area the advantage of vegetable leafminers Liriomyza trifolii in, at the same time the Yangzhou area has 3 annual occurrence peak, which in mid August the population of the largest.2, with RT-PCR and RACE (Rapid-amplification of cDNA ends) technology, from Liriomyza trifolii cloned 4 HSP70 genes, respectively Lthsc70, Lthsp701, Lthsp702 and Lthsp703.4 heat shock protein gene cDNA sequences respectively. 233222612078 and 2296 BP, the length of ORF was 195619381899 and 1923 BP, respectively, encoding 651645632 and 640 amino acids, and the 4 typical domains of heat shock protein has sequence analysis of HSP70 family. Found that the 4 Lthsp70s and HSP70 of other species with high similarity, and the phylogenetic tree was classified into two categories. By comparing the genomic sequence and cDNA sequence, Lthsp701, Lthsp702 and Lthsp703 do not contain introns, while Lthsc70 contains 1 introns. The genome length is 1273 BP. Splice site of intron with the classic GT-AG rule.3 was cloned by RT-PCR 9 Liriomyza trifolii reference gene fragments, including ACTIN, 18S, TUB, AK, EF-1, CAD, RPL32, GAPDH and H3 by qRT-PCR (Quantitative real-time PCR) technology of Liriomyza trifolii stability 9 reference genes under different experimental conditions, the use of reference genes commonly used screening program (NormFinder, geNorm and BestKeeper) and ACt method were ranked for each reference gene, and finally use the RefFinder "program for the comprehensive assessment, and obtained the best in Referencegene scheme. The most suitable combination of reference genes in different developmental stages are as follows: ACTIN and 18S; female was TUB and 18S, ACTIN and GAPDH for high temperature stress; low temperature stress for ACTIN and 18S. finally, to validate the reference gene screening by Lthsp21.7 gene screening, the reference genes on the research objective gene quantitative standards are very.4, using qRT-PCR technology to study the Liriomyza trifolii 4 Hsp70s in high and low temperature stress and different developmental stages under the condition of expression. The results showed that expression of Lthsp70s could be induced by high temperature stress, but the expression of different.Lthsc70 and Lthsp702 on the temperature stress is not sensitive to the expression amount of the highest the temperature (Tmax) expression ratio compared with the control group significantly than Lthsp701 and Lthsp703; 4 Hsp70s expressed at different developmental stages of Liriomyza trifolii, Lthsc70 and Lthsp702 in the hair In the breeding stage, the expression level was significantly different. The expression level of Lthsc70 in the female adults was the highest, while the expression level of Lthsp702 in the larvae and prepupal stage was the highest, while the expression of Lthsp701 and Lthsp703 in different developmental stages was not significant.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433

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