發(fā)布時間：2018-02-16 09:30

  本文關(guān)鍵詞： 斑潛蠅 三葉斑潛蠅 發(fā)生危害 熱激蛋白70 內(nèi)參篩選 基因表達(dá)模式　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：斑潛蠅屬Liriomyza隸屬于雙翅目Diptera、潛蠅科Agromyzidae、植潛蠅亞科Phytomyzidae。斑潛蠅種類繁多,種間競爭與替代迅速,不斷向世界各地蔓延危害。三葉斑潛蠅Liriomyza trifolii,美洲斑潛蠅Liriomyza sativae和南美斑潛蠅Liriomyza huidobrensis是入侵我國危害嚴(yán)重的三種多食性斑潛蠅。近年的調(diào)查顯示,在江蘇地區(qū)發(fā)生危害的主要是美洲斑潛蠅和三葉斑潛蠅,未發(fā)現(xiàn)南美斑潛蠅的危害。此外,物種對溫度脅迫的耐受能力與其它因素一起決定它們的生存、分布、競爭和擴(kuò)散。熱激蛋白被認(rèn)為是與昆蟲溫度耐受性關(guān)系最密切的一類蛋白質(zhì),其中熱激蛋白70(HSP70)家族與溫度脅迫的關(guān)系最為密切,而有關(guān)三葉斑潛蠅的HSP70研究甚少。本研究于2014-2016年對江蘇地區(qū)斑潛蠅發(fā)生危害情況進(jìn)行了調(diào)查;同時克隆了三葉斑潛蠅的4條HSP70基因,并對其表達(dá)模式進(jìn)行了研究,同時還與其近緣斑潛蠅種類的HSP70基因進(jìn)行比較,從分子水平上揭示其抗逆境和生態(tài)適應(yīng)能力,為闡明其入侵及成災(zāi)機(jī)制提供理論依據(jù)。主要研究結(jié)果如下:1、2014-2016年連續(xù)三年對江蘇地區(qū)斑潛蠅的發(fā)生危害進(jìn)行了調(diào)查,結(jié)果表明:江蘇地區(qū)主要發(fā)生危害的是美洲斑潛蠅和三葉斑潛蠅,且美洲斑潛蠅主要在蘇北地區(qū)發(fā)生危害,三葉斑潛蠅主要在蘇南蘇中地區(qū)發(fā)生危害,兩種斑潛蠅共同發(fā)生危害的區(qū)域主要集中在蘇中和沿江的部分地區(qū)。此外,不論在寄主種類、危害程度還是危害面積上,三葉斑潛蠅較美洲斑潛蠅具有更大的競爭優(yōu)勢,而且揚(yáng)州地區(qū)三葉斑潛蠅基本取代美洲斑潛蠅,成為該地區(qū)蔬菜上的優(yōu)勢種斑潛蠅,同時三葉斑潛蠅在揚(yáng)州地區(qū)全年有3個發(fā)生危害高峰,其中8月上中旬的種群數(shù)量最大。2、采用 RT-PCR 和 RACE(Rapid-amplification of cDNA ends)技術(shù),從三葉斑潛蠅中克隆得到4條HSP70基因,分別為Lthsc70、Lthsp701、Lthsp702和Lthsp703。4條熱激蛋白基因cDNA序列全長分別為2332、2261、2078和2296 bp,開放閱讀框長度分別為1956、1938、1899和1923 bp,分別編碼651、645、632和640個氨基酸,同時這4個熱激蛋白都具有HSP70家族的典型結(jié)構(gòu)域。序列分析發(fā)現(xiàn),這4條Lthsp70s與其它物種的hsp70具有很高的相似性,并在系統(tǒng)進(jìn)化樹上被歸為兩類。通過比對基因組序列和cDNA序列發(fā)現(xiàn),Lthsp701、Lthsp702和Lthsp703均不含有內(nèi)含子,而Lthsc70含有1個內(nèi)含子,長度為1273 bp,內(nèi)含子的基因組剪切位點(diǎn)符合經(jīng)典的GT-AG規(guī)則。3、通過RT-PCR技術(shù)克隆了 9條三葉斑潛蠅內(nèi)參基因片段,包括ACTIN,18S,TUB,AK,EF-1,CAD,RPL32,GAPDH和H3 利用 qRT-PCR(Quantitative real-time PCR)技術(shù)研究了三葉斑潛蠅9條內(nèi)參基因在不同實(shí)驗(yàn)條件下的穩(wěn)定性,運(yùn)用常用的內(nèi)參基因篩選程序(NormFinder、geNorm和BestKeeper)以及比較ACt法對各內(nèi)參基因進(jìn)行了排名,最后使用RefFinder網(wǎng)頁程序進(jìn)行綜合評估,得出最佳的內(nèi)參基因方案。最合適的內(nèi)參基因組合如下:不同發(fā)育階段為ACTIN和18S;性別為TUB和18S,高溫脅迫為ACTIN和GAPDH;低溫脅迫為ACTIN和18S。最后,利用目的基因Lthsp21.7對篩選的內(nèi)參基因進(jìn)行驗(yàn)證,結(jié)果表明內(nèi)參基因的篩選對目的基因定量標(biāo)準(zhǔn)化研究非常必須。4、采用qRT-PCR技術(shù)研究了三葉斑潛蠅4條hsp70s在高低溫脅迫和不同發(fā)育階段條件下的表達(dá)情況。結(jié)果表明高低溫脅迫都能誘導(dǎo)Lthsp70s的表達(dá),但表達(dá)情況各不相同。Lthsc70和Lthsp702對溫度脅迫不敏感其表達(dá)量最高時的溫度(Tmax)的表達(dá)倍數(shù)與對照組相比顯著比Lthsp701和Lthsp703低;4條hsp70s在三葉斑潛蠅各發(fā)育階段中均能表達(dá),Lthsc70和Lthsp702在各發(fā)育階段中表達(dá)差異顯著,Lthsc70在雌成蟲中的表達(dá)量最高,Lthsp702在幼蟲和預(yù)蛹期的表達(dá)量最高,而Lthsp701和Lthsp703在各發(fā)育階段表達(dá)差異不顯著。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S433

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