Occurrence And Damage Regularity Of Liriomyza Species In Jiangsu,Cloning And Expression Of HSP70s CDNA In L.Trifolii

Liriomyza belongs to Diptera,Agromyzidae,and Phytomyzidae.There are many different kinds of leafminer species,which interspecific competition and substitution quickly and invade to all over the world.Liriomyza trifolii,Liriomyza sativae and Liriomyza huidobrensis are three invasive insect pests of vegetable crops and ornamental plants in china and elsewhere in the word.The previous survey showed that L.sativae and L.trifolii were the mainly species of leafminer in Jiangsu,while not found of L.huidobrensis.The tolerance ability of species to temperature stress is an important condition for their survival,distribution,competition and diffusion.The expression of hsps has been widely studied and which is one of the best predictors of insect tolerance to temperature stress and the HSP70 represents one of the most highly conserved proteins in HSP family.Compared with its congener species,the study of heat shock proteins in L.trifolii was relatively limited.In this study,we investigated the occurrence and damage of the leaf miner in Jiangsu area duing 2014-2016 and the cloning and expression of hsp70s in L.trifolii under different treatments was also been studied.These results showed,in terms of gene expression levels and compare the differences between L.trifolii and it congener species and to reveal their ability to resist stress and ecological adaptation at the molecular level.The main results are as follows:1.The occurrence and distribution of Liriomyza species were investigated in Jiangsu for consecutive three years since 2014,the results showed:Species of Leafminer of vegetable crops and ornamental plants in Jiangsu were L.sativae and L.trifolii,L.sativae hazards mainly in the northern regions of Jiangsu while L.trifolii is mainly distribted across the central and southern areas of Jiangsu,L.sativae and L.trifolii cooccurrence areas are mainly concentrated in Jiangsu medium and along the Yangtze River.The figure legend of Liriomyza species distribution and damage situation was recovered in the present study.At the same time,we analyzed the host plants difference of two species of leafininer in the Jiangsu and the annual variation of the population of the Liriomyza spp.in Yangzhou.The results showed that regardless of the host plants,the degree of damage or the acreage of damage shown on that the competitive advantage of L.trifolii was stronger than that of the L.sativae,L.trifolii occurred from March to November and there were three peak periods of damage during the year.The most serious damage occurred in early August.Meanwhile,L.sativae has been displaced by the L.trifolii,which was the dominate specie of Liriomyza in Yangzhou.2.Four heat shock protein 70 genes were cloned from L.trifolii by RT-PCR and RACE,and were named Lthsc70?Lthsp701?Lthsp702 and Lthsp703,respectively.The complete cDNA of four HSP70 genes are 2332?2261?2078 and 2296 bp respectively,and their respective opening reading frames are 1956?1938?1899 and 1923 bp,which encode 651?645?632 and 640 amino acids,respectively.Sequence analysis suggested that the four Hsps shared high similarity with those of other species and contained conserved motifs found in HSP70 respective family of other species.At the same time,phylogenetic analysis showed that four HSP70s were divided into two branches in the phylogenetic tree.Lthsc70 contained an intron,1273 bp,located in the coding region.But no introns were found in Lthsp701?Lthsp702 and Lthsp703.And the intron genomic shear sites were consistent with the classical GT-AG rule.3.Nine reference genes(partial sequence)of ACTIN,18S,TUB,AK,EF-1,CAD,RPL32,GAPDH and H3,were cloned from L.trifolii by RT-PCR.And these genes were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper,geNorm,Normfinder and the comparative ACt method by qRT-PCR.Then,we chose a web-based comprehensive tool Reffinder to get the final ranking.The results indicated that the best sets of candidates as reference genes were as follows:Different developmental stages were ACTIN and 18S;Sex was TUB and 18S;high temperature stress was ACTIN and GAPDH,low temperature stress was ACTIN and 18S.Finally,we used Lthsp21.7 as target gene to validate the suitable reference genes under different experimental conditions.So the choice of internal standard is necessary to normalize the target gene expression levels.4.The expression patterns of four Lthsp70s under different development stages and temperature stress were studied by real-time quantitative PCR.The results showed that the expression of all four Lthsp70s could be induced by high and low temperature stress,but the expression patterns were different.Lthsc70 and Lthsp702 were not sensitive to temperature stress,and the expression of Tmax was significantly lower than that of Lthsp701 and Lthsp703.Four Lthsp70s could be expressed in all developmental stages in this study.The expression of Lthsc70 and Lthsp702 have significantly differences in developmental stage and adult female was the highest expression level of Lthsp702,but in Lthsp702 larvae and prepupae were the highest expression level.In Lthsp701 and Lthsp703,there were not significantly different in different development stages.