花绒寄甲成虫3条Mthl基因及Mth-like 2-5干扰效果研究
Three mthl Genes and RNA Interference Effect of Mth-like 2-5 in the Audlt of Dastarcus helophoroides

筛选花绒寄甲成虫高表达的mthl基因，注射目标基因dsRNA，以确定干扰效果并解析其功能。合成花绒寄甲成虫目标基因mthl保守序列的dsRNA，分别注射0.3、1.2 μg/μL和4.8 μg/μL的dsRNA，利用实时荧光定量PCR技术检测目标基因的表达量，记录处理组虫体的体重、存活率和雌虫产卵情况。从花绒寄甲转录组数据库中分别鉴定出Mth-like 11、Mth-like 2-4、Mth-like 2-5基因。注射目标基因Mth-like 2-5的长度约400 bp的dsRNA，对目标基因表达干扰效果显著，但干扰过程中雌雄虫均具有应激表达反应。其中0.3 μg/μL低浓度dsRNA对目标基因干扰效果差，应激高表达反应明显；1.2 μg/μL中等浓度dsRNA对目标基因有显著的干扰效果，应激高表达反应弱、引起的体重下降和死亡率较小，雌虫产卵期推迟；4.8 μg/μL高浓度dsRNA干扰效果显著，引起的体重下降和死亡率较高，雌虫难以产卵。注射Mth-like 2-5 dsRNA可影响Mth-like 2-5基因的正常表达和转录，随dsRNA浓度增大和处理后时间的延长，目标基因表达量、雌雄成虫体重明显降低，死亡率增高，雌成虫产卵期推迟或难以产卵。
The high-expression mthl gene was screened and the target gene dsRNA was injected to determine the interference effect and analyze its function in Dastarcus helophoroides.Synthetic mthl gene target gene conservative sequence of dsRNA was injected in the adult of D.helophoroides with dosages of 0.3,1.2 μg/μL and 4.8 μg/μL dsRNA respectively.Real-time qPCR technique was used to detect the expression of target genes.The body weight of treatment group,survival rate,and the conditions of female egg-laying were recorded.The results showed that,Mth-like 11,Mth-like 2-4 and Mth-like 2-5 genes were identified from the transcriptome database of D.helophoroides.The length of the target gene Mth-like 2-5 was about 400 bp dsRNA,which had a significant effect on the expression of the target gene.Low concentration dsRNA with 0.3 μg/μL had poor interference effect on target genes and obvious high expression response to stress.Medium concentration dsRNA (1.2 μg/μL) had significant interference effect on target genes with weak high expression response to stress,decreased weight and low mortality,and delayed spawning period of female.High concentration dsRNA (4.8 μg/μL) had significant interference effect,resulting in weight loss and high mortality,and female was difficult to lay eggs.Injected Mth-like 2-5 dsRNA could affect the normal expression and transcription of the Mth-like 2-5 genes; with the increase of concentration of dsRNA and the extension of processing time,the target gene expression,male and female adult weight decreased,mortality rate increased,female adult spawning period delayed or was difficult to lay eggs