【作者】 陈建华；

【导师】 张克云；

【作者基本信息】 南京农业大学 ， 动物学， 2017， 硕士

【摘要】 昆虫病原线虫(Entomopathogenicnematodes,EPN)指以昆虫为寄主的致病性线虫,其致病性源自于其肠道内所携带的共生菌,二者呈互惠共生关系。目前已发现的昆虫病原线虫有3大类,分别是小杆科(Rhabditidae)拟异小杆属(Heterorhabditidoides)、异小杆科(Heterorhabditidae)和斯氏线虫科(Steinernematidae)。本研究试材崇明拟异小杆(Heterorhabditidoides chongmingensis)线虫为本实验室在上海崇明岛分离到的EPN新种,其与嗜线虫沙雷氏菌(Serratia nematodiphila)共生。前期研究运用Solexa高通量测序技术对崇明拟异小杆线虫的模式品系DZ0503CMFT(DZ)与其自身共生菌DZ0503SBS1T(S1)和非自身共生菌DR186(186)构建形成的单菌线虫组合DZ-S1和DZ-186的小RNA文库进行测序,两文库中新型miRNA的表达量具有显著差异。本研究选取显著上调的n-miR-10和n-miR-9进行研究,获得以下主要研究结果:扩增差异表达的n-miR-9和n-miR-10含前体序列在内的片段,序列长度分别为293 bp与330 bp。利用分子生物学技术将n-miR-9与n-miR-10的前体与过表达载体重组。构建好的过表达载体以饲喂法转入DZ-S1线虫中,培养3天后提取总RNA并反转成cDNA,采用荧光定量的方法检测候选靶基因表达量变化。结果表明,n-miR-9候选靶基因的表达量差异明显,呈现上调。n-miR-10候选靶基因均有显著性下调。为了进一步鉴定n-miR-9与n-miR-10的靶基因,以DZ-S1线虫的cDNA为模板扩增靶基因。分别扩增出n-miR-9候选靶基因DZMN9-3和n-miR-10的候选靶基因DZMN10-1、DZMN10-3和DZMN10-5,并构建相应的靶基因过表达载体。含有绿色荧光蛋白(Green fluorescent protein,GFP)的靶基因过表达载体与miRNA过表达载体共转染DZ线虫,利用荧光显微镜检测DZ线虫体内绿色荧光变化。结果显示:DZMN9-3对照组在培养3天后DZ体内绿色荧光并无明显差异,而实验组在培养三天后DZ体内绿色荧光有一定程度的减弱,结合荧光定量的结果:我们推测n-miR-9还存在着其他的靶位点,能调控启动子等转录元件来提高基因的表达;n-miR-10靶基因DZMN10-1的实验组DZ线虫体内荧光有明显降低与荧光定量结果相符合,靶基因DZMN10-3与DZMN10-5实验组均无明显变化。将过表达载体pPD95.67::DZMN9-3与pPD95.67::DZMN10-1通过饲喂法转入DZ线虫中发现,DZMN9-3对DZ线虫的发育率具有一定的抑制作用,与n-miR-9对DZ发育率影响结果相一致;DZMN10-1对DZ线虫的体长起抑制作用。更多还原

【Abstract】 Entomopathogenic nematodes(EPNs)are a kind of parasitic nematodes which can use insects as the hosts.The pathogenicity of EPNs is generated by the symbiotic bacteria that have the mutualistic symbiosis relationship with them.To date,three kinds of EPNs have been found,Heterorhabditidoides,Heterorhabditidae and Steinernematidae.DZ0503CMFT(DZ),the type strain of Heterorhabditidoides chongmingensis was isolated from the Chongming Island in Shanghai and has a stablely mutual relationship with its symbiotic bacteria strain DZ0503SBS1T(S1)of Serratia nematodiphila.Previous studies found that the expression of miRNAs in DZ-S1 and DZ-186,the two monoxenic nematode-bacterial complexes,were significantly different revealed by Solexa sequencing technology.In order to understanding the relationship between miRNAs and symbiotic bacteria in the two monoxenic nematode combinations,we obtained the precursor sequences of the two differentially expressed novel miRNA n-miR-9 and n-miR-10 by PCR amplification.Their sequences were 293bp and 330bp,respectively.Overexpression vector of n-miR-9 and n-miR-10 was successfully constructed by molecular biology.The overexpression vector was transfected into DZ-S1 nematode and cultured for 3 days.Total RNA was extracted and inversely transcripted into cDNA.The expression of candidate target genes was detected by qRT-PCR.The results showed that the expression of n-miR-9 candidate target genes was significantly different,showing up-regulation.n-miR-10 candidate target genes were significantly down-regulated.Growth and development experiments showed that n-miR-9 inhibited the development of DZ nematodesFor further identification the target genes of n-miR-9 and n-miR-10,were cloned and inserted into the plasmid pPD95.67 containings green fluorescent protein to construct recombinant plasmid which was then cotransformed into DZ nematodes with the overexpression vector of n-miR-9 and n-miR-10 by feeding method.Results showed that three days later,there was no significant difference of green fluorescence in the control group,whereas the green fluorescence of the experimental group of target genes DZMN9-3 was weakened in the DZ nematodes.We speculated that n-miR-9 also had other target sites,that could regulate the transcription factor such as promoters for improving the expression of genes.N-miR-10 had a significant decrease in the fluorescence of the potential target gene DZMN10-1 in experimental group,which correspond to the results of quantitative.There were no significant changes in the potential target genes DZMN10-3 and DZMN10-5 in the experimental group.Our results indicated that DZMN9-3 inhibited the development of DZ nematodes which was consistent with the effect of n-miR-9 on DZ nematodes’development rate,and DZMN10-1 inhibited the growth of DZ nematodes’ body length by transfecting the over-expression vectors pPD95.67::DZMN9-3 and pPD95.67::DZMN10-1.更多还原