【作者】 王勇；

【导师】 秦利；

【作者基本信息】 沈阳农业大学 ， 有害生物与环境安全， 2014， 博士

【摘要】 柞蚕(Antheraea pernyi Guerin-Meneville,1855)属于鳞翅目大蚕蛾科,是一种重要的泌丝昆虫。我国年产柞蚕茧约7×104t,占世界野蚕丝总产量的90%以上。柞蚕微粒子病的病原物为柞蚕微孢子虫(Nosema pernyi Wen et Ding),是柞蚕生产中的唯一检疫性病害。该病广泛分布于我国柞蚕产区,给柞蚕生产带来了严重的经济损失,近年来柞蚕微粒子病的发生和蔓延呈现出上升趋势,已成为制约柞蚕产业发展的重要因素之一。因此,开展柞蚕微孢子虫的系统分类学研究、基因信息的获取、孢壁蛋白基因的克隆及柞蚕感染微孢子虫后相关基因表达量的应激变化等研究,对于更深层次了解柞蚕微孢子虫的生物学特征、保障柞蚕产业的健康发展具有重要意义。本研究对Percoll纯化的柞蚕微孢子虫进行了光学和电子显微镜的观察；通过提取柞蚕微孢子虫的总RNA,采用SMART法构建全长cDNA文库,并对EST进行分析；通过微管蛋白基因构建系统进化树；首次克隆得到了柞蚕微孢子虫孢壁蛋白NpSWP1基因,并进行了原核表达和Western-blot验证；利用Realtime PCR技术对感染微孢子虫后柞蚕中肠ApTP1和ApHSC70基因的表达谱进行了分析。研究结果如下：1.柞蚕微孢子虫的形态学观察：利用Percoll不连续密度梯度(Percoll浓度分别为25%、50%、75%、100%),15000r-min-1离心30min,能够得到纯净度很高的柞蚕微孢子虫孢子。通过光学显微镜观察并结合CCD系统进行孢子大小的测量,纯化的孢子在光镜下由于折光性四周呈现淡蓝色荧光,孢子个体大小均匀,呈卵圆形,孢子大小4.36±0.42μm×1.49±0.19μm,扫描电镜显示孢子表面光滑,透射电镜能够看出成熟的柞蚕微孢子虫孢子壁分为外壁、内壁和原生质膜,孢子具有双核,10-11圈极丝围绕着细胞核,纵切面显示极丝成同心圆结构。2.柞蚕微孢子虫的系统发育分析：采用RT-PCR、3’RACE(Rapid amplification of cDNAends)等技术克隆得到了柞蚕微孢子虫的α、β和γ-微管蛋白基因,并利用α、β-微管蛋白序列,分别采用NJ、ML法构建进化树。结果显示,微孢子虫类以一个独立群位于真菌群体中,与真菌的虫霉门关系较近,且与担子菌、球囊菌、壶菌、接合菌及部分子囊菌互为姐妹群。从部分微孢子虫的系统发育分析结果可以看出,20种微孢子虫分为2个分支,柞蚕微孢子虫与其他Nosema属聚为一类。3.柞蚕微孢子虫cDNA文库的构建。采用SMART(Switching mechanism at5’-end of RNA transcript)技术构建了柞蚕微孢子虫全长cDNA文库,初始文库滴度4.2×106pfu/mL,文库容量1.0×107pfu,重组率为98.39%。随机挑取32个克隆,经PCR检测插入的片段长度在750-3000bp,平均长度>1000bp。挑选文库中864个克隆进行表达序列标签(EST)测序,得到626条高质量的EST,拼接后得到197条单一基因(unigenes),包含64条重叠群(contigs)和133条单-EST (singlets),冗余度为68.53%。将这些Unigenes与NCBI数据库进行比对,146条Unigenes能够比对到蜜蜂微孢子虫(Nosema ceranae)、家蚕微孢子虫(Nosema bombycis)、兔脑炎微孢子虫(Encephalitozoon cuniculi)等的相关基因。在随机EST测序中克隆获得了一个孢子形成蛋白(sporulation protein)基因,命名为NpSP-1.该基因的cDNA的ORF长度为1014bp,编码337个氨基酸,蛋白质的理论分子质量为39.2kD,等电点5.71。4.柞蚕微孢子虫孢壁蛋白NpSWP1的克隆及原核表达：从已构建的柞蚕微孢子虫cDNA文库中筛选出一条孢壁蛋白EST序列,并结合相近物种进行同源性分析,利用RT-PCR和3’RACE技术克隆得到了一个柞蚕孢壁蛋白基因,命名为NpSWP1(GenBank登录号：KJ573111)。该基因ORF长837bp,编码278个氨基酸,推测的蛋白分子量(Mw)32.02kD,等电点(pI)7.02。通过Blastp分析,与家蚕微孢子虫孢壁蛋白MSWP1的氨基酸序列同源性达到了79%,预测该蛋白也是一种孢子内壁蛋白。将该基因与原核表达载体pEASY(?)-E1Expression vector相连,并转化到感受态细胞Transetta (DE3), SDS-PAGE结果表明,IPTG诱导5h,重组质粒能够表达约32kD的融合蛋白,与预期结果相近。Western-blot检测显示,只有经过诱导的菌液能够杂交产生约32kD的条带。5.柞蚕ApHSC70基因的表达谱分析：从健康和感病柞蚕中肠转录组数据中筛选了一条热休克蛋白HSP70基因,利用RT-PCR技术克隆得到了一个柞蚕热休克蛋白HSP70基因,命名为ApHSC70(GenBank登录号：KJ437496),该基因的开放阅读框(ORF)长1959bp,编码652个氨基酸,预测蛋白质分子质量为71.49kD,等电点pI值为5.38,具有细胞质特征基序。实时荧光定量PCR检测该基因在添食柞蚕微孢子虫后的应激变化,结果显示：在感染的0-12h内表达量变化不大,15h含量升高,21h达到最高。6.柞蚕ApTP1基因的表达谱分析：人工添食柞蚕微孢子虫的条件下,研究柞蚕中肠一个类胰蛋白酶基因对微孢子虫入侵的应激变化。通过RT-PCR技术克隆的到了该基因,命名为ApTP1(GenBank登录号：KF779933),该基因ORF长774bp,编码257个氨基酸,预测前20个氨基酸为信号肽,蛋白质的分子量为27.7kD,等电点为10.5。结构预测显示ApTP1蛋白具有典型的胰蛋白酶功能结构域Tryp-SPc (trypsin-like serineprotease),含有3对二硫键, IVGG保守序列。同源比对及系统进化关系结果表明,柞蚕类胰蛋白酶与天蚕(A ntheraea yamamai)的同源性最高,为93%,与其他昆虫的同源关系在30%-70%之间。原核表达和Western-blot研究表明,融合蛋白的表达及鉴定与预测的28kD大小一致。半定量PCR检测显示ApTP1在柞蚕各发育时期及5龄幼虫的各组织中均表达,其中在柞蚕发育的4个阶段中以幼虫期的表达水平相对较高,各组织中以中肠组织的表达水平相对较高。Realtime PCR检测显示ApTPl基因的表达量在诱导后1-3h内变化不大,6h的表达量最高。更多还原

【Abstract】 The Chinese oak silkworm (Antheraea pernyi Guerin-Meneville,1855) belongs to Lepidoptera:Saturniidae which is the most well known wild silkmoths. The annual output of silkworm cocoons from China is about7×104t accounts for90%of world production. Nosema pernyi (Wen et Ding) is a lethal pathogen of microsporidiosis in Antheraea pernyi which is the only quarantine disease in sericultural production. This disease is widely distributed in most sericultural areas and cost economic losses. In recent years, microsporidiosis occurring and spreading rate rised. Carry on the systematics research, obtaining genes information, cloning spore wall protein gene and the pattern of genetic expressions in midgut of A. pernyi after N. pernyi infection, these researches have significant role in studing biological characteristics of N. pernyi and guarantee the healthy development of sericultrual industry.This research observed purified spores of N. pernyi using light and electron microscope; total RNA was extracted for cDNA library construction using SMART technology and ESTs were analysed; phylogenetic trees were constuctied based on tubulin genes; one spore wall protein of N. pernyi was cloned and prokaryotic expression was successfully induced; Realtime PCR technology were used to investigate the genes of ApTPl and ApHSC70expression patterns in the midgut of A. pernyi after N. pernyi infection. The results were as follows:1. Morphological study of N. pernyi spores. Purified spores of N. pernyi can be obtained using differential centrifugation and Percoll density gradient centrifugation (discontinuous density gradient Percoll:25%,50%,75%and100%) at15000r-min-1for30min. Using light microscope and CCD system, we tested the size of spores. Purified spores were surrounded by blue-fluorescence due to refractivity under light microscope. Uniformity spores were oval like, and the mean length and width measurements for N. pernyi were4.36±0.42μm and1.49±0.19μm, respectively. Using a scanning electron microscope, the spots have a smooth surface. Transmission electron microscopy of longitudinal sections and cross-sections of spores revealed that the spore wall consisted of exospore, endospore and plasma membrane. The diplokaryotic nuclei occupied the center of the spore and were surrounded by10-11turns of the polar tube.2. Phylogenetic study of N. pernyi. Using RT-PCR,3’RACE technology, α,β and y-tubulin genes of N. pernyi were cloned. Neighbour-Joining and Maximum Likelihood were used to construct a phylogenetic tree. Phylogenetic analysis shows that the microsporidia are a single group in the fungi that are most closely related to the Entomophthoromycota, and are sistergroups to the Basidiomycota, Chytridiomycota, Zygomycota, Glomeromycota and some of the Ascomycota. N. pernyi, together with other Nosema spp., belongs to one of two groups in the microsporidia.3. cDNA library of N. pernyi. Using SMART (switching mechanism at5’end of RNA transcript) technology, we constructed a full-length cDNA library of N. pernyi. The identification results of library construction quality showed that the titer of primary library was4.2×106pfu/mL, the library capacity was1.0×107pfu with98.39%recombinant. PCR amplification of32randomly picked clones revealed that the inserted cDNA fragments ranged from750to3000bp with an average length of1000bp. These data indicated that a successful cDNA library of N. pernyi has been constructed in this research. From864randomly selected and sequenced clones,626high-quality ESTs were generated. These ESTs were assembled into197Unigenes including64Contigs (38.49%) and133Singlets (61.51%). The redundancy rate is68.53%. Among these Unigenes which had homology with submitted sequences in the NCBI database, about146Unigenes were similarity to known mRNAs of Nosema ceranae, Nosema bombycis, and Encephalitozoon cuniculi with BLASTn and BLASTx analysis. A full-length gene encode Sporulation Protein (named NpSP-1) has got from the cDNA library. This cDNA contained an ORF of1014bp coding for a protein of337amino acids. The predicted molecular weight and isoelectric point of this protein was39.2kD and5.71, respectively.4. NpSWPl cloning and prokaryotic expression. From the constructed cDNA library of N. pernyi, an EST of annotated spore wall protein was seletcted. Using RT-PCR and3’RACE technology, a spore wall protein gene named NpSWPl (GenBank accession number: KJ573111) from N. pernyi were cloned by screening the EST information and analyzing homological genes. This cDNA contained an ORF of837bp coding for a protein of278amino acids. The predicted molecular weight and isoelectric point of this protein was32.02kD and7.02, respectively. Thought Blastp analysis, the deduced amino acid sequence of NpSWP1shared79%identity with NbSWP1(GenBank accession number:EOB12097) in Nosema bombycis, and inferred NpSWP1to be an endosporal protein. Recombinant plasmid of NpSWP1-E1Vector was transferred into Transetta (DE3) and the target protein was expressed. According to SDS-PAGE result, the molecular weight of the target protein was32kD under the condition of IPTG for5h. Western-blot showed that the target gene was about32kD, and it has been successfully expressed in E. coli.5. Expression pattern analysis of ApHS70. From the transcriptomic research of midgut tissues from healthy larvae and N. pernyi infected larvae, HSP70gene was seletcted. Using RT-PCR technology, a HSP70gene of A. pernyi was cloned (named ApHSC70, GenBank accession numbers:KJ437496). The ORF is1959bp and encodes652amino acids with predicted molecular mass of71.49kD and theoretical isoelectric point of5.38, which contains cytoplasmic characteristics motif. Quantitative Real time PCR (qRT-PCR) was employed to further identify the expression pattern of ApHSC70gene in the midgut of A. pernyi after N. pernyi infected. qRT-PCR indicated that ApHSC70gene content in the midgut of A. pernyi little changed during0-12h, content begin rised at15h and reached the maximum expression at21h.6. Expression pattern analysis of ApTP1. Under the condition of artificial feeding N. pernyi, a trysin-like serine proteases protein expression pattern was studied. Using RT-PCR, ApTP1(GenBank accession number:KF779933) was cloned. This gene contained an ORF of774bp coding for a protein of257amino acids. The predicted molecular weight and isoelectric point of this protein was27.7kD and10.5, respectively. ApTP1had the common characteristics of trypsin-like serine proteases with the catalytic active centers of histidine, aspartic acid and serine. The predicted protein constructed of six conservative cysteines and a20-residue signal peptide sequence. Thought Blastp analysis, the deduced amino acid sequence of ApTP1shared930identity with Antheraea yamamai, the homology of other species ranged from30%to70%. Recombinant plasmid of ApTP1-E1Vector was transferred into E. coli and the expressed protein was28kD the same as forecast. Semiquantitative PCR detection indicated that ApTP1is expressed in all stage and tissues of5th larvae, but the highest expression was the instar of larvae, the tissues of midgut has the highest expression in5th instar. qRT-PCR indicated that ApTP1changed little from1to3h, and6h reach the highest level.更多还原