【作者】 郭建洋；

【导师】 叶恭银； 刘树生；

【作者基本信息】 浙江大学 ， 环境生物学， 2010， 博士

【摘要】 科学家近年来的研究表明,B／Q型烟粉虱成功入侵的原因主要有以下几种：一是生殖干涉,即非对称性交配；二是感染双生病毒,即与病毒的互利互惠；三是烟粉虱对化学杀虫剂的抗性。但引起这些机制的生理和分子途径尚不明确,因此,探讨烟粉虱入侵的生理和分子机制,对于日后有效控制其为害具有深远意义。本研究主要从烟粉虱的生殖发育包括：卵巢形态及发育、精巢发育及卵子发生等为切入点,通过比较不同生物型烟粉虱卵巢内不同发育级别卵子比例、卵子发生过程及卵黄发生动态等的异同,分析引起不同生物型烟粉虱之间生殖差异的生殖基础,并通过克隆卵黄原蛋白(Vitellogenin, Vg)及其受体(Vitellogenin Receptor, VgR)基因,分析比较Vg基因序列和结构,以及检测感染TYLCCNV病毒生物型烟粉虱转录水平的差异,探讨入侵种烟粉虱成功入侵的生殖基础与分子机制。主要结果如下：1烟粉虱内生殖系统形态和卵巢发育的比较分析B型烟粉虱雌虫内生殖系统主要由对称的一对卵巢、一个直径约20μm的受精囊、侧输卵管和中输卵管组成。B型烟粉虱卵巢管属于端滋式发育模式,由12-22根卵巢管组成,可以根据发育时期将烟粉虱的卵巢分为四个不同阶段。根据卵巢内卵子形态和卵黄蛋白沉积的情况,可以把卵子分为四个级别,即Ⅰ、Ⅱ、Ⅲ和Ⅳ级。B烟粉虱的精巢主要由一对对称排列的睾丸、雄性附腺和输精管组成。比较B型和ZHJ1型烟粉虱卵巢发育显示,两种生物型烟粉虱卵巢形态相似,以健康棉花为寄主植物时,生殖发育差异不显著。2烟粉虱卵子发生和精子发生烟粉虱的卵子发生始于伪蛹期,在烟粉虱雌虫开始形成卵巢时,卵原干细胞分别分化成滋养细胞、卵母细胞以及滤泡细胞。红眼期伪蛹已经发现有卵巢管,成虫羽化后12 h就观察到有产卵行为,24 h后解剖出含有成熟卵子的卵巢。48 h解剖出带有受精囊的卵巢。烟粉虱的卵子发生分为滋养期、卵黄发生前期、卵黄发生期和成熟期四个时期。卵母细胞通过滋养细胞运输和自身摄取两种方式进行Vg等营养物质的转运。烟粉虱的精子发生同样始于伪蛹期,精细胞先后经历精原细胞、初级精细胞、次级精细胞和成熟精细胞四个阶段,不同阶段的形态和结构也不同。3烟粉虱Vg单克隆抗体和多克隆抗体的制备通过收集烟粉虱产的新鲜卵作为免疫抗原制备单克隆、多克隆抗体。经小鼠脾细胞与可体外培养并繁殖的入骨髓瘤细胞进行体外融合,利用ELISA筛选阳性克隆并扩大培养后用上清制备腹水,收集腹水经纯化后得到Vg的单克隆抗体。经SDS-PAGE电泳,切取雌性特异性条带,将回收条带电透析后浓缩制备抗原,皮下多点注射雄性白兔,尔后取静脉血经沉淀纯化后获得多克隆抗体。ELISA测定效价发现,单抗效价为1：100,000,多抗的效价为1：68,000。Wertern-blot分析发现抗卵黄蛋白抗体能和几种生物型烟粉虱卵黄蛋白及血淋巴中的雌性特异性蛋白产生免疫反应,但是不能与雄虫提取物起特异性免疫反应。4烟粉虱卵黄发生及Vg特性的分析利用凝胶层析和离子交换层析对烟粉虱卵黄蛋白(Vetellin, Vt)进行了分离纯化,利用非变性凝胶电泳和变性凝胶电泳分析卵黄蛋白分子组成发现,卵黄蛋白是由两个大小为190 kDa相似亚基组成的分子量约380 kDa的大分子蛋白。对烟粉虱Vg性质分析显示,烟粉虱Vg是磷酸化糖基化脂蛋白。利用双抗夹心ELISA对取食健康棉花的烟粉虱不同发育时期Vg含量检测发现,烟粉虱Vg合成始于伪蛹期,羽化后血淋巴中的Vg含量先下降后逐渐上升,卵巢中的Vt基本呈逐渐上升趋势。5烟粉虱Vg基因的克隆及转录表达分析克隆获得B型烟粉虱Vg基因cDNA全长序列为6731 bp,开放阅读框长度为6474bp,共编码2158个氨基酸,预测分子量为242 kDa,等电点为8.9,3’端非编码区为220bp,5’端非编码区为37 bp。其中前16个氨基酸为信号肽,丝氨酸(S)占12.5%,天冬氨酸(N)占12.7%,谷氨酰胺(Q)8.9%。在490个氨基酸有一个RXXR酶切位点,将Vg基因切成编码约50 kDa和190 kDa的大小蛋白。序列相似性比对后发现,烟粉虱Vg基因与玻璃叶蝉Homalodisca coagulata的相似性为86.7%,与叶蜂Athalia rosae的相似性为85.6%,与大鳖负蝽Lethocerus deyrollei的相似性为84.5%。利用荧光定量PCR(Q-PCR)技术检测不同时期Vg基因转录水平分析发现,羽化后烟粉虱Vg表达量呈逐渐升高趋势。6不同生物型烟粉虱Vg基因克隆及分析。ZHJ2型烟粉虱Vg基因全长6721 bp,开放阅读框长度约6549 bp,共编码2183个氨基酸,预测分子量为245 kDa,等电点为8.7,3’非编码区为109 bp,5’非编码区为63bp。其中前22个氨基酸为信号肽,在450个氨基酸有一个RXXR酶切位点,将Vg基因切成编码约50 kDa和190 kDa大小蛋白。丝氨酸(S)占12.5%,天冬氨酸(N)占13.5%,谷氨酰胺(Q)7.1%,丙氨酸(A)9.2%。序列相似性比对后发现,ZHJ-2型烟粉虱Vg基因与菜叶蜂Athalia rosae的相似性为93.9%,与大鳖负蝽Lethocerus deyrollei的相似性为93.5%,与玻璃叶蝉Homalodisca coagulate相似性为89.9%。Q型烟粉虱Vg基因全长6871 bp,开放阅读框长度约6651 bp,共编码2217个氨基酸,预测分子量为246 kDa,等电点为8.9,3’非编码区为107 bp,5’非编码区为43 bp。其中前17个氨基酸为信号肽,在490和790个氨基酸各有一个RXXR酶切位点,可能会将Vg基因切成编码约50 kDa、90 kDa、150 kDa或者190 kDa大小蛋白。丝氨酸(S)占15%,天冬氨酸(N)占12.8%,谷氨酰胺(Q)6.2%,丙氨酸(A)9.2%。序列相似性比对后发现,Q型烟粉虱Vg基因与菜叶蜂A. rosae的相似性较低,仅为63.4%,与大鳖负蝽L. deyrollei的相似性为57.3%,与玻璃叶蝉H. coagulata相似性为55.6%。对三种烟粉虱Vg的核酸序列和氨基酸序列比较分析后发现,入侵种B型／Q型与本地种ZHJ2型烟粉虱相比,在第620-640氨基酸序列间,均比ZHJ2型烟粉虱多一段长度为15个氨基酸的多聚丝氨酸片段。而入侵种Q型与B型、本地种ZHJ2烟粉虱Vg基因序列相比,Q型烟粉虱具有两个酶切位点,且在多聚丝氨酸区多9个重复的GHN功能结构域。三个生物型烟粉虱Vg基因都具有卵黄蛋白典型的功能结构域GCMG和NIIK,以及重复的多聚丝氨酸区,与其它昆虫Vg基因推导氨基酸序列比较,烟粉虱Vg基因不仅含有多个多聚丝氨酸区域,且存在多聚天冬氨酸和谷氨酰胺区,这在昆虫卵黄蛋白基因序列结构上是不多见的。有意思的是在烟粉虱Vg基因序列的氮端多聚丝氨酸区之前,都会出现多个重复的GH/RN功能结构域,其功能还有待进一步研究。7烟粉虱Vg受体基因的克隆及表达分析克隆到B型烟粉虱Vg受体(Vg Receptor, VgR)基因全长cDNA序列,大小5774bp,编码1919个氨基酸,5’端非编码区201 bp,等电点为8.7,分子量大小约201 kDa,跨膜结构域分析显示,VgR跨膜结构位于C-末端的1679-1696个氨基酸位置。烟粉虱的VgR属于低密度脂蛋白受体(Low Density Lipoprotein Receptor, LDLR)家族,具有一些LDLR家族典型的保守结构域,主要包括：配体结合域(Ligand-Binding Domain)、表皮生长因子前体同源域(Epidermal Growth Factor Precursor Homology Domain)、跨膜域(Transmembrane Domain)、O-联糖功能域(O-linked Carbohydrate Domain)及胞质尾域(Cytoplasmic Domain)。同源比对分析显示,B型烟粉虱VgR同美洲大蠊Periplaneta americana相似度达83%,及德国小蠊Blattella germanica相似度达74%,并利用同源建模方法构建了VgR编码蛋白质三维结构。同时用荧光定量PCR (Q-PCR)技术检测分析了B型烟粉虱Vg受体基因转录表达情况。8感染TYLCCNV病毒对烟粉虱生殖力及Vg、VgR基因转录表达的影响对羽化后15 d内的B型烟粉虱雌虫卵巢发育进度观察发现,初羽化的雌虫已经开始卵巢发育,在卵巢内未见成熟的卵子。羽化后2 d发现有卵黄沉积的卵子,发育3-4 d的卵巢内观察到成熟卵子,且随着发育时间的延长,卵巢内成熟卵子数目逐渐增多,至羽化后11-14 d卵巢内成熟卵子数目达到最大。烟粉虱卵巢中的卵子在形态上分为四个级别,分别标记为Ⅰ级、Ⅱ级、Ⅲ级和Ⅳ级,其中Ⅳ级为成熟卵。烟粉虱卵巢时期和卵子发育级别的划分为评价烟粉虱生殖力奠定了基础。比较B型／ZHJ1型烟粉虱取食感染中国番茄黄化曲叶病毒(TYLCCNV)烟草植株与在健康烟草植株上取食2种条件下的卵巢发育进度结果表明,B型烟粉虱在患病植株上取食,卵子发育进度加快,表现在不同发育时间Ⅱ、Ⅲ和Ⅳ级卵子数目显著高于取食未感毒烟草的烟粉虱。然而,观察发现TYLCCNV对土著ZHJ1型烟粉虱卵巢内卵子发育进度却无显著影响。提取同期烟粉虱总RNA,并分别考察了取食健康烟草和感染病毒烟草后的卵黄发生、Vg/VgR基因转录情况。发现了感染病毒后烟粉虱Vg含量、Vg/VgR基因转录水平显著提高。更多还原

【Abstract】 The catastrophic outbreak of biological invasions had made scientists all over the worlds pay attentions to this problem. From the previouse researches, the mechanisms for successful invasion of whiteflies Bemisia tabaci are as follows:first, the asymmetric mating interactions; second, the vector-virus mutualism; third, the resistance of chemical pesticide. However, the physical and molecular mechanisms induced these ecological phenomenon were still poorly understood. It is important for us to verify these mechanisms and find an effective method for the management of this world pest. In our research, the reproductive behaviors in types of whiteflies were observed, the cDNA of vitellogenin and vitellogenin receptor were cloned, the characteristics of genes were compared and the developmental model was observed using RT-PCR. Examination of the difference among three biotypes of whiteflies was aimed to find the molecular and physiological mechanisms of successful invasion. The results are as follows:1 The morphology of reproductive system in whiteflies and comparison of the development of ovariesThe reproductive system of female is mainly composed of a pair of ovary, a spermatheca of 20μm, a pair of lateral oviduct and common oviduct. The ovaries of the whitefly were composed of 12-22 telotrophic ovarioles. Four developmental stages of ovary were divided according to lifespan of whiteflies. The oocytes in ovary were divided into four types, typeⅠ,Ⅱ,ⅢandⅣ. The morphology of male reproductive system was composed of a pair of tesis, male accessory gland and vas deferens. Compared with those ZHJ1 biotype whiteflies, no differences were examined on the development of ovary in the B biotype whiteflies fed on cotton plants.2 The oogenesis and spermiogenesis of Bemisia tabaciThe oogenesis of Bemisia tabaci was observed at the pupal periods. The oogonial stem cell differentiated into oocytes, nursecells and follicle cells during the formation of the ovaries. We detected ovarioles in females during pupal period. The spermatheca could be examined 48 h after eclosion. Four stages during oogenesis could be observed,1:trophocyte stage; 2: previtellogenesis stage; 3:vitellogenesis stage; 4:mature stage. The nutrition was transport to oocytes by receptor mediated way and transferred by the trophocytes. Only one oocyte was examined during oogenesis in ovariole. Sperms were differentiated by the sperm stem cell and the spermiogenesis could be divided into four developmental stages, each stage has their own structure and morphology was different.3 Preparation of the monoclonal and polyclonal antibody of vitellin (Vt) in B. tabaci.In order to study vitellogenesis and its endocrine regulation, we used hybridoma techniques, and developed monoclonal antibodies to Bemisia tabaci egg soluble yolk proteins. The monoclonal antibodie have an IgGl heavy chain and aκlight chain.ELISA was used to determine the titer of antibodies. The results revealed that monoclonal antibodies have a titer of 1:100000 and the polyclonal antibodies were 1:68000. The high specificity and affinity not only to the ovarian vitellin (Vt), but also to female hemolymph vitellogenin (Vg). Western-blot revealed that the two types of antibody had immunological reaction with the others whiteflies Vt and Vg. However, they had no immunological reaction with the male substances.4 The vitellogenesis and characterization of vitellogenin in B. tabaciWith a combination of gel chromatography and ion exchange column, the vitellogenin was purified from the soluble proteins fractions of crude egg extracts. And then, Native-PAGE and SDS-PAGE were applied to analysis the molecular composition of Vg protein, we found that the yolk protein had a native molecular mass of 380 kilodaltons and was composed of two similar subunits with molecular mass of 190 kilodaltons. Carbohydrates, lipids, and phosphorus were found associated with Vt as revealed by the respective stain ability to periodic acid-Schiff’s reagent, Sudan Black B and Methyl Green Solution after gel electrophoresis. We realized that vitellogenin in B. tabaci belonged to phosphoric-glycosylated proteins. We also detected the Vg level in B. tabaci of different developmental stages using the method of sandwich ELISA. It was examined that the vitellogenin in B. tabaci was synthesized in pretended pupal stage and the level of Vg in hemolymph is decreased after eclosion. Increased Vg level was observed in the lateral stage. While the level of Vt in ovary kept increase after eclosion.5 cDNA cloning and expression analysis of vitellogenin in B. tabaciThe cDNA sequence of vitellogenin was cloned from B biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci is 6731 bp with 6474 bp of open reading frame. The molecular weight deduced by amino acid sequence was 242 kDa, coded with 2158 amino acid. The calculated isoelectric point was 8.9. The Vg was cut into 50 kDa and 190 kDa protein by a putative cleavage site of RXXR. Utilizing online software, SignalP,3’non-coding area is 220 bp,5’non-coding area is 37 bp. The signal peptide was consisted of 16 amino acids. The deduced primary structure indicated the content of Ser (S) up to 12.5%, the content of Asp (N) is 12.7%, the content of Glu (Q) is 8.9%. Clustal W and phylogenetic tree analysis showed that Vg in B. tabaci was most closely related with Homalodisca coagulate (86.7%), Athalia rosae (85.6%), Lethocerus deyrollei (84.5%). After we conducted the study on the gene transcription level, increased Vg transctiption gradually in B. tabaci fed on health cotton was observed.6 cDNA cloning and expression analysis of vitellogenin receptor in B. tabaciThe cDNA sequence of vitellogenin receptor (VgR) was cloned from B biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci is 5574 bp. The molecular weight deduced by amino acid sequence was about 201 kDa, coded with 1919 amino acids,5’non-coding was 201 bp. The calculated isoelectric point was 8.7. The transmembrane domain was from 1679 to 1696 amino acid at C-terminal. The VgR belong to the low density lipoprotein receptor (LDLR) family and have some conserved domains, including EGF-C, DXS/KDE, ligand-binding domain, transmembrane and the O-linked carbohydrate domain. Clustal W and phylogenetic tree analysis showed that Vg receptor in B. tabaci was most closely related with Periplaneta americana (83%), Blattella germanica (74%). The transcriptional level of VgR was detected using quantitative RT-PCR.7 cDNA cloning analysis of vitellogenin in Q and ZHJ2 biotype whitelflies B. tabaciThe cDNA sequence of vitellogenin was cloned from Q biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci is 6871 bp with 6651 bp of open reading frame. The molecular weight deduced by amino acid sequence was 246 kDa, coded by 2217 amino acid. The calculated isoelectric point was 8.9. The Vg could be cut into 50 kDa,90 kDa,150 kDa or 190 kDa proteins by two putative cleavage sites of RXXR at either 490 and/or 790 amino acid.3’ non-coding area is 107 bp,5’non-coding area is 43 bp. The signal peptide was consisted of 17 amino acids. The deduced primary structure indicated the content of Ser (S) up to 15%, the Asp (N) was 12.8%, the Glu (Q) was 6.2% and the Ala (A) was 9.2%. Clustal W and phylogenetic tree analysis showed that Vg in Q biotypewas most closely related with Athalia rosae (63.4%), Lethocerus deyrollei (57.3%), Homalodisca coagulate (55.6%).The cDNA sequence of vitellogenin was cloned from ZHJ2 biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci was 6721 bp with 6549 bp of open reading frame. The molecular weight deduced by amino acid sequence was 245 kDa, coded by 2183 amino acid. The calculated isoelectric point was 8.7. The Vg was cut into 50 kDa and 190 kDa protein by a putative cleavage site of RXXR. 3’non-coding area was 109 bp,5’non-coding area was 63 bp. The signal peptide was consisted of 22 amino acids. The deduced primary structure indicated the content of Ser (S) up to 12.5%, the Asp (N) was 13.5%, the Glu (Q) was 7.1% and the Ala (A) was 9.2%. Clustal W and phylogenetic tree analysis showed that Vg in ZHJ2 was most closely related with Athalia rosae (93.9%), Lethocerus deyrollei (93.5%), Homalodisca coagulate (89.9%).Comparison of cDNA sequence of three biotypes of whitefly, the invasive B and Q biotype have more serine (S) at the N-teminal from 620-640. The Q biotype has two putative cleavage sites of RXXR at either 490 and/or 790 amino acid. However, the B and ZHJ2 have only one. The Q biotype has GHN domain while the others have no similar demain. All of the three biotypes whitefly has poly S, Q and N domain at the N-terminal and C-terminal. However, the functions of these domains have not been reported clearly.8 Infection of tomato yellow leaf curl China virus affects the potential fecundity and the transcription level of Vg and VgR gene of its whitefly vector B. tabaci Gennadius (Hemiptera:Aleyrodidae)The application of light microscopy allowed us to verify that the reproductive organ of the whiteflies. The ovary of whitefly formed as soon as the adult emergence. No mature oocytes were observed in females in 1 d and the maximum number of oocytes was observed 11-14 d after emergence. According to main characteristics of oocytes at various development stages and the level of yolk content, the oocytes in ovaries were classified into four developmental types, separately named typeⅠ,Ⅱ,ⅢandⅣ. The type IV were those mature eggs. A rounded bacteriocyte sphere was transferred into oocyte at the terminal of ovariole. We compared the percentage of different type oocytes in the ovaries of two biotypes of whitefly fed on healthy tobacco with virus-infected plants after eclosion. TYLCCNV-infected plant had significant effects on the percentage various types of oocytes in the ovaries of B biotype. The markedly higher percentage of type II and III oocytes was examined in B biotype of whitefly fed on healthy host plants. In contrast, ZHJ-I biotype adult females had similar levels of status of various types of oocyte when fed on healthy and virus-infected plants. The transcription level of Vg and VgR gene was detected using semi-quantitive PCR and RT-PCR, the transcription level was higher in whiteflies fed on virus-infection plant when compare with those whiteflies fed on healthy plant.更多还原