烟粉虱卵黄发生、卵黄蛋白及其受体基因序列的分析
发布时间：2018-07-16 15:53
【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。
[Abstract]:Research in recent years has shown that the main reasons for the successful invasion of B / Q type Bemisia tabaci are as follows: one is the reproductive interference, that is, asymmetric mating; two is the infection of the double biovirus, that is, the mutual benefit of the virus, and the three is the resistance to chemical insecticides by the whitefly. Therefore, the study of the physiological and molecular mechanisms of the invasion of Bemisia tabaci is of profound significance for the effective control of its damage in the future. This study mainly from the form and development of the ovary, the development of the ovary, the development of the spermary and the oogenesis, and the comparison of the egg ratio of different developmental levels in the ovary of different biologic type of Bemisia tabaci. The reproductive basis of reproductive differences between different biotypes of Bemisia tabaci was analyzed. The sequence and structure of the Vg gene were analyzed and compared by cloning the Vitellogenin (Vg) and its receptor (Vitellogenin Receptor, VgR) gene, and the detection of the biotype of the biotype of TYLCCNV virus was detected. The differences in transcriptional level were explored to explore the reproductive basis and molecular mechanism of invasive species of Bemisia tabaci.
Comparative analysis of reproductive system morphology and ovarian development in 1 Bemisia tabaci
The internal reproductive system of female Bemisia tabaci B mainly consists of a symmetrical pair of ovary, a seminal vesicle with a diameter of about 20 mu m, the lateral oviduct and the middle fallopian tube composed of the.B type of the ovaries, which are composed of 12-22 ovarian tubes and can be divided into four different stages according to the development period. The condition of submorphologic and yolk protein deposition can be divided into four levels. The sperma of.B, II, III and IV is mainly composed of a pair of symmetrical arranged testis, male attached glands and vas deferens. The ovarian development of B and ZHJ1 type tobacco whitefly shows that the ovarian morphology of the two species of Bemisia tabaci is similar to that of healthy cotton. In the main plant, the difference of reproductive development is not significant.
2 oogenesis and spermatogenesis of Bemisia tabaci
The eggs of Bemisia tabaci began in the puppet stage. Oocytes were differentiated into trophoblastic cells, oocytes and follicle cells when the females began to form ovaries. The ovaries were found in the pseudochrysalis at the red eye stage. The oviposition was observed at 12 h after adult emergence. After 24 h, the ovaries containing mature ovaries were dissected and dissected by.48 H. The ovaries with the seminal vesicles. The eggs of Bemisia tabaci are divided into the nourishing period, the early stage of the yolk occurrence, the yolk occurrence period and the maturity period of four periods. Oocyte transshipment through the trophoblast transport and self uptake in two ways. The spermatogenesis of Bemisia tabaci also begins in the pseudo pupae period and spermatocytes successively experience spermatogonial cells. There are four stages of primary spermatocyte, secondary spermatocyte and mature spermatocyte, and their morphology and structure are different at different stages.
Preparation of 3 monoclonal antibodies and polyclonal antibodies against Bemisia tabaci Vg
By collecting the fresh eggs produced by Bemisia tabaci as immunogen to prepare monoclonal and polyclonal antibodies, the mouse splenocytes were fused with the myeloma cells which could be cultured and propagated in vitro. The positive clones were screened by ELISA and the ascites were prepared with the supernatant, and the monoclonal antibodies of the Vg were obtained after the purification of the abdominal water. The SDS-P The female specific bands were cut by AGE electrophoresis. The antigen was concentrated by electrified dialysis, and the male rabbits were injected subcutaneously. Then the venous blood was purified and purified to obtain the titer of the polyclonal antibody.ELISA. The titer of the monoclonal antibody was 1:100000. The titer of the polyclonal antibody was 1: 68000.Wertern-blot analysis to find the yolk antibody. It can react with the yolk protein of several biotype Bemisia tabaci and the female specific protein in the hemolymph, but it can not react with the male insect extract specific immune response.
Analysis of the yolk occurrence and Vg characteristics of 4 whitefly
Vetellin (Vt) was purified by gel chromatography and ion exchange chromatography. The analysis of the molecular composition of yolk protein was found by non denaturing gel electrophoresis and denaturing gel electrophoresis. The yolk protein was a large molecular weight of about 380 kDa with two molecular weights of 190 kDa similar subunits. To Vg of Bemisia tabaci. The properties analysis showed that the Vg of whitefly was phosphorylated glycosylated lipoprotein. Using the double anti sandwich ELISA to detect the Vg content in the different developmental stages of the healthy cotton Bemisia tabaci, the Vg synthesis began in the pseudo pupa period. The Vg content in the hemolymph was decreased first and then gradually increased, and the Vt in the ovary increased gradually.
Cloning and transcriptional expression analysis of the Vg gene of 5 whitefly
The full-length sequence of Vg gene cDNA of the B type Bemisia tabaci was 6731 BP, the length of the open reading frame was 6474bp, a total of 2158 amino acids were encoded, the predicted molecular weight was 242 kDa, the isoelectric point was 220bp in the non coding region of the 8.9,3'terminal, the non coding region of the 5' terminal was 37 bp., the first 16 amino acids were signal peptides, the serine (S) was 12.5%, the aspartic acid (N) was 12.7%, and the valley ammonia was 12.7%. Amide (Q) 8.9%. has a RXXR enzyme cut site at 490 amino acids, and the Vg gene is cut into the size protein of about 50 kDa and 190 kDa. Sequence similarity comparison shows that the similarity between the Vg gene of the whitefly and the Homalodisca coagulata of the leafhopper is 86.7%, and the similarity between the Athalia Rosae of the leaf bee and the Athalia Rosae is 85.6%. The similarity of 84.5%. using fluorescence quantitative PCR (Q-PCR) technique to detect the transcriptional level of Vg gene in different periods showed that the Vg expression of whitefly was gradually increasing.
6 cloning and analysis of Vg gene of different biotype Bemisia tabaci.
The Vg gene of Bemisia tabaci ZHJ2 is 6721 BP in length, the length of the open reading frame is about 6549 BP, which encodes 2183 amino acids, the predicted molecular weight is 245 kDa, the isoelectric point is 109 BP in 8.7,3'non coding region, the non coding region of 5' is 63bp. and the first 22 amino acids are signal peptides, and there is a RXXR enzyme cut site in the 450 amino acids, and the Vg gene is cut into about 50 kDa and the Vg gene is cut into about 50 kDa and 190 kDa serine (S) was 12.5%, aspartic acid (N) accounted for 13.5%, glutamine (Q) 7.1%, and alanine (A) 9.2%. sequence similarity comparison found that the similarity between the Vg gene of ZHJ-2 type tobacco whitefly and the Athalia Rosae of the leaf wasp was 93.9%. The similarity between the ZHJ-2 and the Lethocerus deyrollei was 93.5%. The similarity is 89.9%.
The Vg gene of Bemisia tabaci Q has a full length of 6871 BP, the length of the open reading frame is about 6651 BP, encoding a total of 2217 amino acids, the predicted molecular weight is 246 kDa, the isoelectric point is 107 BP in the non coding region of 8.9,3', the non coding region of 5' is 43 bp. and the first 17 amino acids are signal peptides, and there is a RXXR enzyme cutting site in the 490 and 790 amino acids each, and the Vg gene may be cut into the Vg gene. Coding about 50 kDa, 90 kDa, 150 kDa or 190 kDa protein. Serine (S) accounted for 15%, aspartic acid (N) accounted for 12.8%, glutamine (Q) 6.2%, and alanine (A) 9.2%. sequence similarity comparison found that the Vg gene of Q type tobacco whitefly was less similar to 63.4%, and 57.3%, and vitreous leaf. The H. coagulata similarity of cicadas is 55.6%.
After comparison and analysis of nucleic acid sequence and amino acid sequence of three species of whitefly Vg, it was found that the invading species B / Q type was more than the local ZHJ2 type of Bemisia tabaci and was more than 15 amino acid polyserine fragments in the 620-640 amino acid sequence compared to the ZHJ2 type tobacco whitefly. The invasive species Q and B, and the Vg gene sequence of native ZHJ2 In comparison, the Q type of Bemisia tabaci has two enzyme cutting sites and 9 repetitive GHN functional domains in the polyserine region. Three biotype Vg genes of Bemisia tabaci have typical functional domains GCMG and NIIK, and repeated polyserine regions. Compared with other insect Vg genes, the Vg gene of the Bemisia tabaci Vg gene is derived. It not only contains multiple polyserine regions, but also has polyaspartic acid and glutamine region, which is not much in the sequence structure of the insect yolk protein gene. It is interesting that many repetitive GH/RN functional domains appear before the N-terminal polyserine region of the Vg gene sequence of the Bemisia tabaci, and its function remains to be further studied. Research.
Cloning and expression analysis of Vg receptor gene of 7 Bemisia tabaci
The full-length cDNA sequence of the Vg Receptor (Vg Receptor, VgR) gene was cloned, the size 5774bp, the encoding 1919 amino acids, the non coding region of 5'end 201 BP, the isoelectric point 8.7 and the molecular weight about 201 kDa, the transmembrane domain analysis showed that the VgR transmembrane structure was located at the 1679-1696 amino acid position at the end of the C-. The Low Density Lipoprotein Receptor (LDLR) family has some typical conservative domains of the LDLR family, including the ligand binding domain (Ligand-Binding Domain), the epidermal growth factor precursor domain (Epidermal Growth Factor Precursor), the trans membrane domain, and the carbohydrate domain. D Carbohydrate Domain) and cytoplasmic tail region (Cytoplasmic Domain). Homologous analysis showed that the Americana similarity between B type VgR and cockroach of cockroach of American cockroach was 83%, and the Blattella germanica similarity of German cockroach was 74%, and the three-dimensional structure of the VgR coding protein was constructed by using the homologous modeling method. The transcriptional expression of Vg receptor gene in Bemisia tabaci B was detected by technology.
8 effects of TYLCCNV infection on fecundity and transcription of Vg and VgR genes in Bemisia tabaci
The ovarian development of female Bemisia tabaci (Bemisia tabaci) in 15 d after eclosion was observed. It was found that the ovaries were developed and no mature eggs were found in the ovaries. The egg yolk deposited in the 2 D was found, and the mature ovaries were observed in the ovary of 3-4 D, and the number of mature ovaries in the ovary increased with the development time. The number of mature ovaries in the ovary of 11-14 d after eclosion reached the maximum. The ovaries in the ovaries of the Bemisia tabaci were divided into four grades, which were marked as grade I, grade II, grade III and IV respectively, of which the grade IV was mature. The division of the ovary period and the egg development level of the whitefly laid a foundation for evaluating the fertility of the whitefly. The results of ovarian development of type / ZHJ1 type / ZHJ1 Bemisia tabaci (Bemisia tabaci) infected with Chinese tomato yellow leaf curl virus (TYLCCNV) tobacco plants and feeding on healthy tobacco plants showed that the B type of Bemisia tabaci was fed on the sick plants, and the development of eggs was accelerated. The number of eggs of grade III and IV was significantly higher than that of the number of grade III and IV. However, it was observed that TYLCCNV had no significant effect on the development of ovaries in the aboriginal ZHJ1 - type tobacco whitefly. The total RNA of the tobacco whitefly was extracted at the same time, and the yolk occurrence and the Vg/VgR gene transfer in the healthy tobacco and infected tobacco were investigated. The Vg content of the infected whitefly was found. The transcriptional level of Vg/VgR gene was significantly increased.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R384.3
本文编号：2126879
[Abstract]:Research in recent years has shown that the main reasons for the successful invasion of B / Q type Bemisia tabaci are as follows: one is the reproductive interference, that is, asymmetric mating; two is the infection of the double biovirus, that is, the mutual benefit of the virus, and the three is the resistance to chemical insecticides by the whitefly. Therefore, the study of the physiological and molecular mechanisms of the invasion of Bemisia tabaci is of profound significance for the effective control of its damage in the future. This study mainly from the form and development of the ovary, the development of the ovary, the development of the spermary and the oogenesis, and the comparison of the egg ratio of different developmental levels in the ovary of different biologic type of Bemisia tabaci. The reproductive basis of reproductive differences between different biotypes of Bemisia tabaci was analyzed. The sequence and structure of the Vg gene were analyzed and compared by cloning the Vitellogenin (Vg) and its receptor (Vitellogenin Receptor, VgR) gene, and the detection of the biotype of the biotype of TYLCCNV virus was detected. The differences in transcriptional level were explored to explore the reproductive basis and molecular mechanism of invasive species of Bemisia tabaci.
Comparative analysis of reproductive system morphology and ovarian development in 1 Bemisia tabaci
The internal reproductive system of female Bemisia tabaci B mainly consists of a symmetrical pair of ovary, a seminal vesicle with a diameter of about 20 mu m, the lateral oviduct and the middle fallopian tube composed of the.B type of the ovaries, which are composed of 12-22 ovarian tubes and can be divided into four different stages according to the development period. The condition of submorphologic and yolk protein deposition can be divided into four levels. The sperma of.B, II, III and IV is mainly composed of a pair of symmetrical arranged testis, male attached glands and vas deferens. The ovarian development of B and ZHJ1 type tobacco whitefly shows that the ovarian morphology of the two species of Bemisia tabaci is similar to that of healthy cotton. In the main plant, the difference of reproductive development is not significant.
2 oogenesis and spermatogenesis of Bemisia tabaci
The eggs of Bemisia tabaci began in the puppet stage. Oocytes were differentiated into trophoblastic cells, oocytes and follicle cells when the females began to form ovaries. The ovaries were found in the pseudochrysalis at the red eye stage. The oviposition was observed at 12 h after adult emergence. After 24 h, the ovaries containing mature ovaries were dissected and dissected by.48 H. The ovaries with the seminal vesicles. The eggs of Bemisia tabaci are divided into the nourishing period, the early stage of the yolk occurrence, the yolk occurrence period and the maturity period of four periods. Oocyte transshipment through the trophoblast transport and self uptake in two ways. The spermatogenesis of Bemisia tabaci also begins in the pseudo pupae period and spermatocytes successively experience spermatogonial cells. There are four stages of primary spermatocyte, secondary spermatocyte and mature spermatocyte, and their morphology and structure are different at different stages.
Preparation of 3 monoclonal antibodies and polyclonal antibodies against Bemisia tabaci Vg
By collecting the fresh eggs produced by Bemisia tabaci as immunogen to prepare monoclonal and polyclonal antibodies, the mouse splenocytes were fused with the myeloma cells which could be cultured and propagated in vitro. The positive clones were screened by ELISA and the ascites were prepared with the supernatant, and the monoclonal antibodies of the Vg were obtained after the purification of the abdominal water. The SDS-P The female specific bands were cut by AGE electrophoresis. The antigen was concentrated by electrified dialysis, and the male rabbits were injected subcutaneously. Then the venous blood was purified and purified to obtain the titer of the polyclonal antibody.ELISA. The titer of the monoclonal antibody was 1:100000. The titer of the polyclonal antibody was 1: 68000.Wertern-blot analysis to find the yolk antibody. It can react with the yolk protein of several biotype Bemisia tabaci and the female specific protein in the hemolymph, but it can not react with the male insect extract specific immune response.
Analysis of the yolk occurrence and Vg characteristics of 4 whitefly
Vetellin (Vt) was purified by gel chromatography and ion exchange chromatography. The analysis of the molecular composition of yolk protein was found by non denaturing gel electrophoresis and denaturing gel electrophoresis. The yolk protein was a large molecular weight of about 380 kDa with two molecular weights of 190 kDa similar subunits. To Vg of Bemisia tabaci. The properties analysis showed that the Vg of whitefly was phosphorylated glycosylated lipoprotein. Using the double anti sandwich ELISA to detect the Vg content in the different developmental stages of the healthy cotton Bemisia tabaci, the Vg synthesis began in the pseudo pupa period. The Vg content in the hemolymph was decreased first and then gradually increased, and the Vt in the ovary increased gradually.
Cloning and transcriptional expression analysis of the Vg gene of 5 whitefly
The full-length sequence of Vg gene cDNA of the B type Bemisia tabaci was 6731 BP, the length of the open reading frame was 6474bp, a total of 2158 amino acids were encoded, the predicted molecular weight was 242 kDa, the isoelectric point was 220bp in the non coding region of the 8.9,3'terminal, the non coding region of the 5' terminal was 37 bp., the first 16 amino acids were signal peptides, the serine (S) was 12.5%, the aspartic acid (N) was 12.7%, and the valley ammonia was 12.7%. Amide (Q) 8.9%. has a RXXR enzyme cut site at 490 amino acids, and the Vg gene is cut into the size protein of about 50 kDa and 190 kDa. Sequence similarity comparison shows that the similarity between the Vg gene of the whitefly and the Homalodisca coagulata of the leafhopper is 86.7%, and the similarity between the Athalia Rosae of the leaf bee and the Athalia Rosae is 85.6%. The similarity of 84.5%. using fluorescence quantitative PCR (Q-PCR) technique to detect the transcriptional level of Vg gene in different periods showed that the Vg expression of whitefly was gradually increasing.
6 cloning and analysis of Vg gene of different biotype Bemisia tabaci.
The Vg gene of Bemisia tabaci ZHJ2 is 6721 BP in length, the length of the open reading frame is about 6549 BP, which encodes 2183 amino acids, the predicted molecular weight is 245 kDa, the isoelectric point is 109 BP in 8.7,3'non coding region, the non coding region of 5' is 63bp. and the first 22 amino acids are signal peptides, and there is a RXXR enzyme cut site in the 450 amino acids, and the Vg gene is cut into about 50 kDa and the Vg gene is cut into about 50 kDa and 190 kDa serine (S) was 12.5%, aspartic acid (N) accounted for 13.5%, glutamine (Q) 7.1%, and alanine (A) 9.2%. sequence similarity comparison found that the similarity between the Vg gene of ZHJ-2 type tobacco whitefly and the Athalia Rosae of the leaf wasp was 93.9%. The similarity between the ZHJ-2 and the Lethocerus deyrollei was 93.5%. The similarity is 89.9%.
The Vg gene of Bemisia tabaci Q has a full length of 6871 BP, the length of the open reading frame is about 6651 BP, encoding a total of 2217 amino acids, the predicted molecular weight is 246 kDa, the isoelectric point is 107 BP in the non coding region of 8.9,3', the non coding region of 5' is 43 bp. and the first 17 amino acids are signal peptides, and there is a RXXR enzyme cutting site in the 490 and 790 amino acids each, and the Vg gene may be cut into the Vg gene. Coding about 50 kDa, 90 kDa, 150 kDa or 190 kDa protein. Serine (S) accounted for 15%, aspartic acid (N) accounted for 12.8%, glutamine (Q) 6.2%, and alanine (A) 9.2%. sequence similarity comparison found that the Vg gene of Q type tobacco whitefly was less similar to 63.4%, and 57.3%, and vitreous leaf. The H. coagulata similarity of cicadas is 55.6%.
After comparison and analysis of nucleic acid sequence and amino acid sequence of three species of whitefly Vg, it was found that the invading species B / Q type was more than the local ZHJ2 type of Bemisia tabaci and was more than 15 amino acid polyserine fragments in the 620-640 amino acid sequence compared to the ZHJ2 type tobacco whitefly. The invasive species Q and B, and the Vg gene sequence of native ZHJ2 In comparison, the Q type of Bemisia tabaci has two enzyme cutting sites and 9 repetitive GHN functional domains in the polyserine region. Three biotype Vg genes of Bemisia tabaci have typical functional domains GCMG and NIIK, and repeated polyserine regions. Compared with other insect Vg genes, the Vg gene of the Bemisia tabaci Vg gene is derived. It not only contains multiple polyserine regions, but also has polyaspartic acid and glutamine region, which is not much in the sequence structure of the insect yolk protein gene. It is interesting that many repetitive GH/RN functional domains appear before the N-terminal polyserine region of the Vg gene sequence of the Bemisia tabaci, and its function remains to be further studied. Research.
Cloning and expression analysis of Vg receptor gene of 7 Bemisia tabaci
The full-length cDNA sequence of the Vg Receptor (Vg Receptor, VgR) gene was cloned, the size 5774bp, the encoding 1919 amino acids, the non coding region of 5'end 201 BP, the isoelectric point 8.7 and the molecular weight about 201 kDa, the transmembrane domain analysis showed that the VgR transmembrane structure was located at the 1679-1696 amino acid position at the end of the C-. The Low Density Lipoprotein Receptor (LDLR) family has some typical conservative domains of the LDLR family, including the ligand binding domain (Ligand-Binding Domain), the epidermal growth factor precursor domain (Epidermal Growth Factor Precursor), the trans membrane domain, and the carbohydrate domain. D Carbohydrate Domain) and cytoplasmic tail region (Cytoplasmic Domain). Homologous analysis showed that the Americana similarity between B type VgR and cockroach of cockroach of American cockroach was 83%, and the Blattella germanica similarity of German cockroach was 74%, and the three-dimensional structure of the VgR coding protein was constructed by using the homologous modeling method. The transcriptional expression of Vg receptor gene in Bemisia tabaci B was detected by technology.
8 effects of TYLCCNV infection on fecundity and transcription of Vg and VgR genes in Bemisia tabaci
The ovarian development of female Bemisia tabaci (Bemisia tabaci) in 15 d after eclosion was observed. It was found that the ovaries were developed and no mature eggs were found in the ovaries. The egg yolk deposited in the 2 D was found, and the mature ovaries were observed in the ovary of 3-4 D, and the number of mature ovaries in the ovary increased with the development time. The number of mature ovaries in the ovary of 11-14 d after eclosion reached the maximum. The ovaries in the ovaries of the Bemisia tabaci were divided into four grades, which were marked as grade I, grade II, grade III and IV respectively, of which the grade IV was mature. The division of the ovary period and the egg development level of the whitefly laid a foundation for evaluating the fertility of the whitefly. The results of ovarian development of type / ZHJ1 type / ZHJ1 Bemisia tabaci (Bemisia tabaci) infected with Chinese tomato yellow leaf curl virus (TYLCCNV) tobacco plants and feeding on healthy tobacco plants showed that the B type of Bemisia tabaci was fed on the sick plants, and the development of eggs was accelerated. The number of eggs of grade III and IV was significantly higher than that of the number of grade III and IV. However, it was observed that TYLCCNV had no significant effect on the development of ovaries in the aboriginal ZHJ1 - type tobacco whitefly. The total RNA of the tobacco whitefly was extracted at the same time, and the yolk occurrence and the Vg/VgR gene transfer in the healthy tobacco and infected tobacco were investigated. The Vg content of the infected whitefly was found. The transcriptional level of Vg/VgR gene was significantly increased.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R384.3
【引证文献】
相关硕士学位论文 前1条
1 刘佳;含有Bph15基因的水稻对褐飞虱卵巢发育相关基因表达的调控[D];华中农业大学;2012年
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