【作者】 柴春利；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2007， 博士

【摘要】 胚胎发育是生物学研究中最重要的基本问题之一，所有的多细胞生物都是由一个单细胞——受精卵发育而来。受精卵通过一系列的分裂和分化形成不同形态和功能的细胞，然后进一步构建各种组织和器官，最后建成一个胚胎有机体。整个发育过程都是大量的基因按照一定的时间、空间和次序表达的结果，其中许多调控机制在胚胎发育阶段就已经决定，因此，研究胚胎发育调控机制对于阐明生物的生长、发育和起源进化具有重要意义。昆虫是目前地球上最繁盛的物种类群，了解昆虫的生活方式，探讨它们怎样成功地在地球的任何一个角落繁衍生存是一项非常重要的研究工作。家蚕是一种完全依靠人类饲养生存的经济昆虫，是重要的模式昆虫之一，有非常重要的产业价值，并且有着很好的研究基础并保持有大量的突变体及繁育系。对家蚕的胚胎发育相关基因进行研究，不仅可以加速人们对家蚕胚胎发育调控机制的分子水平研究，也可以促进人们对其它昆虫胚胎发育机制的了解，并可为昆虫分化多样性的分子机制研究提供线索。借鉴模式生物果蝇中的先进研究成果，利用家蚕的全基因组序列、EST以及基因芯片数据，我们对家蚕胚胎发育相关基因进行了生物信息学分析，同时采用克隆、表达、RT-PCR、免疫组织化学染色和RNA干涉等技术对部分基因的结构、转录水平及蛋白质水平的时空表达以及功能进行了分析和研究。获得的主要结果如下：1．家蚕发育相关基因的生物信息学分析基于序列的相似性，在家蚕基因组数据中检索参与果蝇胚胎发育调控级联的86个重要基因的同源基因，结果表明，除6个母性基因在家蚕中没有找出同源基因外，其余基因在家蚕中均存在同源基因，并且其中一部分具有相当高的相似性。这一结果表明参与胚胎发育调控级联的大多数基因在家蚕和果蝇间较为保守。利用果蝇及其它物种中已知的homeodomain序列对家蚕基因组中的homeobox同源基因进行了检索和分析，结果表明，家蚕中存在92个homeobox基因，所编码的homeodomain蛋白分属于21个不同的家族和亚家族。大多数在其它物种中得到鉴定的homeodomain家族在家蚕中都存在，如ABD-B，Antp，Lab，Cad，CUT,Ems，EN，Eve，H2.0，NK-2，ZFH，PARIED，SIX，LIM，POU，ro，msh和TALE等。其中，有23个基因所编码的homeodomain蛋白属于PARIED家族，是基因数目最多的一个家族；有12个基因所编码的homeodomain蛋白属于Antp家族，为第二大家族。同果蝇相比，家蚕中一些家族成员的数目有所增加，如果蝇的Lab亚家族只有1个基因成员，在家蚕中增加到了6个；还有些有所减少，如家蚕LIM和POU家族中的基因数目分别减少了2个和3个。生物信息学分析结果表明，参与果蝇胚胎发育调控的大部分基因在家蚕中都存在同源基因，推测家蚕的胚胎发育调控机制同果蝇相似。而分析所鉴定的参与家蚕胚胎发育调控级联的同源基因以及homeobox同源基因为进一步的功能研究提供了可靠的数据和信息。以此为基础，我们选择了部分基因进行克隆、表达及功能研究，其中包括两个母性基因(vasa和mago nashi)，一群家蚕特异的homeobox基因和一个同源异型基因(AbdB)。2．家蚕vasa基因的结构和mRNA表达型研究Vasa mRNA和VASA蛋白都是果蝇生殖质的主要成分，其同源基因已经在许多物种中得到鉴定。利用家蚕的基因组数据和EST数据，我们对家蚕vasa基因的结构进行了分析。结果表明，家蚕vasa基因由13个外显子组成，在第二和第三外显子间插入了一个mariner型转座子，散布距离约10Kb，同黑腹果蝇的vasa基因相比增加了6个外显子，复杂度明显增加。RT-PCR检测结果表明，家蚕vasa基因在胚胎发育的各个时期均有表达，但在5龄第3天幼虫中，只在生殖腺中特异性表达，在其它组织中不表达。这一结果进一步证实了前人的研究结果，说明家蚕的vasa基因同其它物种vasa同源基因的功能相似，也是生殖系特异的成分之一。以vasa基因作为一个标志基因，我们合成地高辛标记的cDNA探针，在探索中建立了家蚕的胚胎及组织原位杂交技术体系。原位杂交结果表明，vasa基因在家蚕幼虫中只在生殖系来源的细胞中表达，在其它的组织细胞(如结缔组织)中无表达。3．家蚕mago nashi基因的发育表达研究Mago nashi是一个进化中高度保守的基因，它所编码的MAGO蛋白是EJC(exon-exon junction complex)复合体的成分之一，负责mRNA从细胞核到细胞质中的运输过程，同时也是NMD途径(nonsense-mediated mRNA decay)的重要成分之一，参与剪接后mRNA的质量控制。我们克隆了家蚕的mago nashi基因，对其结构和时空表达模式进行了分析和检测。结果表明，家蚕mago nashi基因由2个外显子组成，编码区长度为441bp，编码146个氨基酸。对EST数据的分析结果表明在其5’端可能存在选择性剪接。RT—PCR检测和组织芯片数据分析结果表明，家蚕mago nashi基因在胚胎、幼虫、蛹和蛾期都有表达，在5龄第3天幼虫的各组织中也均有表达，但在生殖腺和丝腺中的表达量高于其它组织。我们制备了家蚕MAGO蛋白的多克隆抗体，对MAGO蛋白在生殖腺和丝腺中的表达分布进行了免疫组化检测。结果表明，家蚕mago nashi基因在生殖腺和丝腺的细胞核和细胞质中均有表达，且在生殖腺细胞核中的表达强于细胞质中。推测家蚕MAGO蛋白同果蝇中的同源体功能相似，是EJC复合体的重要成分，参与mRNA的运输和定位。4．家蚕HOX1-like cluster(Hlc)基因的鉴定及其结构和进化分析在进行生物信息学分析的过程中，我们发现了scaffold001640中存在家蚕特有的一串homeobox基因，呈串联重复状，在基因组注释中的编号分别为Bmb008349，Bmb008350，Bmb008351，Bmb008352，Bmb008354和Bmb008355。这群基因的散布距离约为35Kb，每个基因编码homeodomain结构域1～3个不等，因为它们所编码的homeodomain序列同脊椎动物HOX基因群蛋白的homeodomain序列较为相似，所以将这群基因命名为HOX-like cluster(Hlc)基因，并按照基因编号的顺序，将其中的每个基因分别命名为Hlc-1，Hlc-2，Hlc-3，Hlc-4，Hlc-5和Hlc-6。除Hlc-5基因的转录方向相反外，其它5个基因的转录方向均相同。除Hlc-6～Hlc-5和Hlc-5～Hlc-6两段基因间区外，Hlc群基因的其余部分均得到了进一步的克隆验证。结合早期的染色体原位杂交定位和新完成的家蚕基因组9×覆盖度的序列拼接结果，我们分析发现这群基因位于家蚕第六染色体上传统HOX群基因Proboscipedia(Pb)和zerknüllt(zen)之间。家蚕HOX基因群的这种特殊结构在其它物种中未见报道。对家蚕、果蝇、按蚊、蜜蜂以及人中HOX群基因的系统发生关系进行了分析，结果表明，Hlc群基因首先自己聚为一类，然后再同其它物种来源的zen基因聚在一起，最后和其它HOX基因相聚。推测Hlc群基因起源于Bmzen基因。芯片数据分析及RT-PCR检测结果表明，Hlc-6基因在雌蛹的发育中有表达，在雌蛾中的表达量达最高，但在雄蛹和雄蛾以及产后的卵中检测不到任何表达，表明这一基因是一个母性基因。5．家蚕Abdominal-B(AbdB)基因的克隆、mRNA表达型及功能研究AbdB基因决定昆虫腹部体节后部及末端的分化。早期研究表明Abd-B基因在果蝇生殖腺的发育中起重要作用，并且可以调控色素基因的表达，引起雌雄个体形态上的差异。根据生物信息学分析结果，我们设计引物克隆了家蚕的AbdB基因，对其在不同发育时期的表达进行了检测，并合成RNA双链探针，采用RNAi技术对该基因的功能进行了深入研究。RT-PCR检测结果表明，家蚕AbdB基因在产卵后30h左右开始表达，并且表达量逐渐增强，在产卵后7d表达量达到最高，而后逐渐降低。利用芯片数据对家蚕AbdB基因在蛹、蛾发育时期的表达进行分析，结果表明，该基因在整个蛹、蛾发育期均有表达，且在上簇后12h和上簇后7d，在雌、雄个体之间的表达水平存在显著差异。取雌、雄蛹的生殖腺材料进一步对上簇后7d AbdB基因在雌、雄蛹中的表达差异进行了验证，结果同前面分析一致。推测AbdB基因与雌、雄蛹生殖腺的发育有关。制备DIG标记的AbdB RNA探针，以上簇两天的蛹输卵管为材料进行原位杂交，结果表明，AbdB基因在输卵管腔内、卵间隙内有表达，特别是在输卵管的末端有高表达，但是在正发育形成的卵中无表达。对家蚕胚胎期AbdB基因的RNAi研究结果表明，AbdB基因在家蚕胚胎发育中起重要作用，能够决定第10～13体节的特征。该基因的沉默可以引起原本不长腹足的体节产生过剩附肢，并可以影响尾角和气门的发育。突变表型的产生对探针浓度有一定的剂量依赖性。对家蚕5龄末期幼虫AbdB基因的表达进行干涉研究，发现在AbdB基因沉默的情况下，蛹输卵管不能够正常生长伸长，未发育成熟的卵粒在输卵管的末端集聚，导致形成畸形。未成熟卵中的卵母细胞和滋养细胞也发育异常，呈空泡状和碎片状。这一结果表明AbdB基因在家蚕蛹输卵管的发育中起重要作用。更多还原

【Abstract】 Embryonic development is one of the most important basic questions in biology. All of the multicellular organisms are developed from a single cell, zygote. Zygote cleavages and differentiates into cells with different morphology and function. Various tissues and organs are subsquently formed, consquently develops a embryonic organism. The whole embryonic developmental course results from the ordinal expression of a large number of genes according time and space. Many developmental mechanisms have been determined in the embryonic stage. Therefore, study on the regulative mechanism of embryonic development is important for elucidate principles of biologic growth, development and evolution.Insects are the most prosperous life form on the planet with wide distribution. It is interesting and important to explore the secret how they can survive and reproduce anywhere around the world. Silkworm is an important economic insect which is raised indoors, also is one of the most noted model insects with solid research basis and numerous mutant lines and breed lines. Therefore, study on silkworm embryonic development-related genes can not only accelerate researches in embryonic developmental mechanism of silkworm at the molecular level, but also promote understandings to developmental regulatory mechanism of others insects and provide clues for studies on diversity of insects.Learning from advanced achievements in Drosophila, we performed a homology search on embryonic development-related genes in the silkworm genome by bioinformatics analysis. The structure, mRNA and protein spatio-temporal expression profile and function of several homologous genes have been analyzed by clone and expression technique, RT-PCR, immunohistochemistry and RNAi techniques based on genomic sequence, EST and gene chip data of silkworm. The major findings are as follows.1. Bioinformatics Analysis of Silkworm Development-Related GenesAccording to sequence homology, homologous genes for 86 genes participating in embryonic development genetic hierarchy of Drosaphila were searched in silkworm genome, including 43 maternal genes, 12 gap genes, 9 pair rule genes, 13 segment polarity genes and 9 homeotic genes. Results show that all but 6 maternal genes have the homologues in silkworm genome and some of them have high similarity. This result implies that genes involved in developmental regulation are conservative between Drosaphila and silkworm.We searched homeobox homologues in silkworm genome by utilizing known homeodomain sequences discovered in Drosaphila and other species. Results show that there are 92 homeobox genes exist in silkworm genome which encoded different homeodomain proteins belong to 21 different families and sub-families. Most of these homeodomain proteins families in other species also exists in silkworm, such as ABD-B, Antp, Lab, Cad, CUT, Ems, EN, Eve, H2.0, NK-2, ZFH, PARIED, SIX, LIM, POU, ro, msh and TALE. Among these genes, 23 genes belong to PARIED family, forming the largest family; 12 genes belong to Antp family, the second largest family. Comparing with Drosaphila, the number of members in some families in silkworm varied, for example, Lab family had only one member in Drosaphila, while 6 in silkworm; 7 and 6 members in LIM and POU family in Drosaphila, respectively, while 5 and 3 in silkworm corresponding family, respectively.From the above, most of homologues of genes which participate in cascade regulation during the embryonic development of Drosaphila can be found in silkworm, inferring that there was a similar mechanism in their embryonic development. It provides a reliable basis for further functional researchment of silkworm genes. Based on the work, 2 maternal genes vasa and mago nashi, a group of silkworm specific homeobox genes and one homeotic gene AbdB were cloned, and their expression pattern and functions were investigated.2. Structure and expression pattern of silkworm vasa geneBoth vasa mRNA and VASA protein are important components in the germ plasm of Drosaphila, and its homologous genes have been identified in many other species including silkworm. We analyzed the structure of silkworm vasa gene with silkworm genome and EST data. The results showed that silkworm vasa gene consists of 13 extrons with distribution distance of about 10 Kb. A mariner-like transposon was found between the second and the third extron. Compared with Drosaphila vasa gene, the number of extrons in silkworm vasa gene is greatly increased from 7 to 13 which indicate a higher complexity in the silkworm vasa gene. Results of RT-PCR showes that silkworm vasa gene is expressed through the embryonic development, while only in the gonad of day 3 of the fifth instar, not detected in other tissues. The result also further proved the results of previous studies. Therefore, silkworm vasa gene is one of the germ line specific genes just like its orthologous gene in other species. By taking the DIG-labeled vasa probes as a marker, we performed in situ hybridizations on embryoes and gonads of silkworm.in purpose of establishing in situ hybridization technical system. The findings showed that silkworm vasa gene expressed only in the cells originated from germ line and no expressions were detected in other tissues （for example, connective tissue）.3. Developmental expression of silkworm mago nashi geneMago nashi is a highly conserved gene during evolution. MAGO, the protein it encoded, is one of the components of EJC （exon-exon junction complex） complex, responsible for transportation and localization of mRNAs from nuclear to cytoplasm and it is also one of the members in NMD （nonsense-mediated mRNA decay） pathway. We cloned the silkworm mago nashi homologous gene, and detected its temporal and spacial expression pattern in silkworm embryos and tissues from larva of 3d of fifth instar. The results showed that silkworm mago nashi consisted of 2 extrons, with coding region 441bp in length coding for 146 amino acids. Analysis of EST revealed there are probable alternative splices at its 5’ end. RT-PCR detection and analysis of gene chip data showed that silkworm mago nashi gene expressed in all the stages detected during silkworm development, including embryo, larva, pupa and moth. However, higher expression could be visualized in gonads and silk gland compaired with other tissues. The expression of silkworm mago nashi gene in gonads and silk gland was examined by immunohistochemistry using MAGO polyclonal-antibody. The results showed that silkworm mago nashi expressed in both tissues. In gonads, there is a stronger signal in nuclear than cytoplasm. It is implied that the silkworm MAGO just as its homologue in Drosaphila, is one of the important component of EJC complex, and in charging of transportation and location of mRNA.4. Identification of HOX1-like cluster （Hlc） genes and its phylogenetic analysisDuring the previous analysis, we found a cluster of silkworm specific homeobox genes which appeared as tandem duplications in scaffold001640. Gene IDs for each of them are Bmb008349, Bmb008350, Bmb008351, Bmb008352, Bmb008354 and Bmb008355, respectively. Their distribution distance is about 35Kb, and each of them was predicted to encode 1-3 homeodomains. Because of the similarity with homeodomain sequences of mammal HOX proteins, we called this cluster genes as HOX-like cluster （Hlc）, and each of them was named Hlc-1, Hlc-2, Hlc-3, Hlc-4, Hlc-5 and Hlc-6, respectively. All of the Hlc genes had the same transcription direction except Hlc-5. Almost all the sequences of Hlc genes were further validated via cloning and sequencing except 2 gaps between Hlc-4 and Hlc-5, Hlc-5 and Hlc-6. Combining with the previous FISH results and the new finished silkworm genome sequence （9×coverage）, we found the Hlc genes were located on the 6th chromosome between traditional HOX genes Proboscipedia （Pb） and zerkn（u|¨）llt （zen）. It is never reported in other species for such a special structure of HOX genes like silkworm. We deduce there might be a large gap existing between HOX gene labial （lab） and Pb.The phylogenetic relationships of HOX cluster genes from silkworm, fruitfly, mosquitio, honeybee and human were analyzed by using MEGA3 software. Resutls show that all the members of Hlc genes clustered first, and then with zen （HOX3） homologous genes, then with other HOX genes. We deduce the Hlc genes originated from Bmzen gene.The gene chip data and results of RT-PCR detection revealed Hlc-6 gene expressed during development of female pupa, and reached the maximum in female moth while no signals were detected in the male ones. So it might be a maternal gene.5. Cloning, Expression Profile and Function of silkworm Abdominal-B （AbdB） geneThe products of AbdB gene were reported to play an essential role in determining the differentiation of abdominal posterior and terminal segments in insects. Previous studies also proved that AbdB protein is of vital importance during the development of gonad in Drosophila. Specific expression of AbdB gene in different abdominal segments could regulate the expression of pigment genes leading to the morphologic difference between the male and the female in Drosaphila. No former studies on silkworm AbdB homologous gene. Depending on the results of bioinformatics analysis, we designed primers for silkworm AbdB gene and then cloned this gene. Next, we detected its expression of different stages during silkworm development. Results of RT-PCR showed the expression of AbdB gene started around 30 h after egg laying in silkworm embryo, then its expression increased gradually, reached the maximum 7 d after egg laying, then decreased. Analysis of gene chip data revealed that the silkworm AbdB gene is expressed contiuously during the whole stages of pupa development and there is an evident difference on expression of AbdB in males and the females at 12 h after cocooning and 7 d after cocooning. This result is partially confirmed by relative real time PCR. In situ hybridization results showed that AbdB gene expressed in the oviduct and the region between eggs, especially at the end of the oviduct with strong signal, while didn’t detect any signals in the developing eggs. We deduce that silkworm AbdB gene play important roles in development of embryo and pupa gonads.In order to know the function of silkworm AbdB gene during embryonic development and pupa, we prepared dsRNA probes and performed RNAi experiments in silkworm embryos and larva. Results revealed that AbdB gene is essential for the formation of structures in 10^th -13^th segment during embryonic development. Silence of AbdB gene could lead to excessive appendage sprout, and interfere with development of caudal horn and valve. The appearance of mutants is dose-dependent. The result of AbdB gene RNAi in late 5^th instar larva showed AbdB was important for development of oviduct during pupa development. Silence of AbdB gene led to abnormality of oviduct and gathering of unfledged eggs at the end of the oviduct. Nurse cells and oocytes in the unfledged eggs developed abnormally with vacuoles and fragments inside.更多还原