| 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 10th -13th 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 5th 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.
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