Study On The Wing Pattern Genes In The Silkworm, Bombyx Mori

Insects are the earliest species which could fly over the world, also are the most prosperous organism on the earth with wide distribution at present. The wings of insects make them taking many advantages in predation, mate choice, expanding distribution and safe-keeping, and so on. This is the most important reason for them to become the most prosperous organism. The characteristic of wings is the important basis for the researches of classify and evolution. Following the development of molecular biology, a great amount of outcomes about the development of insect wing had been obtained. It indicated that the basic mechanism in wing growth is quite conserved among different insects. However, the shapes and functions of insect wings have presented many different types after a long evolution course, mainly as a result of natural selection. Which genetic information changed during the evolution? Their molecular regulation mechanism is still unknown. More deeply researches will be required to answer these questions.Silkworm is the typical model insects in Lepidoptera, is also the only one lepidopterous insect which has been completed the genome sequencing at present. The study of wing development genes in the silkworm is significance for insect physiology, developmental biology, evolution, and so on. B. mori has taken place many notably changes during more than 5,000 years artificial breeding, for example, silkworm can not fly, and only some traces for eyespots and bands can be found on the corresponding position of wing. So, the wing of silkworm is really a rare model for the study of degerated wing patterns. Therefore, study on the regulative mechanism of silkworm wing development is important for prevention of Lepidoptera pests.Learning from advanced achievements about wing development in Drosophila, we performed a homology search on wing pattern genes in the silkworm genome by bioinformatics analysis based on the genome sequences, ESTs and the gene chip data. The spatio-temporal expression profiles and functions of several homologous genes have been analyzed by gene clone, RT-PCR, in situ hybridization and RNAi techniques. The major findings are as follows:1. Bioinformatics Analysis of Silkworm Wing Pattern Genes Antero-posterior axis and dorsal-ventral axis are the mainly two gene-regulatory hierarchies for the wing pattern determination in Drosophila, mainly includes 22 genes. Through the homology search, we found 21 genes have the homologous in the Bombyx mori genome except vestigial (vg). Most of them also have been reported in the butterfly. It indicated that the gene-regulatory hierarchy in wing pattern is quite conserved between Lepidoptera and the Diptera.The gene sequence analysis indicated that the similarities of functional domain of corresponding wing pattern genes are very high between Bombyx mori and Drosophila. It further proved that they are playing more similar role in the wing development. However, partial genes (such as Ubx, Dll and so on) also display some differences outside the functional domain. We presumed that the differences of gene structures may be resulted from the differences of gene functions and the regulative ways to adapt for the environment during in the evolution process.2. The morphogenesis and gene expression pattern of silkworm wingWe investigated the development of the wing discs in size and form during 5^th instar larvae and pupal priod of N4. We found that the wing disc growth was slow in the prophase of the larva, while reach a peak just as wandering, and the number of trachea in the disc increased obviously. The wing disc spread out in the end of W2 day, the size of the wing bud nearly quite with that of in pupa. We also found that the pupal stage was a perfecting period of the wing buds, including the formation of vein and scales, with a little change of the size. We found the firstborn veins present on 2d pupa, the firstborn scales present on 3d pupa. The veins and scales were completely formed on 6d pupa. While, we performed the paraffin wax tissue slice on the wing discs of 5^th larva and investigated the changes of internal structure during wing growth.Based on the results of biological information analysis of the wing pattern genes, we investigated the expression profiles of these genes during in wing growth. The results showed that wing pattern genes expressed massively in wing. The expression of most genes showed increasing gradually in larval stage and reducing after pupated, nearly could not be detected in moth. In a word, the expression peaks of these genes mainly present in the stage from the end of larva stage to the initial of pupation, such as, wg, hh, srf,fng and so on. The results of morphous observation indicated that it was just the period during which wing disc taken placed immense changes in morphous. We presumed that the high expressions of these genes were related with the changes of morphous. In addition, some genes were also showed high expression after pupate, such as inv,fng, cut, wg, and so on. Maybe they were also required in the perfecting period of the wing buds, as formation of veins and scales.3. The research of Bmwnt-1 in wing blade developmentRefers to the 38 Wnt protein sequences of human, Drosophila, sea anemone, we found 9 Wnt genes in the Bombyx mori genome through the homology search. Based on sequence similarity, they were named as Bmwnt1,Bmwnt4A,Bmwnt5B,Bmwnt6,Bmwnt7,Bmwnt10A, Bmwnt10B, Bmwnt11 and Bmwnt11A. The numbers of Wnt genes are different in various species. There are 19 Wnt genes in human and 12 Wnt genes in sea anemone. However, there are fewer Wnt genes in insects, such as Bombyx mori, Drosophila and Tribolium castaneum. We presumed that the Wnt genes might lost in the evolution, especially in insect. However, the genome structures of Wnt genes were quite conserved. They are organized in tandem arrays in the genome. The Bombyx mori has 2 Wnt gene bunches, Wnt11-Wnt1-Wnt6-Wnt10 and Wnt7- Wnt5B- Wnt4. They were located at 4 and 28 chromosomes, respectively.The Bmwnt-1 gene belongs to the Wnt1 subtype. The analysis of protein structures of Wnt1 genes in Diptera and the Lepidoptera showed that they have conservative structural domains in the different species, but the gene structures also showed some specificity between Diptera and the Lepidoptera.In this study, we examined the expression profile of the Bmwnt-1 gene during wing development in the silkworm by semi-quantitative RT-PCR. The results showed that it expressed at high abundance in the wing disc from 5^th instar larvae to W2, and reduced gradually after pupate. We used RNAi to reduce the expression of Bmwnt-1 in wing disc and then assayed Bmwnt-1 expression and examined morph after injection. We observed the correlation between reduce of Bmwnt-1 expression and the partly absent of wing blade. It indicated that Bmwnt-1 play important role in the wing blade formation in silkworm.4. The research of AS-C complex in silkwormWe identified four AS-C homologue genes in the silkworm, proneural genes BmASH, BmASH2, BmASH3, and neural precursor gene Bmase.Phylogenetic analysis showed that the ancestral AS-C gene has independently divided into proneural-like and ase-like functions in the insect groups. Most of insects only have a proneural gene and a neural precursor gene. However, there are four AS-C genes in most of Dipteral insects resulted form gene duplication. In our study, we found that the silkworm also has four AS-C genes. It was the first found of another insect besides Diptera which present four AS-C genes. Though the numbers of AS-C genes are different in various species, the AS-C complex and its neighboring genes showed a conserved syntenic structure that the neural precursor gene lied downstream of the proneural genes, a yellow gene and a cyt P450 reside at two ends of the AS-C complex, the yellow gene lies upstream of the complex and the cyt P450 resides the downstream in most insects.To investigate the spatial expression patterns of the AS-C genes, we used semi-quantitative RT-PCR to analyze total RNA samples of 12 silkworm tissues on the sixth day of the 5^th instar larva. The results indicated that the expression levels of the four silkworm AS-C genes were obviously higher in wing disc than in other tissues. So we examined the expression profiles of the four AS-C genes during wing development, the results indicated that the four AS-C genes were all expressed at high abundance in wing disc, but their expression profiles were slightly different. The expression profiles of BmASH2 and BmASH3 were nearly the same. In butterfly, the expression of JcASH1 (AS-C homolog in the butterfly) was detected the following 24 hours after pupate, the critical period when the first scale precursors are formed. In silkworm, the peak level of the BmASH appeared at the onset of pupation, which was the time of the first scale precursors formed in butterfly. These results strongly suggested that the expression of BmASH in wing discs was mostly correlated with the development of the scales in silkworm. We used RNAi to reduce the expression of BmASH in wing disc at the late stage of the larvae and then assayed scale precursor formation molecularly by BmASH expression after injection and by morphological examination. We observed the correlation between reduce of BmASH expression and the absence of scales. It demonstrated that BmASH was essential for the scales differentiation and formation in silkworm.5. The function study of BmSRFgeneWe cloned the serum response factor (BmSRF) in silkworm. The results of expression profile and in situ hybridization of BmSRF gene in wing disc indicated that it play important role in the wing development. The expression of BmSRF gene reached the peak at P1, P2. We used dsRNA to reduce the expression of BmSRF in wing disc at the stage of the larvae. The expression of BmSRF after injection was nearly half of that in controls. And we observed the mutants defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost, and there were more scales in the mutants. It demonstrated that BmSRF was essential for the formation of veins and interveins and the apposition of dorsal and ventral wing epithelia in silkworm.After silence of BmSRF gene, the silkworm presented the mutant phenotype that failure of apposition of dorsal and ventral wing epithelia. This phenotype was similar with that of cf mutant in silkworm. It caught our attentions to find the reason of the formation of mutant phenotype in cf. we dissected and observed the wing discs in cf mutant and normal. We found that there were more tracheas in cf than normal, and they appeared more disorderly. Furthermore, we found that the tracheas elongated and fused when the migration on the W1 stage in cf mutant by sliced, thus, the tracheas in the normal were just migrating on the same time. In view of this, we used real-time PCR to examine the expressions of BmSRF gene in mutant cf and wild type during wing development. The result showed that the expression level of BmSRF gene in cf mutant is higher than that in wild type, especially on the first day of wandering (W1). It was consistent with the results of phenotype examination in the two materials. All of that indicated that the W1 stage is the most important period in which formed cf mutant, and the excessive expression of BmSRF gene perhaps was one of the most important reasons caused the cf mutant.