The SAGE Construction Between Males And Females Of Yellow Cocoon Sex-limited Strain, Ysh, And The Research On Differently Expressed Genes In Silkworm, Bombyx Mori

The domesticated silkworm, Bombyx mori, serves as an importantly economic insect. As is known to all, male silkworms are better than females in respect of physical qualities, synthetic efficiency of silk proteins and silk qualities. At present, the regulatory mechanisms are not thoroughly clear. The sex-limited heredity is widely obtained in Bombyx mori, in which, the cocoon and color sex-limited inherited method, like the stripe sex-limited, is applied to separate males and females in silkworm breeding. Natural colored-cocoons are genetic resources with a promising and significant application future. Genetic controls related to silk color kinds, chroma, color fastness, color well degrees are influenced not only by tens main genes (predicted) of pigment combination and transportation, but more complicated gene nets. Molecular genetics suggests that it hasn’t been confirmed of the related genes yet. The yellow cocoon sex-limited strain (female cocoons are yellow while male cocoons are white) provides excellent materials to research differently expressed genes between yellow and white cocoons.This study constructed LongSAGE libraries of Ysh strain and screened differently expressed genes. In this study, the spatio-temporal expression patterns and gene regulation changes after the treatment with hormone and food limitation were investigated, meanwhile, the activities of total proteinase and trypsin were measured with two representative genes researched at the level of proteins by constructing apEST electronic expression profile, querying online BmMDB and some experimental technologies, such as RT-PCR, Real-Time PCR, immunohistochemistry and Western blotting. The main results are as follows:1 apEST provides the platform to obtain and analyze electronic expression profilesGene electronic expression profile analysis platform(apEST)was constructed based on the data resources of Bombyx mori dbEST( http://jysw.suda.edu.cn/bombyx/Download.html). (316,223 ESTs by Mar. 20, 2011). Compared with other platforms providing gene electronic expression profile analysis, the reliability and feasibility of ours have been ensured. This platform can provide advantageous references to unearth special genes, which has been widely used in our laboratory.2 The construction of Long SAGE libraries between the males and females of the yellow cocoon sex-limited strain, YshAccording to the tissue specificity and the stage when pigment is rapidly accumulated in larvae, the Long SAGE libraries between the males and females were constructed based on the materials of the midgut and silkgland of the fifth instar larvae. A total number of 32,099 tags were obtained, of which 202 differently expressed tags were singled out. Among these tags, 108 were up-regulated in females while 94 were up-regulated in males. And 76 of these tags were annotated with the help of reference libraries and the improved GLGI methods, which created a platform for cocoon color-related genes exploration, especially for new ones besides main genes involved in the pigment combination and transportation. Since a large quantity of sexual different tags are provided by SAGE libraries, it is valuable to clone the full length of these tags and research the functions of them which will definitely contribute to characterize the mechanisms of the character difference between males and females of the yellow cocoon sex-limited strain, Ysh.3 Serine protease (SPs) and Serine protease inhibitor (Serpins) belong to interactional enzymesDuring early and medium stages of 5th instar lavae of Ysh,the especially expressed mRNA levels of SPs precursor gene (Spp) in the midgut was higher than the last stages of the fifth instar larvae as well as the molting stages which was the same of the activity of trypsin. Meanwhile, proteins could not be detected in the molting stages. Spp is transiently regulated by the hunger treatment suggesting that the food induced the secretion of SPs, which participated in protein digestion and absorption, thereby started the expression of SPs genes. When feeding stopped, the secretion of proteinases stopped, either. Thus, the expression character of SPs provided molecular evidence that SPs participated in food protein digestion. The SPs gene expression level in female is higher than that of male in the midgut of the last stages of the fifth instar larvae, suggesting that the females need more protein nutrition during oviposition.The specifically expressed activity of Serpins showed in the silkgland of the middle and late stage of the fifth instar, confirming the physiological phenomenon that a large number of Serpins was needed to protect mass of accumulated silk materials in the silkgland from the degradation by proteinase bofore spinning. The differences between Serpins types needed in different periods and tissues (MSG and PSG) reflected the complicated synthesizing and storing regulation nets of silk materials in Bombyx mori. However, the expression and translation time of the serine protease inhibitor 1 gene (Spi1) lasted longer in the male silkgland than that in female, suggesting that this phenomenon might to be related to the advantage of males on both quality and quantity of silk materials compared with females.The fifth larvae on day 3 were treated by the exogenous molting hormone (MH), and SPs and Serpins presented different regulation mechanisms. Juvenile hormone analogue (JHA) had a similar positive regulation effect on expression of these two genes.The effect of hormones on gene expression is mainly related to the development stages and hormone doses.The down-regulation of Spi1 gene occurred later than Spp gene after hunger treatment on the larvae, suggesting that the direct influence by food on Serpins was slower than SPs, and also showing that the reaction of food supply and demand to the silkgland was later than the influence on the midgut. The tissue expression specificity of SPs and Serpins genes provided a proof that they were involved in transcription regulation of the corresponding tissues. Moreover, both of the expression differences during the developmental period and regulation changes after the hormone treatment reflected the time sequence and particularity of these gene functions. Many members in Serpins participated in the activity regulation of SPs via inhibiting SPs isoenzymes to keep balance of homeostasis.4 Cloning and functional research on putative cuticle protein gene, CPH45A cDNA sequence of 676 bp was cloned according to the tag‘GCTTCCGCCGTGCCTGC’(Tag.1161)in SAGE libraries containing only one exon with no intron. The length of deduced protein was 165aa, located on the nscaf 2767 of the 19th chromosome of Bombyx mori. This gene specifically expressed in the midgut and owning more than 42% homology with 4 protein sequences of CPH family of Bombyx mori. According to the existing gene name order of CPH gene in Bombyx mori, the novel gene was named putative cuticle protein gene 45, CPH45. The expression level of CPH45 in males was apparently higher than that in females of Ysh, which was negatively regulated by exogenous MH and positively regulated by JHA during the middle stage of the 5th instar. Males were more sensitive to exogenous insect hormones. The expression level of CPH45 appear to be an obvious effect on suggesting that it did not directly participate in food digestion. Functional research showed that CPH45 had an influence on maintaining shape of the midgut of Bombyx mori. The expression differences of CPH45 in different silkworm strains showed that this gene might be implicated in different pathways.5 Cloning and characterization of juvenile hormone methyltransgerase gene, JHAMT2A cDNA sequence, 1007 bp, was cloned according to the tag‘GAAACAGCACGCACGCT’(Tag.1221)in SAGE libraries. This gene contains four exons, located on the nscaf 2 993 of the 12th chromosome of Bombyx mori. The deduced protein sequence is 266aa with a functional locus of methyltransgerase super family. It owns 38% homology with JHAMT in Manduca sexta and therefore the cloning gene was named as juvenile hormone methyltransgerase gene JHAMT of Bombyx mori, JHAMT2. The JHAMT2 specially expressed in the midgut, especially in the middle midgut. During different developmental stages, it had a relatively higher expression level in the 4th instar larvas, while it only expressed on day 3 during the whole fifth larvae. The sexual differences were reflected by a longer continuous expression time in males than that in females. In molecular level, the period expression features of JHAMT2 verified that the synthesis of JH was relatively exuberant in the 4th instar larvae while inhibited after day 3 of the fifth instar larvae. Meanwhile, the requirement of JHAMT, catalyzing the synthesis of JH, reduced somewhat.