Studies On Comparative Proteomics And MicroRNAs Expression Profiling Of Silkworm (Bombyx Mori)Posterior Silk Gland

As a typical representative Lepidoptera insect, silkworm Bombyx mori not only has irreplaceable economic traits, but more importantly, as a second model insect only next to Drosophila, it possesses almost all the biological characteristic of other insects. Such outstanding features collectively support a sustainable economy industry, and moreover, a large number of multidisciplinary participation is also attracted. All of these have contributed lots of valuable findings to the researches on silkworm in recent years. Especially, the successful accomplishment of silkworm genome project provides us critical theoretical basis and technological platform to further study in the post-genomic era. There are many research contents in post genomic era. For instance, the accurate annotation to the genome is one of the important tasks. It has been proved that organisms are complex systems, and its gene expression is regulated by many factors. To some extent, the expression levels of mRNA do not represent the protein levels. To solve this problem and further reveal the complex mechanisms in organisms which are under physiological or pathological conditions, we should not only rely on the study of proteins, but also to explore other regulatory factors, such as microRNA (miRNA). This study focused on comparative proteomics and expression profile of miRNAs in the posterior silk glands of Bombyx mori and obtained several preliminary findings as follows:1. Comparative proteomic and phosphoproteomic analysis of the silkworm posterior silk gland under high temperature treatmentThe proteins from the posterior silk gland of silkworm hybrids and their parents reared under high temperatures were studied by using comparative proteomic and phosphoproteomic analysis. A total of82.07%.6.17%and11.76%protein spots showed additivity. overdominance and underdominance patterns, respectively. Fifteen differentially expressed protein spots were identified by peptide mass fingerprinting. Among these, four spots, including sHSPs and prohibitin protein that were directly relevant to heat response, were identified. Eleven protein spots were found to play an important role in silk synthesis, and nine protein spots expressed phosphorylation states. According to Gene ontology (GO) and KEGG pathway analysis, these nine spots played an important role in stress-induced signal transduction. Expression of most silk synthesis-related proteins was reduced, whereas stress-responsive proteins increased with heat exposure time in three breeds. Furthermore, most proteins showed under-or overdominance in the hybrids compared to the parents. The results suggested that high temperature could alter the expression of proteins related to silk synthesis and heat response in silkworm. Moreover, differentially expressed proteins occurring in the hybrid and its parents may be the main explanation of the observed heterosis.2. Bioinformatics analysis and screening of miRNA prediction programsAlthough thousands of candidate novel miRNAs can be found by deep-sequencing technology, many pseudo-miRNAs might be produced and bring challenge to further miRNAs identification. Based on the previous research, three SVM-based predictions were selected due to their good performance on distinguishing real-or pseudo-miRNAs. Because all of them were developed based on the known human pre-miRNA characteristics, it sounds reasonable to compare the feature differences between humans and insects, and then select a more suitable program according to their predicting performances on insects. In the present work, we have systematically analyzed, utilizing bioinformatics tools, the featural differences between human and insect pre-miRNAs, as well as differences across24insect species. Results showed that the nucleotide composition, sequence length, nucleotides preference and secondary structure features between human and insects were different. Subsequently, with the aid of three available SVM-based prediction programs, pre-miRNA sequences were evaluated and given corresponding scores. Thus it was found that of2633sequences from the24chosen insect species.2229(84.7%) were successfully recognized by the Mirident classifier, higher than Triplet-SVM (72.5%) and PMirP (72.6%). In contrast, four species, including the domestic silkworm moth, the fruit fly. Drosophila melanogaster Meigen, the honeybee. Apis mellifera L. and the Red Flour beetle, Tribolium castaneum (Herbst). were found to be largely responsible for the poor performance of some sequence matching. Compared with other species. B. mori especially showed the worst performance with the lowest average MFE index (0.73). Collectively these results pave the way for understanding specificity and diversity of miRNA precursors in insects, and lay the foundation for the further development of more suitable algorisms for insects.3. miRNAs profiling in posterior silk gland of silkworm identified by solexa deep-sequencingIn order to study the miRNAs expression profiling of silkworm posterior silk gland, we sequenced six small RNA libraries and obtained293known miRNAs. We found that all the known miRNAs were divided into65miRNA families with the exception of undefined miRNAs, in which,40families were conserved in insects, the left families were only discovered in silkworm. From phylogenetic analysis, those conserved miRNAs were widely distributed in over14species from invertebrates to vertebrates. Result showed that these conserved miRNAs might serve as potential phylogenetic markers and rapid evolutionary signaling molecules. At meantime, we analyzed different expression profiling of two strains with different silk-synthesis traits. One named J1is a strain of silkworm which can normally produce silk, the other termed R1which is the mutant of J1, lack of the ability of spinning cocoon. We found more down-regulated miRNAs in J1compared with it counterpart. We also analyzed the different expression profiles of two developmental stages, specifically,4th-instar molting to5th-instar day-2and5th-instar day-3to5th-instar day-8before spinning. Result showed that the miRNAs expression of stage2obviously declined compared to stage1. Two individual experiments confirmed that silk synthesis related genes might be regulated by miRNAs, which influenced the expression profile of silk proteins. Meanwhile, we employed mireap to predict the novel miRNAs in six libraries with the result of1.373new findings. Utilizing mirident program,432pre-miRNA sequences and414non-redundant miRNAs were successfully recognized to be novel miRNAs. Among these novel miRNAs, we found50pairs of miRNA/miRNA*duplex, which largely enriched the miRNA database. Furthermore, Utilizing RNAhybrid to predict the target genes of differentially expressed miRNAs, we found that many down-regulated miRNAs in J1or stage2were in connection with metabolism and synthesis, which further illustrated the likely function which miRNAs played in silk synthesis.4. Identification of miRNAs in5th-3day of PSG by microarrayWith the aid of microarray.333mature miRNAs were confirmed, and in which,213miRNAs were known miRNAs, and the left, including13conserved in Drosophila, were novel. We also analyzed their different expression profiling between genders and breeds. Results showed that there were no significant differences in genders and in breeds. There were28differentially expressed miRNAs in male and female of Q. Twenty of them were up-regulated in female silkworms, while only8of them were up-regulated in male. On the other hand, sixteen differentially expressed miRNAs were identified in two genders of B, in which, seven were up-regualted in female, while the left in male. Meantime, we also compared the differences between two breeds and observed that4miRNAs up-regulated in Q, contrary to8up-regulated in B. Nevertheless, the newly identified miRNAs largely enrich the repertoire of silkworm miRNAs. Moreover, comparative analysis of known and novel miRNAs indicated that the expression level of miRNAs might be the key reason to interpret different yields of cocoon.All of the results shed light to further illustration of stress response and heteosis occurring in organisms under high tempreture stress. The newly identified miRNAs provided first hand data for extending the miRNA database, and more important, these findings might pave the way for elucidating likely function of miRNAs in the silk production of posterior silk gland at final moth of silkworm.