The SAGE Construction Of The Reciprocal Cross F-1Generation Of The Silkworm, Bombyx Mori, And Theresearch On Space-time Expressions Of Differently Expressed Genes

Bombyx mori is the only certificated model insect of Lepidoptera by theInternational Invertebrate Association and it is of great value in economy. Breeding byhybridization is an important way to improve the efficiency. The heredity of silkwormcan be divided into nuclear heredity and cytoplasm heredity. Generally, different typesof parent combinations would influence the heredity of practical traits, which are maintargets for breeding, as well as main indexes for breeding. Therefore, the difference ofreciprocal cross on breeding is tremendously important for practical breeding andbreeding efficiency.In the study, four samples of male and female reciprocal cross offspring by strain75Xin and7532are used to investigate the mechanism of reciprocal-cross disparity inBombyx mori. SAGE is employed to construct4database of F1by reciprocal cross, so asto screen the genes with significant disparity between samples. By BmMDB, NCBI andRT-PCR, the spatial and temporal expression profiles of these genes in reciprocal crossprogeny are investigated. The expression levels of5genes are compared between roomtemperature and high temperature. Finally, the tags without any annotations butsignificantly different are elongated by GLGI to excavate useful information. Theresults are as follows:1. Analysis and comparison on SAGE libraries of the reciprocal-crossoffspring in the same genderSAGE can be used to investigate gene expression profiles, and also it is veryimportant in promoting unknown genes, especially those with low copies. After librariesof F1in the same gender by reciprocal cross are compared, genes with significantdisparity are screened. Then the results of male and male F1of75Xin×7532arecompared. Among the results,298genes of75Xin×753males are up-regulated while 46down-regulated;453genes of75Xin×753females are up-regulated while240down-regulated. Analyzing50transcripts with high abundance selected, we find thatmost of the highly-expressed genes are housekeeping genes, such as ribosomal proteinsgene, cell-structure-related genes or genes responsible for metabolism.Most of the genes in backcross offspring F1(7532×75Xin), treated under hightemperature, were up-regulated, meanwhile, disparate genes in backcross offspring F1(7532×75Xin), engaged in metabolic pathways, oxidative phosphorylation pathway andsignaling pathway, are also up-regulated. The result above demonstrats that backcrossoffspring F1(7532×75Xin) may fulfill a rapid adaption to the xin surrounding whenfaced up with inverse conditions. This process would consume tons of basic materialswithin the body to follow the physiological changes, so materials supposed to besynthesized into fibroin would be impaired tremendously, sustaining the fact thatcocoon amount of backcross F1(7532×75Xin) is less than that of normal-cross F1(75Xin×7532). However, in normal-cross F1(75Xin×7532), cocoon protecting geneslike serine protease inhibitor and so forth are remarkably up-regulated, indicating that75Xin×7532can greatly insure the amount of cocoon. This is consistent with thestatistical data of reciprocal cross experiments under high temperature.2. Space-time expressions of five different expression genesFive differentially expressed genes are selected from the comparative SAGElibraries between the reciprocal cross F1generations of the same sex. The expressionlevels of mitochondrial thioredoxin2(BGIBMGA012029) and glutathione peroxidase(BGIBMGA011438) show no significant difference between the male reciprocal crossF1generation, but the female orthogonal F1generation higher than Anti-cross progeny. Theexpression levels of Bombyxin E-1precursor (BGIBMGA000667) and antimicrobialpeptides (BGIBMGA000023) are significantly higher in Anti-cross progeny thanorthogonal F1generation between the same sexes. The expression level of hypotheticalprotein (BGIBMGA003920) is higher in the male Anti-cross F1generation thanorthogonal progeny, while has no significant difference between the female reciprocalcross F1generation. So the Space-time expressions of the five genes are furtherresearched. Part of glutathione peroxidase (BGIBMGA011438) and hypothetical protein(BGIBMGA003920) in BmMDB are matched in tissue expression, indicating that theexpression of the two genes is not influenced by temperature or varieties. It alsoapproves the result of RT-PCR. Bombyxin E-1precursor (BGIBMGA000667) andantibacterial peptide (BGIBMGA000023) are not committed with any spatial andtemporal investigation previously. The expression levels of these4genes are allimpaired and then ascend from day1to day7of the5thinstar. The expression ofmitochondrial thioredoxin2(BGIBMGA012029) in Dazao strain is almost ignored inBmMDB, while in our study it is highly expressed on day6of the5thinstar, indicatingthat the gene was remarkably up-regulated in reciprocal cross progeny.Moreover, investigation on the5genes in fatbody, midgut, and silkgland underroom temperature and high temperature proves that these genes within three tissues areall significantly up-regulated, of which antimicrobial peptides (BGIBMGA000023) andmitochondrial thioredoxin2(BGIBMGA012029) stand out. Antimicrobial peptides isclosely related to the immunity of silkworm body, while mitochondrial thioredoxin2(BGIBMGA012029) is against the accumulation of oxygen radical caused by hightemperature, so as to protect and sustain the normal physiological processes within thebody. Therefore, high temperature would stimulate the body to act to the environmentalstress.3. Disparate tags can be annotated by GLGI amplificationTheoretically, long sequences (200bp-1000bp) can be obtained by GLGIamplification, but shorter sequences would be obtained in practical experiments. Thistechnology can be a highly efficient for excavating unknown genes in the constructedSAGE libraries. In the experiment,12elongated sequences of the16tags selected aredetected, contributing to excavating unknown genes in SAGE libraries.