Screening And Cloning Of Differentially Expressed Genes Between Wildtype And White 2 Egg Mutant Of Silkworm, Bombyx Mori

Silkworm, Bombyx mori is an important economic insect in China since the ancient times, Recently, it becomes an important organism model of lepidopteran insects. Its transgenic technology shows significant value in the fields of breeding new varieties, bioreactor and basic scientific development. So far breeding new variety by transgenic method will become the key point in the field of sericulture research, thus the development of detection method of positive transformation will be the core technology in this research field.The color of wild type silkworm egg is yellow-white at the beginning, about 20h later, gradually become darker and darker,and dark brown finally. The color of the white egg 2 (w-2) mutant of silkworm is yellow-white with a little light red, and eyes of moth are white. So it's easy to distinguish w-2 silkworm from wild type by egg color and ommateum,also the white egg 2 trait is regular hereditary. w-2 has no negative influences on its hatching, vitality and economic characters,thus it is suitable for marker gene for transgenic silkworm research. Now the understanding of molecular mechanism of the w-2 of silkworm eggs is still not clear.In this study, we use the established near-isogenic line of w-2 of silkworm,whole genomic gene chip and Real-time Quantitative RT-PCR to explore the functional genes for characteristics of w-2 of silkworm. The result of the experiment will be helpful to elucidate the molecular mechanism of w-2, and be useful for developing new marker for the transgenic silkworm.The main conclusions we made in this study are as follows: 1. The differentially expressed genes high-throughput explored by silkworm whole genomic gene chipThe silkworm whole genomic gene chip which contains 23000 genes is constructed by CapitalBio corporation and southwest university jointly, is used to find the differrentially expressed genes between white egg 2 and normal eggs of silkworm.Using an criterion of ratio≥2.0 or ratio≤0.5, We obtained 163 genes, in expression of multiple between normal eggs and white eggs that develop to the 24th h, and when silkworm eggs develop to the and 48th h, we obtained 186. The biggest difference expression level gene is sw04840.When silkworm eggs develop to the 24th h and 48th h, the expression level of sw04840 gene in wild type silkworm is 32.4675 and 22.8311 folds respectively comparing to that in white egg 2 mutant.2. Analysis of differentially expressed genes by Real-time fluorescence quantitative RT-PCR.According to the results obtained by the whole genomic gene chip, we use Real-time fluorescence quantitative RT-PCR to identify some larger differrentially expressed genes and some differrentially expressed genes located on the 10th linkage group. The results show that the degree of concordance between data generated by the two methods is high. According to the results obtained by Real-time fluorescence quantitative RT-PCR, gene sw04840 is also the biggest difference expression level gene. When silkworm eggs develop to the 24th h and 48th h, the expression level of sw04840 gene in wild type silkworm is 424.61 and 797.86 folds respectively comparing to that in white egg 2 mutant.Further analysis showed that this gene is an abundantly expressed gene in wild type strain eggs, when wild type strain eggs develop to the 24h and 48h, the expression level of sw04840 gene relative to Bmactin A3 is about 1.92times and 0.73 times respectively. In addition, when silkworm eggs develop to the 48th h, the expression level of sw00132 and sw04534 between white egg 2 and normal eggs of silkworm is also reached several hundred times, and when silkworm eggs develop to the 24th h, the biggest difference expression level gene is sw13775 in up-regulate genes in white egg 2.We select this four gene as candidate genes.3. In silico cloning and RACE elongation of the candidate genes.For the above four candidate genes, we use in silico extention technology with their probe sequence as seed sequence to extend their sequence, and with assembly with the sequencing results of 3'RACE elongation,we obtained more sequence information of them.. Sequence analysis revealed that all of them contains a long open reading frame(ORF), and gene sw13775, sw00132 and sw04840 contains full 3'end sequence.4. Analysis the sequence of the candidate genes and the protein that encoded by themWe analysis the gene structure of the four candidate genes,the results show that the gene sw13775 and gene sw00132 contain more than one exons,but the gene sw04840 and the gene sw04534 contains only one.and in database of silkworm genome assembly we did not find any sequence highly homologous with sw04840, indicate that sw04840 is a new gene which is significant difference expressed between white egg 2 and normal eggs.The analysis of deduced amino acid of the above four candidate genes with blastx shows that gene sw13775 corresponds to the alcohol dehydrogenase of silkworm; sw00132 highly homology with the aldehyde dehydrogenase 7 family, member A1. But we can't find protein homology with protein encoded by sw04534 and sw04840. Bioinformatics analysis show that sw04534 is likely to be an endocellular enzyme and protein encoded by sw04840 probably not a enzyme.