Screening And Cloning Of The Silkworm White Eggs Near-isogenic Lines Of Differentially Expressed Genes

Silkworm,Bombyx mori is an important economic insect, also an important organism model of lepidopteran insects.With the advance research on the genome and functional genomic of silkworm,Bombyx mori,the transgenic technology of silkworm plays important roles in basic research of lepidopteran insect and silk production industry.The development of detection method of positive transgenic silkworm will be the core technology in this research field.If curtain qualitative traits, such as egg color, eye color, markings, etc. can be employed to select the positive transgenic silkworm, which we can easily visual observe, combined with RNA interference technology, it will improve the screening efficiency and help the development and application of the transgenic technology of silkworm.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 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,suppression subtractive hybridization and cDNA microarray 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 construction of silkworm SSH cDNA library and sequence analysisIn this study,the direction subtractive suppression hybridization library (SSH) of the near-isogenic line w-2 silkworm strain was constructed to explore the molecular mechanism of w-2 of silkworm.300 clones were selected randomly from SSH library to make cDNA microarray,and 107 clones among them were sequenced to confirm the library capacity.Homogenous analysis of these sequences in silkworm genome database and elongation by electronic clone shows that all the sequences are high homogenous with the sequences of silkworm published in database. The library enrichment is suitable for cDNA microarray manufacture.2. The differentially expressed genes identified by cDNA microarray analysisBased on SSH cDNA library we constructed,300 cDNA clones were selected randomly to manufacture the cDNA microarray.We use cDNA microarray technology to find the differentially expressed genes in w-2 of silkworm.At last we get 11 differently expressed genes from the combination of SSH and cDNA microarray analysis.3. The homology analysis of the differentially expressed genes11 differently expressed genes were identified by cDNA microarray analysis.We study these differentially expression genes by blasting in database of silkworm genome and EST,and try to amplify the full sequences of these differentially expression genes by RACE, finally we got 7 partial long sequences among these 11 differentially expression genes.Five sequences of these 11 differentially expression genes are unknown genes,2 sequences have a high homology with the hypothetical protein genes,4 sequences have a high homology with the known functional genes.Allied species are Bombyx mori, Tribolium castaneum, Anopheles gambiae, Plasmodium falciparum, zea mays, and Chironomus tentans.4. Analysis of differentially expressed genes by Real-time fluorescence quantitative RT-PCRThe Semi-quantitative RT-PCR which is more sensitive than cDNA microarray was performed to confirm the differently expressed genes identified by the cDNA microarray analysis,the degree of concordance between data generated by the two methods is high,the folds of differently expression level are from 0.1 to several thousands respectively based on Semi-quantitative RT-PCR.The biggest difference expression level gene is J241 among 11 differentially expression genes.When silkworm eggs develop to the 24th h and 48th h, the expression level of J241 gene in wild type silkworm is 5753 and 7951 folds respectively comparing to that in white egg 2 mutant.When wild type strain eggs develop to the 24h and 48h, the expression level of J241 gene relative to Bmactin A3 is about 0.7 times.While in the white eggs,the expression level of J241 gene relative to Bmactin A3 is very low,almost did not express.5. Molecular cloning and analysis of J241We gain the partial sequence 137bp of J241 from the SSH cDNA library.Then 3'end sequence 240bp of J241 was identified by Rapid Amplification of cDNA Ends.Based on bioinformatics analysis,we suppose the J241sequence may consist of two exon sequences, supposed as first exon and second exon.We amplify J241 using different primers from two exons inside or outside. The result shows that the splicing ways of J241 in wild type strain and w-2 eggs are very different. Only one PCR product was amplified in the w-2 egg,while two PCR products in wild type egg.The sequence of wild type consists of two sequences, one is wild type specific sequence and another is w-2 sequence, the wild type specific sequence is about 900bp. Interestingly in the middle of wild type specific sequence sequence,a 239bp unique sequence is inserted compared with the w-2 egg sequence.6. The analysis of the different expression level of J241 in wild type and w-2 mutantThe results of Real-time fluorescence quantitative RT-PCR shows that the w-2 egg sequence expressed in wild type and w-2 eggs at low and slight different level, but the wild type specific sequence only abundantly expressed in wild type, not in w-2 eggs. So we concluded that the wild type specific sequence type is a significant difference expressed gene in wild type and w-2 mutant.