| Insects are widely distributed animals, occupying ocean, plain and mountain areas. Different insects have different feeding habits, some are herbivores, others are carnivores. But the characteristic that captured the most attention of people, both scientific researchers and nature-loving collectors are the countless and dazzling array of insect body colors. Insects have long been used as model organisms for biological studies, from fruitflies in the kin eyes of Morgen to red flour beetles in the capable hands of modern scientists, all of them have contributed to better understanding the magic of nature.Silkworm is a holometabolous insect, it has been reared in China for silk production for more than 5000 years, and has been studied as scientific material for more than a century. Silkworm is a model insect of lepidoptera, and the 500 or so mutations provide researchers with excellent materials for gene function studies and developmental process investigations. Body color mutants account for a big part of them. Three pigment pathways mainly determine the overall body color of silkworm, ommochrome pathway, pteridine pathway and melanin pathway, each has lots of mutation correspond to, and many of them have already been cloned, including:w-3oe, w-2, and re for the ommochrome pathway, lemon for the pteridine pathway, bts, ch, so, mln, sch for the melanin pathway, etc. Some other chemicals also contribute to the overall body color of silkworm, such as uric acid and carotenoid, and mutations involve these that has been studied include oq, og, ow, os for uric acid, and Y and C for carotenoid.The light body color (lbc) mutant is an autosome recessive mutant of silkworm, it is located on the 5th chromosome, its phenotype is:develop normally during embryo and 1st instar stage, turns totally white after first molting. By microarray analysis and linkage analysis, we identified the gene responsible for the Ibc mutant, and investigated its expression patterns. We also cloned the full-length of the candidate gene by RACE and carried out preliminary function verifications. Our main findings are as follows:1. Microarray analysis to narrow down the candidates for the Ibc mutation.Because lbc mutant was identified by researchers at the Silkworm Gene Bank of Southwest University and has not been mapped on classic linkage map of silkworm, we decided to identify the responsible gene using microarray analysis. Result showed that there were 677genes that had evident differences in expression levels between Dazao and lbc. We further screened these genes first by their spacial expression profiles, and lower the number to sixteen. Then we analyzed these sixteen genes according to the functions of their protein product, and only ten genes were left. We designed primers and amplified the CDS of these ten gene in Dazao and lbc, and found that only one gene---Ⅴshowed polymorphism between the two. We selected this gene as a hopeful candidate for Ibc mutation.2. Linkage analysis of lbcWe amplified the genomic sequences ofⅤin Dazao and lbc and found polymorphism. F1 individuals were obtained from parent strains Dazao(+/+) and lbc mutant(lbc/lbc). F2 individuals generated from sibling mating of F1 were used for linkage analysis. UsingⅤgenomic polymorphism as marker, we carried out linkage analysis. Result showed that there was no recombination between the Ibc locus and theⅤgenomic marker. To further prove thatⅤis the gene responsible for the lbc mutation, we amplified the CDS ofⅤin thirteen different strains, electrophoresis showed that the amplified product of Ibc was the only fragment that was lagged behind.3. Cloning ofⅤCDSWe designed primers based on the predicted CDS ofⅤ, and performed cloning in wild-type and lbc mutant. After sequencing, we found that the sequence of Dazao was identical to that listed in SilkDB, however, a 27bp insertion was found in the CDS of lbc. We also found six single nucleotide substitutions in the lbc mutant, four before the insertion and two behind. We translated the CDS sequence of lbc and found that the six substitutions did not lead to amino acid change. However, although the 27bp insertion did not cause a frameshift, it did lead to a nine amino acid insertion in its encoded protein. Using TMHMM on-line prediction model, we found that this insertion may have disrupted the third transmembrane domain of the predicted protein.4. Cloning of full-length ofⅤWe cloned the full-length ofⅤin Dazao and 3’UTR of lbc by RACE (rapid amplification of cDNA ends), we assembled the 5’UTR and 3’UTR to the CDSs of Dazao and lbc. After sequence analysis, we found that the full-length cDNA of V was 693bp, with a 67bp 5’UTR and a 110bp 3’UTR. And, we also found a 9bp insertion in the 3’UTR of the Ibc mutant.5. Expression profile analysis of VWe carried out temporal and spacial expression analyses of V. Result demonstrated that V was highly expressed in larval head, cuticle and fat body (spacial expression analysis). In temporal profile analysis, strong expression was detected at the beginning of the 4th instar, day 2 of the 4th instar, beginning of the 5th instar to day 5 of the 5th instar and 4 days after spinning, but expression was almost not detectable from day 2 of the 4th instar to the beginning of the 5th instar, and it is the same from day 5 of the 5th instar to moth (with the exception of day 4 after spinning).6. qRT-PCR analysis of Bmlbc and key genes in melanin pathway and amino acid analysis in Dazao and IbcWe quantified the expression levels of Bmlbc and key genes of melanin pathway by qRT-PCR, including PAH, TH, DDC and yellow. Result showed that the expression levels of PAH, TH and DDC were 3 times, 30 times, and 2 times higher in lbc than in Dazao, respectively, while expression of Bmlbc in lbc was about 1/20th that in Dazao. However, expression level of yellow showed no significant difference between Dazao and lbc. In the amino acid analysis, result demonstrated that the precursor of melanin----phenylalanine and tyrosine existed at much higher levels in lbc than in Dazao, with ratios of lbc to Dazao of 39.3 and 14.7, respectively. We also carried out experiment to find out the relationship between Bmlbc(V) and 20E, and we found that this gene was suppressed by 20E. |
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