Research On The Expression Regulation Of Silk Gland Related Genes In Bombyx Mori Induced By Ecdysone

In general, for real-time quantitative polymerase chain reaction （qPCR）,normalization strategies use differente reference genes as a control and to avoid differencesin RNA quality, quantity and reverse transcription efficiency between samples. Theexpression of ideal housekeeping gene should not vary in response to differentexperimental conditions and all kinds of tissue, cells. However, results have been reportedthat the mRNA level of reference genes can vary considerably. Without appropriatenormalization, the expression profile of a target gene could be misinterpreted.In this study, the expression level of seven commonly used reference genes （Actin3,GAPDH,28SrRNA, RPL3, α-Tubulin, UBC and TBP） were detected at differentdevelopment time points and in response to treatment with20-Hydroxyecdysone （20E）according to MIQE. The expression stability was analyzed using geNorm and NormFinderprogram. We found the most stable reference genes among the different tissues andbetween the different treatments. Furthermore, we used the RT-PCR technique to measurethe transcriptional level of Fib-H, Fib-L, P²⁵, Ser-1, SGF-1and Fmbp-1in the posteriorsilk gland, middle silk gland and fat body of Bombyx mori5thinstar larvae fed on mulberryleaves that were immersed in ecdysone solution. Through the comparison of thetranscriptional level between the tissues, we analyzed the expression regulationcharacteristics of silkworm larvae tissues and the Fib-H, Fib-L, P²⁵, Ser-1, SGF-1andFmbp-1gene in molting hormone metabolic process. Results are as follows:1. Stability of candidate reference genes in normal tissuesWe used the RT-PCR technique to measure the transcriptional level of seven referencegenes at different development time points under normal rearing and analyzed theexpression stability using geNorm and NormFinder program. We found that the most stablereference genes were different among different tissues. In normal tissues, α-Tubulin,GAPDH in middle silk gland, GAPDH, UBC in posterior silk gland, α-Tubulin, UBC in fatbody were identified as the most stable genes. 2. Stability of candidate reference genes by induction of molting hormoneWe analyzed the expression stability of seven reference genes by the induction ofmolting hormone using geNorm and NormFinder program. In response to the treatmentwith20-Hydroxyecdysone （20E）, α-Tubulin,28SrRNA in middle silk gland, GAPDH,28SrRNA in posterior silk gland, α-Tubulin, UBC in fat body were identified as the moststable genes.3. mRNA transcriptional activity variations by induction of molting hormoneThe expression level of Fib-H, Fib-L, P²⁵, Ser-1all have a decrease in mid-silk glandand posterior silk gland by induction of molting hormone. This reveals that20E inhibits theexpression of Fib-H, Fib-L, P²⁵and Ser-1at the early stage of Bombyx mori5thinstarlarvae. The expression level of SGF-1and Fmbp-1increased at the different time points inmid-silk gland and posterior silk gland by induction of molting hormone. This reveals that20E can stimulate the expression of SGF-1and Fmbp-1. The expression of fibroin geneneeds many regulatory factors, and the complex networks of gene expression regulationneed further study.In this study, the expression level of seven commonly used reference genes weredetected at different development time points and in response to treatment with20Eaccording to MIQE. We found that the most stable reference genes were different amongthe different tissues and between the different treatments. The selection of reference genesis critical for expression studies in RT-PCR and depends on the experimental system.Without appropriate normalization, the expression profile of a target gene could bemisinterpreted. This has certain guiding significance on the research of methods of mRNAquantitation about Silkworm gene.