The Research For Cytochrome P450 Genes In The Silkworm, Bombyx Mori And Its Quantitative Method

Cytochrome P450 monooxygenases, known as multi-functional oxidase(MFO),are a very important enzymatic system, which has been found in all living organisms systems examined. Cytochrome P450 gene originated from a common ancestor 35 million years ago,is one of the oldest and the largest super-gene families. In the process of life, they catalyse and regulate the metabolism and transform of endogenous compounds or the activation and degradation of exogenous compounds. There are around 100 or so P450s in insect genome. Their functions involve growth, development, feeding, etc., which not merely involves a series of endogenous materials such as juvenile hormone(JH) and its analogue, ecdysone, biologic pheromone, but also involve the supersession to exogenous many kinds of compounds such as insecticide, plant secondary materials, environmental pollutant.Bombyx mori is one of model organisms in Lepidoptera. This research take the Bombyx mori as the object of study, and mark the genomic sequence analysis of CPY305B1 gene in the Bombyx mori. Through the current real-time quantitative PCR study of silkworm commonly used endogenous reference gene analysis, it has proposed the new quantitative PCR method, and using this method, has conducted the quantitative research to the Bombyx mori and the Bombyx mandarina part of P450 genes. The main results are as followed.1. Cloning and sequence analysis of cytochrome CPY305B1 gene in the Bombyx moriIn order to study the gene structure of CYP305B1, the genomic sequence of this P450 gene was cloned by reverse PCR method. The complete genomic sequence of CYP305B1 was obatined after sequencing and assembling. Sequence analysis showed the first intron is located in the 5′UTR of this gene. Comparison of the genomic sequence of CYP305B1 with that of CYP305B1V1 from Bombyx mandarina demonstrated that the 370 bp sequence between 770 bp and 400 bp upstream of the 5′UTR of these two P450 genes shared only 40.4% identity to each other. The biggest differences between the two genes lies in the first and sixth introns. In the first intron, there is an additional insertion sequence of 330 bp before the 340 bp sequence upstream of the translation initiation codon ATG of CYP305B1V1 compared with CYP305B1, and the 230 bp sequences between 340 bp and 110 bp upstream of the translation initiation codon of the two genes showed only 46.9% identity to each other. In the sixth intron, there is an extra insertion sequence of about 300 bp in CYP305B1 compared with CYP305B1V1. This research will help us to research the mechanism of transcriptional regulation of this P450 gene.2. The comparative study of real-time quantitative PCR methodsIn order to compare the current commonly used endogenous reference genes actin-3, GAPDH, 28s rRNA, We determined the gene transcription levels of actin-3, GAPDH, 28s rRNA in different tissues of Bombyx mori with IFP2 mRNA and DNA mixed in the experiment type variety as the spike-in gene.Simultaneously it has determined the transcription level of FibH,the FibL gene in middle silk gland, Ser-1 gene in the the posterior silk gland, and comparison with the consequences determined with actin-3,GAPDH,28s rRNA.As the results shown , actin-3, GAPDH and 28s rRNA are very different in each tissue of Bombyx mori.A3 gene in the posterior silk gland transcription level is in the highest (1,098),the lowest in the mid-gut(9.9);GAPDH gene transcription level in the fat body is the highest(10,475), the lowest in the mid-gut (6.57). 28S rRNA gene transcription level of the highest is in the fat body (97,321), the lowest in the mid-gut (2,178).With actin-3, GAPDH, 28s rRNA measured , FibH, FibL genes in the middle silk gland,the Ser-1 gene transcription level in the posterior silk gland,there is a large gap between any two.And adding IFP2 into a mixture of mRNA and DNA at the same time, as a tracking method of spike-in gene,we have measured the transcription level of gene FibH, FibL genes in the MSG and the Ser-1 gene in the PSG.The transcription level (106,453 and 192,029) of gene FibL and FibH in PSG, and the transcription level (196,539) of Ser-1 gene in MSG compare the level closer to the actual transcription.Adding a mixture of mRNA and DNA at the same time,as a tracking method of spike-in gene,known as the dual-sipke-in quantitative PCR. The obtained result is called the gene apparent transcription activity.3. Pesticides-induced apparent transcription levels of P450 geneIt has applied of dual-spike-in method of quantitative PCR to measure the apparent transcription levels of P450 gene in the Bombyx mandarina and Bombyx mori different varieties. At the same time, it has determined Bombyx mori L11 and Bombyx mandarina different P450 gene apparent transcription activity under the induction of the pesticide DDVP(dimethyl dichloro vinyl phosphate) and DKDD (Decamethrin Kothrin Decis Deltamethrin)The results show that P450 gene mainly has the higher transcription level in the middle gut, followed by the fat body; Bombyx mori Haoyue P450 genes also have a higher transcription in the silk gland.The apparent transcription level of P450 gene in the Bombyx mandarina is the highest in silk gland, followed by the mid-gut.Induced by DDVP and DKDD,the transcription level of P450 gene in the mid-gut of Bombyx mori L11 increases significantly, followed by that of the fat body.CYP9A19 and CYP9A22 in Bombyx mori L11 are significantly raised.CYP9A19 and CYP9A22 in Bombyx mandarina are significantly raised, and reach a higher transcriptional activity under the induction of DDKD.However,the transcription of the DDKP-induced P450 gene measured in the Bombyx mandarina is not detected in a higher level.4. Fluoride-induced apparent transcription levels of P450 gene in Bombyx mori Using the dual-sipke-in quantitative PCR, the apparent transcription levels of P450 gene under the induction of NaF are measured.The results indicated that CYP9A19 and CYP9A22 have the higher apparent transcriptional levels in the middle gut, but CYP305B1 and CYP4M5 have the higher apparent transcriptional levels in the fat body(FB). In this study,As shown in the NaF-induced time curve,there is tissue speciality. The induction in the Mid-gut and fat body is called the positive induction,and it reflects the resistance of Bombyx mori to NaF,and that in the malpighian tubules is called the negative induction, it reflects the poison effect of NaF to Bombyx mori. In this study,we proposed a new opinion that we should consider these two aspects in study of resistance of Bombyx mori in the future.