| Fluorosis is a serious harm to human health. It is one of the main endemic disease and it has serious threat. It can not only cause dental fluorosis, skeletal fluorosis hard tissue disorders such extensive damage, but also the body can cause varying degrees of extensive soft tissue damage. Clarify the pathogenesis of fluorosis is one of the hot issues of today's research.Cytochrome P450 enzymes are also said to be mixed function oxidases, they are complex family of heme-containing enzymes,and they are found in all aerobic organisms. Cytochrome P450 is one of the oldest and largest gene superfamilies. It is evolved from a common ancestor who has existed for more than 3.5 billion years. Cytochrome P450s have Many functions including the metabolism of both endogenous substrates and xenobiotics. P450 enzymes metabolize insecticides, poisonous substance in bioactivation or in detoxification, and the latter process being enhanced in many strains with resistence to insecticides. Furthermore, P450 metabolism of certain chemicals is often the key to the adaptation of insect herbivores.Silkworm(Bombyx mori) is the model organism for Lepidoptera, the second biggest order in insects. On basis of the completing genome sequence database of silkworm, coupled with its ESTs and the whole genome microarray. We analyzed the sequence structure and studied on the cloning and function of orthologous Bmcyp6ul and Bmcyp6a8. The main results present as follows.1. Silkworm study of fluoride-resistantWe studied how mulberry silkworm add fluoride concentration and methods of fluoride detection. Mulberry soak with 200 ug/mL NaF for 15 minutes, air dry. The leaf grinding, adding 1mol/L of Hcl soak 1 hour (mild concussion), pH5-6 of TISAB buffer volume to 50ml, fluoride concentration of fluoride ion selective electrode detection, can detect the actual leaf fluoride concentrations.Screen of silkworm of fluoride-resistant strains T6 and sensitive strains 734. It is detected that the threshold of resistant fluoride T6 is 200 ug/mL. Mulberry leaves treated with 200ug/mL NaF of for 25min was feeding to the larvae at the first day of forth instar of silkworms. Fluoride-resistant strains were found although the weight rising rate is less than clean water control group, but still be able to collect a steady growth in first; while simultaneously feeding the 734 the same conditions but it is unusually slow weight rising, and to the late 4th instar, gradually to die, eventually can not molt, and all died.Examined the amount of mulberry leaves treated with NaF of the larvaes. Compared with the control group, T6, and 734 of the amount of mulberry leaves treated with fluoride had decrease the amount of mulberry leaves. But the larvae of 734 reduced too fast to grow normallyl; Tested NaF on the amount of litterof Silkworm. Statistics show that 734F excrement was significantly lower than other groups. NaF caused difficulties on silkworm excretion.Examined the fluoride concentration of fluorosis silkworm body and its organizations.734F discarded the highest amount of F from 48h to 72h, valued 36ug. F content did not increase in litter, though 734F was fed with the same F treated leaves everyday. F concentration of silkworm litter in T6F kept increasing till the 7th in fifth star. Analysis showed that Fluoride excretion of the silkworm is an important fluoride-resistant pathway. The F concentration in the tissues of the fluorosis larvae was measured respectively in order to see how strain T6 resists F. It was found that the F concentration was the highest in the malpighian tubule and its value was 103.5ug/mL. The next was in head which F concentration value was 43.3ug/mL. We detected the head, the malpighian tubule, the hemocyte, the fat body, the integument, the midgut, the ovary and the silk gland, but F were highly found in the head and the malpighian tubule. Head organization is not sensitive sodium fluoride is another important pathway of resistant fluoride as well as fluoride metabolism in silkworm. Detection fluoride concentration of fluorosis silkworm moth eggs, the fluoride concentration is found that the fluoride concentration of silkworm moth eggs is equal to moth and the 7th day of fifth instar of larvae approximatively.Detection of the protain content and acetylcholinesterase activity of the larvae treated with fluoride and the control groups. The results showed that compared with the control group, the protein content in head treated with fluoride is significantly higher. and while acetylcholinesterase activity was significantly lower regularity. Analysis showed that silkworm AChE content and activity is one of the important target.of silkworm resistance to fluoride. AChE protein increases make up for the lack of activity maybe causes the head organization of fluoride-resistant strains insensitivity to fluoride. 2. Bmcyp6ul cloning of silkworm and function of geneThe sequence homology with Drosophila cyp6ul gene of silkworm was obtained by bioinformatics.Its ORF is 1476bp, encoding 491 amino acids. It is deduced that its molecular weight is 56.15 kD and isoelectric point is 9.23. The cDNA of the testis of silkworm larvae third day in fifth instar as a template, using PCR primers designed to amplify, a treaty of 1500 bp bands was obtained, the size of the ORF sequence is close to silkworm cyp6ul predictive value, and named Bmcyp6ul. (GenBank Accession "Number:HM130560). Homologous analysis showed that it is more closely related to cyp6AS13 of Apis mellifera (56%), cyp72A82 of Arabidopsis thaliana (48%) and cyp3A7 of Homo sapiens (50%). Microarray data analysis shows that Bmcyp6ul gene in larvae of third day in 5th instar has only low levels of expression in the testis. It suggests the gene functional specificity.3. Bmcyp6a8 cloning of Silkworm and function of geneBased on the bioinformation of the genomic sequences database of Bombyx mori, a new gene was successf ully cloned from the head mRNA of B. mori by RT-PCR. Its ORF (open reading frame) includes a segment 1572 bp in length and encodes 523 amino acid residues, with the deduced molecular weight of 61.52 kD and isoelectric point of 8.17. Alignment of the cDNA with the silkworm genomic sequences revealed that there is only one 1706bp intron. It is designated Bmcyp6a8 (GenBank accession number:GQ241737). Compare with the other Paralogous gene of cytochrome P450 subfamily 6 in silkworm, only the Bmcyp6a8 has gene expression of the posterior silk gland. It suggests the gene functional specificity. |
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