【作者】 曾媛琴；

【导师】 朱勇；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 氟中毒是严重危害人体健康的主要地方病之一,该病危害严重,不仅能够造成氟斑牙、氟骨症等硬组织的广泛损伤,还可引起全身软组织不同程度的广泛损害。阐明氟中毒致病机制,是当今研究的热点问题之一。细胞色素P450酶系也就是多功能氧化酶(MFO),是所有需氧生物体都存在的代谢酶系。细胞色素P450基因由35亿年前的一个共同祖先进化而成,是最古老和最庞大的超基因家族之一。它对代谢与转化内源物质和活化与降解外源化合物进行催化和调控。昆虫中的细胞色素P450酶系作用涉及生长、发育、取食等过程,其对异生物质的代谢特性导致了昆虫对杀虫剂的抗药性和对有毒物质的耐受性。家蚕(Bombyx mori是鳞翅目昆虫的模式生物,鳞翅目是昆虫中的第二大目。本研究基于家蚕基因组精细图和EST、全基因组基因芯片数据,对家蚕细胞色素P450基因Bmcyp6ul和Bmcyp6a8基因进行了克隆和生物信息学研究。本论文的主要研究结果如下：1.家蚕耐氟性研究分析研究了家蚕添氟方法及桑叶氟浓度检测方法。桑叶用200ug/mLNaF浸渍15分钟,自然晾干。将桑叶研磨,加入1mol/L的HCl浸泡1小时(轻微震荡),pH5-6的TISAB缓冲液定容至50ml,氟离子选择电极检测氟浓度,能够精细地检测出桑叶的氟浓度值。筛选了家蚕耐氟性品种T6和敏感性品种734,检测出耐氟阈值200ug/mL。用200 ug/mL的NaF浸渍桑叶15min,4龄起蚕开始饲喂家蚕,发现耐氟性品种T6虽然体重增长速率低于清水对照组,但是仍然能够一直稳步成长至上蔟：而同时同等条件喂养的734却体重增长异常缓慢,至4龄后期,渐渐死亡,最终不能进入眠期,全部死亡。检测了NaF对家蚕食桑量的影响。相对于对照组,T6和734的添氟组食桑量都有所减少,但是734食桑量减少量异常,严重影响了正常生长；检测了NaF对家蚕蚕沙量的影响。统计显示,添氟组蚕沙量明显低于对照组。除了食桑量减少造成的蚕沙量减少,NaF造成家蚕排泄困难。检测了氟中毒家蚕体内氟含量和各组织的氟含量。统计显示耐氟品种T6和敏感品种734的蚕沙氟含量一直上升；敏感品种734排氟量在到达36ug后就直线下降,一直在36ug徘徊；T6排氟量到达233ug后开始下降。分析显示氟排泄是家蚕耐氟性的一种重要途径。检测了氟中毒家蚕T6各组织的氟含量,包括头、血液、表皮,中肠、马氏管、丝腺、精巢(卵巢),发现头部组织和马氏管中氟离子含量最高。头部组织对氟化钠的不敏感性与氟代谢相配合是家蚕耐氟性另一重要途径。检测了氟中毒家蚕蛾期和蚕卵的氟含量,发现蛾的氟含量约等于5龄7天的氟含量。检测了家蚕添氟组与对照组的乙酰胆碱酯酶含量和活性变化。结果显示,与对照组相比,添氟组头部酶原蛋白含量明显升高,乙酰胆碱酯酶活性呈规律性的明显降低。分析显示,家蚕乙酰胆碱酯酶是家蚕耐氟性的重要靶标之一。乙酰胆碱酯酶酶原蛋白量的增加弥补了活性的缺失,可能是耐氟品种头部组织对氟不敏感性的原因。2.家蚕细胞色素P450第六家族基因的克隆及功能研究采用生物信息学方法获得与果蝇cyp6ul基因同源的家蚕cyp6ul基因序列,实验结果表明,该基因ORF为1476 bp,编码491个氨基酸,推定的蛋白质分子质量为56.15 kD,等电点为9.23。(GenBank登录号：HM130560)。同源性分析表明：Bmcyp6ul基因与蜜蜂同源基因cyp6AS13的相似性为56%：与拟南芥cyp72A82的相似性为48%；与人的cyp3A7基因的相似性为50%。芯片数据分析表明,Bmcyp6ul基因在家蚕5龄第3天幼虫组织表达量很低,只在精巢组织稍有表达,推测该基因具有功能特异性。依据家蚕基因组数据,通过BLASTP比对,RT-PCR克隆了家蚕Bmcyp6a8基因。结果表明,该基因ORF为1572bp,编码523个氨基酸,推定的蛋白质分子质量为61.52kD,等电点为8.17,只有1个内含子。生物信息学分析显示,该基因位于第16号染色体,与野蚕cyp6AE8的相似性较高,同源性为93%,与棉铃虫的同源性为57%,与人的cyp3A43的同源性为33%。GenBank登录号为：GQ241737。从NCBI上下载人、果蝇、蜜蜂、斑马鱼、线虫的基因组与Bmcyp6a8进行比对,发现同源性基因全部是P450基因,构建进化树,Bmcyp6a8与蜜蜂第六家族基因聚集成簇。芯片表达图谱显示,Bmcyp6a8基因具有功能特异性。更多还原

【Abstract】 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.更多还原