【作者】 李兵；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 家蚕（Bombyx mori）和野桑蚕（Bombyx mandarina）具有共同的起源,家蚕的人工驯化历史已经有5 700多年,经过长期的人工选择,家蚕的产丝性能得到了大幅度的提高,但对环境的适应性出现了退化的趋势;野桑蚕长期栖息在野外的桑树上,经过自然选择和农药的压力选择,其对野外环境的抗性,特别是对农药的抗性表现增强的趋势。一方面,家蚕由于对农药的抗性弱,每年都造成大量的农药中毒现象,有的蚕区已经放弃秋蚕的饲养;另一方面,野桑蚕由于对农药的抗性在增强,严重危害桑叶的生产,成为制约蚕丝业健康发展的一对矛盾。为了比较研究家蚕和野桑蚕对有机磷农药抗性差异的分子基础,本文以家蚕品种大造和苏州系统的野桑蚕为研究材料,比较研究了家蚕和野桑蚕对辛硫磷农药的敏感性、乙酰胆碱酯酶（acetylcholinesterase, AChE）的活性、两种类型乙酰胆碱酯酶基因（acetylcholinesterase gene, ace）的结构、ace在农药诱导下的表达特征及家蚕和野桑蚕的ace2的体外表达产物对毒扁豆碱的敏感性。以家蚕和野桑蚕的各龄起蚕为实验材料,采用浸叶法,研究了1⁵龄家蚕和野桑蚕对辛硫磷农药的抗性的差异。结果表明,小蚕期家蚕和野桑蚕对辛硫磷的敏感性差异相对较小,随着龄期的增大,家蚕和野桑蚕对辛硫磷农药的敏感性差异较大,野桑蚕4龄LC50为家蚕的4.43倍,5龄为家蚕的4.02倍。为了研究家蚕和野桑蚕的AChE的活性及毒扁豆碱对其抑制中量(I₅₀)差异,本文以家蚕和野桑蚕2龄起蚕及5龄第3 d的头、中肠、脂肪体和丝腺为实验材料,测定了AChE的酶活性和I₅₀。结果表明,野桑蚕2龄起蚕的酶活是家蚕的1.65倍;5龄第3 d野桑蚕和家蚕的头、中肠、脂肪体和丝腺的酶活之比分别为:1.90倍、2.23倍、2.76倍、2.78倍;毒扁豆碱对家蚕和野桑蚕AChE的I₅₀分别为5.02×10^-7 M和5.23×10^-7 M。构建了野桑蚕脑组织的cDNA文库,经鉴定文库的滴度达3.5×10⁵ pfu/mL,文库插入片段的平均大小为1.2 kb。从文库的测序结果中获得野桑蚕化学感受蛋白基因（CSP3）完整的开放阅读框（登录号:EU439267）并对其进行了序列分析。结果表明:该基因读码框由384个核苷酸组成,编码127个氨基酸,分子量为14.6 kD。通过对该基因编码的氨基酸和其它17种昆虫的CSP编码的氨基酸进行进化分析,发现该基因与家蚕的CSP3的同源性最高,达96.85%。该基因的发现对于研究家蚕和野桑蚕对化学农药的敏感性具有重要意义。利用RT-PCR方法,克隆了家蚕乙酰胆碱酯酶基因（Bm-ace1、Bm-ace2）。序列分析表明:Bm-ace1的ORF包含碱基2 025 bp,编码683个氨基酸,推导其表达蛋白的分子量为76.955 kD,等电点（pI）为6.36;Bm-ace2的ORF包含碱基1 917 bp,编码638个氨基酸,推导其表达蛋白的分子量为71.675 kD,等电点（pI）为5.49;Bm-ace1和Bm-ace2均具有乙酰胆碱酯酶基因（ace）的特征性区域。进化分析表明:Bm-ace1（ABY50088）和来源于中国野桑蚕的Bmm-ace1（ABM66370）的同源性最高,达99.71%, Bm-ace2（ABY50089）和来源于中国家蚕的Bm-ace2（NP₀01037366）的同源关系最近,为99.84%。依据家蚕基因组信息,将Bm-ace1和Bm-ace2分别定位于第15号和第9号染色体,为深入研究家蚕对有机磷农药的抗性机制打下基础。为了进一步研究野桑蚕和家蚕对辛硫磷农药的抗性差异的分子基础,采用了RT-PCR、RACE（rapid-amplification of cDNA ends）方法,克隆了野桑蚕两种AChE基因的cDNA全长,命名为Bmm-ace1和Bmm-ace2,对其进行了序列分析。研究结果表明:Bmm-ace1和家蚕ace1（Bm-ace1）编码的氨基酸数均为681,同源性达99.71%,存在2个氨基酸的突变（G664S、S307P）,野桑蚕ace2（Bmm-ace2）和家蚕ace2（Bm-ace2）编码的氨基酸数为634,同源性达99.37%,存在4个氨基酸的突变（M18I、N233S、I310V、G621S）。进化分析表明,ace2在物种之间相对保守。分析Bmm-ace1的cDNA 5′非翻译序列（5′UTR）区域,与家蚕ace1基因（Bm-ace1）的cDNA 5′UTR（242个碱基）相比,Bmm-ace1的5′UTR包含有231个碱基,存在2个位点的点突变和一段连续11个碱基的缺失。通过克隆和测序分析野桑蚕基因组中Bmm-ace1的5′UTR片段,表明野桑蚕和家蚕ace1基因的RNA剪切都遵循GT-AG规则,但在其cDNA 5′UTR的RNA剪接过程中存在差异,野桑蚕在其cDNA上游+33处存在比家蚕少剪接的序列TGATTTGAAGG,而在其基因组中5′UTR的TGATTTGAAGG序列下游-4位点缺失ACAGA序列。研究结果可为深入探讨ace1基因和抗药性的关系提供新的信息。为了比较研究家蚕和野桑蚕ace1和ace2基因在幼虫各发育阶段、各组织及其在辛硫磷农药诱导后的表达特征。本实验采用了半定量RT-PCR方法,研究了ace1、ace2在野桑蚕和家蚕的1⁵龄各发育阶段、5龄第3 d的血淋巴、脑组织、中肠、脂肪体和丝腺各组织的表达特点;并且研究了在不同浓度的辛硫磷添食后的表达特征,并研究了高于致死中量浓度的辛硫磷诱导后,ace1和ace2在各组织的表达特点。发育表达分析表明,从1龄到5龄家蚕Bm-ace1和Bm-ace2的表达量都表现先下降后上升的趋势,其中Bm-ace1的最低表达量在3龄、Bm-ace2的最低表达量在2龄,野桑蚕从1龄到5龄Bmm-ace1和Bmm-ace2的表达量都表现逐步下降的趋势,其中Bmm-ace1的下降趋势较Bmm-ace2明显,和家蚕呈现不同的表达特征。组织表达分析表明,ace1只在家蚕和野桑蚕的脑组织和脂肪体中表达,ace2在家蚕和野桑蚕的所研究的组织中都有表达,ace1和ace2在脑组织和脂肪体内过量表达;ace1在家蚕和野桑蚕的脑组织表达量一致,而ace2在野桑蚕的脑组织表达量为家蚕的4.17倍。辛硫磷添食后的表达表明,2龄家蚕和野桑蚕在小于致死中量浓度时,随着浓度的上升,ace1的表达量都在增加,ace2在家蚕体内有增加的趋势,但在野桑蚕体内维持较高的表达量,总体表达量在不断下降;在高于致死中量的辛硫磷诱导下,ace1和ace2的表达量都在减少,但Bmm-ace2的减少量较少。高于致死中量的辛硫磷诱导后的组织表达分析表明,在脂肪体的表达量都存在减少的趋势,Bmm-ace1、Bm-ace1、Bm-ace2分别减少了82.67%、81.11%、84.50%,Bmm-ace2的表达减少程度较低,只有30.56%。为了比较研究Bm-ace2和Bmm-ace2基因的表达产物的生物活性及其对农药代谢的差异,分别将Bm-ace2和Bmm-ace2克隆至杆状病毒转移载体pFastBacHTB中获得pFast-Bm-ace2和pFast-Bmm-ace2,分别将其转化DH10Bac感受态细胞后,用PCR方法检测证实所分离的重组病毒DNA中含有目的片段的基因。用脂质体法转染家蚕BmN细胞,分别获得重组病毒。SDS-PAGE和Western Blot分析表明,Bm-ace2和Bmm-ace2均被正确表达。对表达产物进行纯化后,测定的活性分别为1 847.94 mOD/ （min.mg）和1 892.17 mOD/ （min.mg）,毒扁豆碱对二者的抵制中量分别为:I₅₀为1.73×10^-7M和1.87×10^-7M。研究结果表明,Bmm-ace2的过量表达和突变是导致家蚕和野桑蚕对辛硫磷农药抗性差异的主要原因。本研究结果为家蚕的抗性品种选育和开发针对鳞翅目害虫的高效杀虫剂奠定了理论基础。更多还原

【Abstract】 Domesticated silkworm （Bombyx mori） and wild silkworm （Bombyx mandarina） originated from a same ancestor. Domesticated silkworm has been tamed for 5700 years. Although the silk production of Bombyx mori has been significantly increased after long-term artificial selection, its adaptability to environment presents the tendency to degradation. Since Bombyx mandarina inhabits in mulberry garden outdoors, its resistance against the external environment especially to the pesticides shows an increasing tendency year by year as a result of natural selection and insecticide pressure. On one side, due to the susceptibility of Bombyx mori to insecticides, many of them were intoxicated and even some areas have given up the rearing of Bombyx mori in autumn. On the other side, because of the increasing insecticide resistance of Bombyx mandarina, it has decreased the production of mulberry leaves significantly. In conclusion, these factors have caused a series of problems that restrict the normal development of silk industry. Bombyx mori （Dazao） and Bombyx mandarina （Suzhou strain） were used as in this paper to study the molecular mechanisms of the organophosphate resistance differences in Bombyx mori and Bombyx mandarina. The susceptibility of Bombyx mori and Bombyx mandarina to organophosphate was investigated, as well as the enzymatic activities of acetylcholinesterase （AChE）, the gene structures of two kinds of ace genes, the inductive expression patterns by organophosphate, and the susceptibility of the heterologous expression products of ace2 gene to eserine.Newly molted Bombyx mori and Bombyx mandarina of all instars were used to investigate the phoxim insecticide resistance differences between them by leaf dipping method. The results showed that phoxim resistance differences between them at early stages are comparatively smaller; but as the instars increased, the resistance differences change significantly with the LC50 of Bombyx mandarina 4.43-fold higher than that of Bombyx mori at the stage of 4th instar, and 4.02-fold higher at the stage of 5th instar.The 2nd instar newly molted silkworms and the brain, midgut, fat body, and silk gland of the third day 5th instar Bombyx mori and Bombyx mandarina were collected to assay the differences of enzymatic activities of AChE and I₅₀ inhibited by eserine between them. The results indicated that the enzymatic activities of the newly molted 2nd instar Bombyx mandarina is 1.65-fold compared with that of Bombyx mori; the ratios of enzymatic activities of the brain, midgut, fat body, and silk gland of third day 5th instar Bombyx mandarina and Bombyx mori are 1.90, 2.23, 2.76, and 2.78-fold respectively; the AChE-I₅₀ values of Bombyx mori and Bombyx mandarina tested by eserine are 5.02×10^-7M and 5.23×10^-7M respectively.We have constructed a cDNA library of brain tissue from Bombyx mandarina. The library qualification evaluation showed the titer of primary cDNA library was 3.5×105 pfu/mL, average inserted size was 1.2 kb. The ORF of chemosensory proteins gene （CSP3）（GenBank Accession No. EU439267） was found through searching cDNA library and was sequenced. Results showed that the CSP3 is 384 bp and encodes a protein of 127 amino acids with predicted molecular weight of 14.6 kD. The amino acids sequence analysis of CSP3 between CSP3 and 17 insect CSP indicated that the CSP3 has 96.85% identities with Bombyx mori CSP3. The detection of the gene has important meaning to research sensitivity to chemistry agrochemical of Bombyx mori and Bombyx mandarina.Acetylcholinesterase genes （Bm-ace1, Bm-ace2） were cloned from Bombyx mori by RT-PCR. Sequence analysis showed that Bm-ace1 contains a 2 025 bp ORF, which encodes a protein of 683 amino acids with predicted molecular weight of 76.955 kD and pI of 6.36; Bm-ace2 contains a 1 917 bp ORF, which encodes a protein of 638 amino acids with predicted molecular weight of 71.675 kD and pI of 5.49, respectively. Both of the two acetylcholinesterase genes contain the characteristic domains. A clustering analysis showed that Bm-ace1（ABY50088）shared highest similarity with Bmm-ace1（ABM66370） from Chinese Bombyx mandarina, which was 99.71%, Bm-ace2 （ABY50089） shared highest similarity with Bm-ace2 （NP₀01037366） from Bombyx mori, which was 99.84%. Bm-ace1 and Bm-ace2 are located on 15th, 9th chromosome, respectively, according to the information of Bombyx mori genome. This research will help understand the resistance mechanism of Bombyx mori to organophosphorous insecticides.RT-PCR and RACE （rapid-amplification of cDNA ends） were used to study the molecular mechanism of the resistance differences of organophosphorus insecticides between Bombyx mori and Bombyx mandarina. Two full-lenth AChE genes of Bombyx mandarina were cloned and named Bmm-ace1 and Bmm-ace2 respectively. The sequence analysis showed that Bmm-ace1 and ace1 （Bm-ace1） encoded 681 amino acids, with the homology of 99.71%, and existed two mutated amino acids（G664S、S307P）. Bombyx mandarina ace2（Bmm-ace2） and Bombyx mori ace2（Bm-ace2）encoded 634 amino acid with the homology of 99.37%, and existed four mutated amino acid （M18I、N233S、I310V、G621S）. The clustering analysis indicates that the ace2 is relatively conserved in the species. By analyzing the 5’UTR （untranslated region ） of the cDNA of Bmm-ace1,and comparing with the 5’URT of cDNA of Bm-ace1, we found that the 5’URT of Bmm-ace1 was 231 bp in length, which is shorter than that of Bmace1 （242 bp）, and there are 2 nucleotide mutations and a loss of 11 consecutive base pairs in 5′UTR of Bmm-ace1. Cloning and sequencing of the genome sequence that contains 5′UTR of Bmm-ace1 indicated that both of the Bmm-ace1 and Bm-ace1 follow the GT-AG rule in their exon-intron boundaries, but there are some differences in their splicing patterns in 5′UTRs. The genome sequence of Bmm-ace1 contains the lost 11 consecutive base pairs （TGATTTGAAGG） for Bm-ace1 at +33 site of Bmm-ace1 cDNA sequence, but exists a loss of five nucleotides （ACAGA） at -4 site compared to TGATTTGAAGG nucleotides. This research provides some basic information for deeply understanding the relationship between Bmm-ace1 gene and insecticide resistance.The expression patterns of ace1 and ace2 genes at different development stages in all the five larval instars, and various tissues such as hemolymph, brain, midgut, fat body, and silk gland of third day 5th instar larvae of Bombyx mori and Bombyx mandarina were investigated by using semi-quantitative RT-PCR method, as well as the expression profiles of the larvae exposed to phoxim of different concentrations, and ace1 and ace2 at different tissues of third day 5th instar larvae exposed to the phoxim at a concentration higher than I₅₀. The results showed that the expression of Bm-ace1 and Bm-ace2 decreases first, then increases from 1st instar to 5th instar stage, with the lowest expression of Bm-ace1 at the 3rd instar, and Bm-ace2 at 2nd instar. The expression of Bmm-ace1and Bmm-ace2 decreased gradually from 1st instar to 5th instar stage, with Bmm-ace1 decreasing greatly than Bmm-ace2, which is different from that of Bombyx mori. Tissue expression analysis showed that ace1 genes were expressed only in the brains and fat bodies of Bombyx mori and Bombyx mandarina, while ace2 genes were expressed in all the tissues tested. ace1 and ace2 were expressed highly in brains and fat bodies. The expression of ace1 genes in the brains of Bombyx mori and Bombyx mandarina was nearly identical, while the expression of ace2 genes was 4.17-fold higher in the brain of Bombyx mandarina than that of Bombyx mori. The results of the inductive expression by phoxim showed that below the value of I₅₀, the expression of aec1 genes increased along with the concentrations of phoxim in both Bombyx mori and Bombyx mandarina, while ace2 increased in Bombyx mori, but mained high expression and decreased in Bombyx mandarina. When phoxim was applied at higher concentrations than I₅₀, the expression of ace1 and ace2 decreased in both Bombyx mori and Bombyx mandarina, with Bmm-ace2 decreasing less than others. The results of the tissue expression induced by the phoxim of higher concentrations than I₅₀ showed that these genes all decreased in fat bodies, with Bmm-ace1, Bm-ace1, Bm-ace2 decreasing 82.67%, 81.11%, 84.50% respectively, and Bmm-ace2 decreasing the least at 30.56%.To compare the bioactivities of the expression products of Bm-ace2 and Bmm-ace2 and the differences in insecticide metabolism, Bm-ace2 and Bmm-ace2 were cloned into the transfer vector pFastBacHTB to obtain recombinant vectors pFast-Bm-ace2 and pFast-Bmm-ace2. Then they were transformed into DH10Bac competent cells, and recombinant bacmids were verified by PCR analysis. BmN cells were transfected by the recombinant bacmid using lipofectin to get recombinant viruses. SDS-PAGE and Western blot analysis showed that Bm-ace2 and Bmm-ace2 were expressed successfully. After purification of these expression products, the enzymatic activities of the two proteins were detected as 1 847.94 mOD/ （min.mg） and 1 892.17 mOD/ （min.mg）, respectively, and the I₅₀ of Bm-ace2 and Bmm-ace2 to eserine were 1.73×10^-7M and 1.87×10^-7M, respectively.The results showed that overexpression and mutation of Bmm-ace2 are the main reasons for the differences of phoxim resistance in Bombyx mori and Bombyx mandarina. This research provide insights into the breeding of resistant strains of Bombyx mori and the development of highly effective insecticides against Lepidopteral pests.更多还原