家蚕新突变体二龄不眠蚕基因的定位克隆及功能研究

发布时间：2018-08-02 19:53

【摘要】：家蚕(Bombyx mori)作为一种重要的经济型鳞翅目昆虫,有着特殊的研究价值,已经成为重要的实验材料,前期积累了大量的关于基因的功能研究和相关的基础研究成果,使家蚕已经发展成为进行鳞翅目研究的模式生物。家蚕为完全变态发育昆虫,蜕皮是其生长、发育、变态的重要生理过程,是家蚕个体生长发育成熟所必需的。家蚕二龄不眠蚕突变体(non-molting in the 2nd instar,nm2)发育到二龄将眠期后不能蜕皮而死亡,是在家蚕品种资源C603中发现的一种新不眠蚕突变体。本研究对该突变体进行遗传分析,采用图位克隆的方法对nm2基因进行定位克隆,构建遗传连锁图,进一步采用qRT-PCR、2-DE、RNAi、蜕皮激素和环己酰亚胺添食等方法对候选基因进行表达分析和功能验证,以阐述该突变体发生的分子机制。主要的研究结果如下:一、nm2突变体的遗传分析家蚕二龄不眠蚕突变体是从家蚕品种资源C603中分离获得的一个自然突变体,该突变体在1龄期能全部正常就眠,发育到2龄将眠期皮肤光滑有光泽,进食量减少,不能正常入眠蜕皮,维持二龄将眠蚕状态6~8d后陆续死亡。遗传分析表明该突变是由常染色体上的一个隐性遗传基因控制的,具有隐性纯合(即nm2nm2)致死性。二、nm2突变基因的定位构建P1、P2、F1、BC1F和BC1M群体,用P1、F1和P2筛选获得家蚕每个连锁群上的SSR多态性分子标记,然后分别用有分离的BC1F和BC1M群体中正常型和突变型2种个体进行验证。将BC1F群体中同一蛾区的10个二龄不眠蚕突变体和10个正常个体用作连锁分析,结果表明nm2基因位于家蚕第5连锁群;用BC1M群体中594个突变型个体进行精细定位,结果显示nm2基因位于多态性标记S2529-27与S2529-32之间,这两个多态性标记之间相距约275.6kb,其中有13个候选基因。三、nm2突变基因的鉴定采用RT-PCR方法将13个候选基因进行转录水平表达的比较分析,BMgn002601和BMgn002602在正常型个体和突变个体间有表达差异。为进一步确定引起二龄不眠的主效基因,克隆了两个基因的ORF,发现BMgn002601的ORF在野生型和突变体之间不存在差异,而BMgn002602的ORF在其功能区缺失了217bp。对BMgn002602的RNAi试验显示BMgn002601在转录水平的表达受到BMgn002602表达的影响,BMgn002601在转录水平的表达随着BMgn002602的表达下降而升高,而且对BMgn002602进行RNAi后,二龄家蚕入眠时间可推迟48h-72h。因此初步推断BMgn002602是引起nm2突变的主效基因。BMgn002602为一个编码家蚕表皮蛋白的基因(BmCPG10)。在克隆获得BmCPG10基因ORF序列的基础上,利用RACE和克隆测序技术获得了其3’UTR、5’UTR序列和BMgn002602基因的启动子序列,在突变型家蚕中该基因5’UTR和启动子与野生型一致,3’UTR序列与野生型存在差异。四、nm2突变基因的表达及功能验证采用荧光定量PCR的方法测定了BmCPG10基因在野生型家蚕体内的发育时期表达谱和组织表达谱。BmCPG10基因在家蚕各个发育时期均有表达,在幼虫起蚕或盛食期高,而在每龄的眠期或将眠期其表达量均降低;BmCPG10基因在表皮、头部及气管有较高的表达,在中肠、马氏管、前胸腺、血液中的也有少量的表达。ELISA的方法测定了nm2突变体和野生型家蚕体内蜕皮激素的滴度,发现突变体内蜕皮激素的滴度显著低于野生型,用20E、胆固醇和7-脱氢胆固醇对突变体进行添食,可以使大部分突变体在二龄入眠蜕皮,但拯救后的突变体大都只能发育至四龄。野生型二龄盛食期家蚕添食20E、环己酰亚胺综合分析发现BmCPG10的表达与蜕皮激素的滴度有负相关的趋势。纵观上述研究,我们推测BmCPG10基因突变后可能影响了家蚕对植物甾醇类的吸收利用,造成突变体内合成蜕皮激素的原材料胆固醇不足,引起蜕皮激素滴度降低,导致家蚕不能正常入眠蜕皮。五、突变型和野生型二龄将眠蚕表皮蛋白质组学比较分析采用蛋白质双向电泳技术分析了nm2突变体和野生型家蚕表皮蛋白质的表达差异,其中一个蛋白点在突变体中的表达量高于野生型,经质谱鉴定,该蛋白点是由BmCP-like基因编码的一种半胱氨酸蛋白酶样的蛋白,荧光定量PCR分析显示该基因在突变体中转录水平的相对表达量也显著高于野生型,与蛋白质水平的表达一致;时期表达谱表明BmCP-like基因转录水平的表达在家蚕1-3龄期的起蚕和眠蚕高,而在将眠蚕中低,但在4龄盛食期较高,将眠和眠蚕中低;组织表达谱显示BmCP-like基因在表皮中的表达量较高,而在中肠和血淋巴中的表达量较低;克隆测序发现突变体中该基因ORF的序列与野生型一致,在野生型家蚕二龄盛食期对该基因进行RNAi,造成较高的死亡率,存活下来的两头家蚕也在注射点周围形成黑斑,推断该基因可能参与到家蚕的免疫系统,其高表达可能是为了抵御BmCPG10基因突变造成的不利于家蚕的生长因素。本研究通过对二龄不眠蚕突变产生的分子机制解析,有助于深入了解蜕皮激素对家蚕生长发育的调控机制,并为利用家蚕功能基因防治鳞翅目害虫提供新思路。
[Abstract]:Silkworm (Bombyx mori), as an important economic Lepidoptera, has a special research value and has become an important experimental material. A large number of functional studies on genes and related basic research achievements have been accumulated in the earlier period. The silkworm has been developed into a model organism for the study of Lepidoptera. Breeding insects, molting is the important physiological process of its growth, development and metamorphosis, which is necessary for the growth and development of the silkworm. The two instar non-molting in the 2nd instar (Nm2) of silkworm is developed to death after the dormancy period of the silkworm. It is a new dormant silkworm mutant found in the silkworm species resource C603. The genetic analysis of the mutant was carried out. The Nm2 gene was located and cloned by the method of graph cloning, and the genetic linkage map was constructed. The expression analysis and functional verification of the candidate genes were carried out by using qRT-PCR, 2-DE, RNAi, ecdysone and cycloheximide, in order to explain the molecular mechanism of the mutant. The results are as follows: 1. The genetic analysis of the Nm2 mutant of the silkworm, the two instar silkworm mutant is a natural mutant obtained from the silkworm variety resource C603. The mutant can sleep all normal at the 1 age, and the skin is smooth and luster at the age of 2. The genetic analysis showed that the mutation was controlled by a recessive gene on autosomes with a recessive homozygous gene (nm2nm2) lethal. Two, Nm2 mutation gene was located to construct P1, P2, F1, BC1F and BC1M populations, and the SSR polymorphism molecular markers on each chain of the silkworm were screened by P1, F1 and P2, and then respectively, respectively. 2 types of normal and mutant individuals in the isolated BC1F and BC1M populations were tested. 10 two age non sleeping silkworm mutants and 10 normal individuals in the same moth region of the BC1F population were used as linkage analysis. The results showed that the Nm2 gene was located in the fifth linkage group of the silkworm, and the fine location of the 594 mutant individuals in the BC1M population showed the Nm2 base. Between the polymorphic markers S2529-27 and S2529-32, the two polymorphic markers were about 275.6kb, including 13 candidate genes. Three, the Nm2 mutation gene was identified using the RT-PCR method to compare the transcriptional level of the 13 candidate genes, and BMgn002601 and BMgn002602 were expressed between the normal and the mutant individuals. In order to further determine the main effect genes that cause two years of sleep, the ORF of two genes was cloned, and the ORF of the BMgn002601 was found to be no difference between the wild type and the mutant, while the BMgn002602 ORF in its functional area was missing the RNAi test of BMgn002602 in the BMgn002602, and the expression of BMgn002601 at the transcriptional level was reflected by the BMgn002602 expression. The expression of BMgn002601 at the transcriptional level increased with the decrease of BMgn002602 expression, and after RNAi of BMgn002602, the sleeping time of the two age silkworm could be delayed by 48h-72h. and therefore preliminarily deduced that BMgn002602 was the main gene of Nm2 mutation,.BMgn002602, a gene encoding the pekp of the silkworm, which was cloned to obtain BmCPG10. On the basis of the gene ORF sequence, the 3 'UTR, 5' UTR sequence and the promoter sequence of the BMgn002602 gene were obtained by RACE and cloned sequencing. In the mutant silkworm, the gene 5 'UTR and the promoter are in accordance with the wild type, and the 3' UTR sequence is different from the wild type. Four, the expression of the Nm2 mutation gene and its functional verification use fluorescence quantitative PC. The expression profiles and tissue expression profiles of the BmCPG10 gene in the wild type silkworm were measured by R. The.BmCPG10 gene was expressed in the silkworm, silkworm, silkworm, silkworm and the silkworm, while the larvae were high in the silkworm or high feeding period. The expression of the gene was reduced in the dormancy period or in the dormancy period of the silkworm, and the BmCPG10 based on the epidermis, the head and the trachea was highly expressed. In the midgut, martensite, martensite, anterior thymus, and blood, a small amount of.ELISA was also used to determine the titer of ecdysone in the Nm2 mutant and the wild type silkworm. It was found that the titer of the ecdysone in the mutant was significantly lower than that in the wild type. The mutants were fed with 20E, cholesterol and 7- dehydrogenase cholesterol, which could make most of the mutants in two When the age was sleeping and molting, but most of the mutants after the rescue could only develop to four years old. The wild type two years old silkworm feeding 20E, cycloheximide comprehensive analysis found that the expression of BmCPG10 and the titer of ecdysone have a negative correlation. We speculate that the mutation of BmCPG10 gene may affect the silkworm sterols. Absorption and utilization resulted in the deficiency of cholesterol in the raw material of ecdysone in the mutant body and the decrease of the titer of ecdysone, which resulted in the inability of the silkworm to sleep molting normally. Five, the comparative analysis of the proteomics of the mutant and wild type two years old silkworm epidermis analyzed the Nm2 mutants and the eggs of the wild type silkworm. The expression of white matter in the mutant was higher than that of the wild type. The protein point was a cysteine protease like protein encoded by the BmCP-like gene by mass spectrometry. The fluorescence quantitative PCR analysis showed that the relative expression of the gene in the mutant was significantly higher than that of the wild type, and the protein was significantly higher than that of the wild type. The expression of the level of the BmCP-like gene showed that the expression of the transcriptional level of the gene was higher in the 1-3 instar of the silkworm, but lower in the dormant silkworm, but higher in the 4 age feeding period, and lower in the sleeping and sleeping silkworms. The expression of the BmCP-like gene in the epidermis was higher, while the expression in the midgut and hemolymph was expressed in the tissue expression spectrum. It was found that the sequence of ORF in the mutant was consistent with the wild type, and the gene was RNAi in the two age of the wild type silkworm, which resulted in a high death rate. The two silkworm, which survived, also formed a black spot around the injection point. It was concluded that the gene may be involved in the immune system of the silkworm. The high expression of the gene may be for the sake of the silkworm. This study is helpful to understand the regulation mechanism of ecdysone on the growth and development of silkworm, and provide new ideas for the use of the functional genes of the silkworm to prevent and control the insect pests of the Bombyx mori by analyzing the molecular mechanism of the mutation of the silkworm, which is caused by the mutation of the BmCPG10 gene.
【学位授予单位】：江苏科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S881.2

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