【摘要】：家蠶(Bombyx mori)作為一種重要的經(jīng)濟(jì)型鱗翅目昆蟲,有著特殊的研究?jī)r(jià)值,已經(jīng)成為重要的實(shí)驗(yàn)材料,前期積累了大量的關(guān)于基因的功能研究和相關(guān)的基礎(chǔ)研究成果,使家蠶已經(jīng)發(fā)展成為進(jìn)行鱗翅目研究的模式生物。家蠶為完全變態(tài)發(fā)育昆蟲,蛻皮是其生長(zhǎng)、發(fā)育、變態(tài)的重要生理過(guò)程,是家蠶個(gè)體生長(zhǎng)發(fā)育成熟所必需的。家蠶二齡不眠蠶突變體(non-molting in the 2nd instar,nm2)發(fā)育到二齡將眠期后不能蛻皮而死亡,是在家蠶品種資源C603中發(fā)現(xiàn)的一種新不眠蠶突變體。本研究對(duì)該突變體進(jìn)行遺傳分析,采用圖位克隆的方法對(duì)nm2基因進(jìn)行定位克隆,構(gòu)建遺傳連鎖圖,進(jìn)一步采用qRT-PCR、2-DE、RNAi、蛻皮激素和環(huán)己酰亞胺添食等方法對(duì)候選基因進(jìn)行表達(dá)分析和功能驗(yàn)證,以闡述該突變體發(fā)生的分子機(jī)制。主要的研究結(jié)果如下:一、nm2突變體的遺傳分析家蠶二齡不眠蠶突變體是從家蠶品種資源C603中分離獲得的一個(gè)自然突變體,該突變體在1齡期能全部正常就眠,發(fā)育到2齡將眠期皮膚光滑有光澤,進(jìn)食量減少,不能正常入眠蛻皮,維持二齡將眠蠶狀態(tài)6~8d后陸續(xù)死亡。遺傳分析表明該突變是由常染色體上的一個(gè)隱性遺傳基因控制的,具有隱性純合(即nm2nm2)致死性。二、nm2突變基因的定位構(gòu)建P1、P2、F1、BC1F和BC1M群體,用P1、F1和P2篩選獲得家蠶每個(gè)連鎖群上的SSR多態(tài)性分子標(biāo)記,然后分別用有分離的BC1F和BC1M群體中正常型和突變型2種個(gè)體進(jìn)行驗(yàn)證。將BC1F群體中同一蛾區(qū)的10個(gè)二齡不眠蠶突變體和10個(gè)正常個(gè)體用作連鎖分析,結(jié)果表明nm2基因位于家蠶第5連鎖群;用BC1M群體中594個(gè)突變型個(gè)體進(jìn)行精細(xì)定位,結(jié)果顯示nm2基因位于多態(tài)性標(biāo)記S2529-27與S2529-32之間,這兩個(gè)多態(tài)性標(biāo)記之間相距約275.6kb,其中有13個(gè)候選基因。三、nm2突變基因的鑒定采用RT-PCR方法將13個(gè)候選基因進(jìn)行轉(zhuǎn)錄水平表達(dá)的比較分析,BMgn002601和BMgn002602在正常型個(gè)體和突變個(gè)體間有表達(dá)差異。為進(jìn)一步確定引起二齡不眠的主效基因,克隆了兩個(gè)基因的ORF,發(fā)現(xiàn)BMgn002601的ORF在野生型和突變體之間不存在差異,而BMgn002602的ORF在其功能區(qū)缺失了217bp。對(duì)BMgn002602的RNAi試驗(yàn)顯示BMgn002601在轉(zhuǎn)錄水平的表達(dá)受到BMgn002602表達(dá)的影響,BMgn002601在轉(zhuǎn)錄水平的表達(dá)隨著BMgn002602的表達(dá)下降而升高,而且對(duì)BMgn002602進(jìn)行RNAi后,二齡家蠶入眠時(shí)間可推遲48h-72h。因此初步推斷BMgn002602是引起nm2突變的主效基因。BMgn002602為一個(gè)編碼家蠶表皮蛋白的基因(BmCPG10)。在克隆獲得BmCPG10基因ORF序列的基礎(chǔ)上,利用RACE和克隆測(cè)序技術(shù)獲得了其3’UTR、5’UTR序列和BMgn002602基因的啟動(dòng)子序列,在突變型家蠶中該基因5’UTR和啟動(dòng)子與野生型一致,3’UTR序列與野生型存在差異。四、nm2突變基因的表達(dá)及功能驗(yàn)證采用熒光定量PCR的方法測(cè)定了BmCPG10基因在野生型家蠶體內(nèi)的發(fā)育時(shí)期表達(dá)譜和組織表達(dá)譜。BmCPG10基因在家蠶各個(gè)發(fā)育時(shí)期均有表達(dá),在幼蟲起蠶或盛食期高,而在每齡的眠期或?qū)⒚咂谄浔磉_(dá)量均降低;BmCPG10基因在表皮、頭部及氣管有較高的表達(dá),在中腸、馬氏管、前胸腺、血液中的也有少量的表達(dá)。ELISA的方法測(cè)定了nm2突變體和野生型家蠶體內(nèi)蛻皮激素的滴度,發(fā)現(xiàn)突變體內(nèi)蛻皮激素的滴度顯著低于野生型,用20E、膽固醇和7-脫氫膽固醇對(duì)突變體進(jìn)行添食,可以使大部分突變體在二齡入眠蛻皮,但拯救后的突變體大都只能發(fā)育至四齡。野生型二齡盛食期家蠶添食20E、環(huán)己酰亞胺綜合分析發(fā)現(xiàn)BmCPG10的表達(dá)與蛻皮激素的滴度有負(fù)相關(guān)的趨勢(shì)�？v觀上述研究,我們推測(cè)BmCPG10基因突變后可能影響了家蠶對(duì)植物甾醇類的吸收利用,造成突變體內(nèi)合成蛻皮激素的原材料膽固醇不足,引起蛻皮激素滴度降低,導(dǎo)致家蠶不能正常入眠蛻皮。五、突變型和野生型二齡將眠蠶表皮蛋白質(zhì)組學(xué)比較分析采用蛋白質(zhì)雙向電泳技術(shù)分析了nm2突變體和野生型家蠶表皮蛋白質(zhì)的表達(dá)差異,其中一個(gè)蛋白點(diǎn)在突變體中的表達(dá)量高于野生型,經(jīng)質(zhì)譜鑒定,該蛋白點(diǎn)是由BmCP-like基因編碼的一種半胱氨酸蛋白酶樣的蛋白,熒光定量PCR分析顯示該基因在突變體中轉(zhuǎn)錄水平的相對(duì)表達(dá)量也顯著高于野生型,與蛋白質(zhì)水平的表達(dá)一致;時(shí)期表達(dá)譜表明BmCP-like基因轉(zhuǎn)錄水平的表達(dá)在家蠶1-3齡期的起蠶和眠蠶高,而在將眠蠶中低,但在4齡盛食期較高,將眠和眠蠶中低;組織表達(dá)譜顯示BmCP-like基因在表皮中的表達(dá)量較高,而在中腸和血淋巴中的表達(dá)量較低;克隆測(cè)序發(fā)現(xiàn)突變體中該基因ORF的序列與野生型一致,在野生型家蠶二齡盛食期對(duì)該基因進(jìn)行RNAi,造成較高的死亡率,存活下來(lái)的兩頭家蠶也在注射點(diǎn)周圍形成黑斑,推斷該基因可能參與到家蠶的免疫系統(tǒng),其高表達(dá)可能是為了抵御BmCPG10基因突變?cè)斐傻牟焕诩倚Q的生長(zhǎng)因素。本研究通過(guò)對(duì)二齡不眠蠶突變產(chǎn)生的分子機(jī)制解析,有助于深入了解蛻皮激素對(duì)家蠶生長(zhǎng)發(fā)育的調(diào)控機(jī)制,并為利用家蠶功能基因防治鱗翅目害蟲提供新思路。
[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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S881.2

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