【作者】 乔梁；

【导师】 鲁成； 代方银；

【作者基本信息】 西南大学 ， 遗传学， 2012， 博士

【摘要】 家蚕不仅是重要的农业经济昆虫；其适中的身型,较短的生长周期,较高的繁殖率等先天优势与完备的基因组数据,分子标记连锁图谱,遗传变异图谱以及百余年经典遗传学研究所积累的大量突变资源使之成为鳞翅目的模式生物。家蚕突变基因资源的类型众多,涉及体色,躯体附肢发育,体型体态,变态发育,氨基酸代谢,尿酸盐代谢,卵色卵型,致死等；同时,已记载的孟德尔突变超过450个,其中约300余个突变被定位于家蚕各连锁群。如此丰富的突变资源在养蚕缫丝业,家蚕基础生物学研究的模式动物化,家蚕生物反应器,甚至是生物学基础研究等领域扮演着重要的角色。家蚕突变体中所占比例非常大的两个类群是体色和体型突变。在鳞翅目昆虫中,体色和体型模式对其生存和繁衍具有重要的意义。研究家蚕体色和体型突变体的形成机制不仅有助于理解鳞翅目昆虫色素及形态发育的代谢和调控网络,还能够为家蚕功能基因的利用(如家蚕实用种的育种应用或鳞翅目害虫防治)提供思路。因此,我们选择家蚕代表性黑化突变暗化型及幼虫体型代表性突变石蚕作为研究对象,利用定位克隆技术分离这两个突变体的候选基因,并对候选基因进行相关的功能研究。所取得的主要研究结果如下：1.暗化型和石蚕突变体的分子定位及候选基因筛查我们利用SSR分子标记对mln和st位点进行了初步定位,分别获得了包含mln和st位点的分子标记连锁图谱。其中,标记S1807与mln位点紧密连锁；与st位点最近的标记S0809虽然与其图距为49.6cM,但是整张图谱中其他标记之间的相对位置及距离与他们在基因组上的位置和间距吻合,适于进行对st位点的进一步分析。根据分子连锁图中突变基因所在位点与其最近分子标记间的距离和家蚕分子连锁图谱与物理图谱中图距的对应关系进行换算,推测突变基因大概所在的基因组物理位置,并结合生物信息学分析筛查候选区域内可能的候选基因。对于mln来说,在候选区域内,我们关注一个预测基因BGIBMGA008538.该基因在果蝇中的同源体AANAT1能够催化多巴胺等单胺类物质,而多巴胺为黑色素的前体物质,同时该基因的组织芯片数据显示其在幼虫头部高表达,而头部是mln表型最明显的部位；时期芯片数据显示该基因在蛹后期及蛾期高量表达,而mln突变在蛹后期至蛾期明显黑化,若mln中BGIBMGA008538基因功能缺失,存在的无法消耗多巴胺的可能性时,便有可能导致多巴胺过量累积,而形成黑化；另外,基于该预测基因的基因组片段所设计的多态性标记P3C在定位群体中的分型结果显示其与mln位点间没有发生重组；进一步的,对P3C的测序结果显示,在mln中,预测基因BGIBMGA008538的第三外显子存在序列缺失,所以我们将BGIBMGA008538确定为mln突变的候选基因。对于st突变来说,我们通过遗传图距与物理图距的换算,推算最近标记S0809与st位点相距约为14.88Mb,并结合分子图谱中标记间的排序,标记所在位置,家蚕第8号染色体物理图谱中scaffold和gap的长度,我们推测st位点可能落在nscaf2827。进一步关联st的表型,我们推测st突变可能与表皮蛋白相关。我们对nscaf2827上三个预测的表皮蛋白基因进行芯片分析,基因分型分析及RT-PCR分析,结果显示,预测基因G2在幼虫期较为特异的表达；该基因与st位点间没有发生重组且基于其cDNA序列的扩增产物在野生型与st突变中差异明显。因此,我们将G2基因确定为st突变的候选基因。2.暗化型和石蚕突变体候选基因的克隆及其在野生型与突变中的序列差异分析我们克隆了mln与st突变候选基因的全长cDNA及差异所在的基因组序列,并且分析比较它们在野生型与突变中的差异。结果显示：野生型大造的cDNA扩增出一条1460bp的片段,而mln突变中扩增出两条片段,分别为1407bp和1253bp。我们将此3条转录本的测序结果结合前章大造与大造mln中P3C扩增片段的测序结果联合分析,结果表明：突变型缺失了96bp的外显子(67bp)内含子混合序列,同时又插入了29bp的内含子序列。大造中的Bm-iAANAT有5个外显子并编码261个氨基酸残基,具有完整的乙酰基转移酶结构域。大造-mln中的第一种异常的转录本type-1的cDNA缺少除前2bp以外的整个第4外显子,并移码形成了提前终止密码TAG。第二种异常转录本Type-2缺少第4外显子的后67bp,另外插入了15bp的内含子序列。Type-2的终止密码子位于正常转录产物的3’UTR处。所以,type-1与type-2都编码了潜在的异常蛋白质,与野生型相比,均破坏了乙酰转移结构域。st突变的候选基因Bm-st全长547bp,包含4个外显子,其中5’UTR为38bp,3’UTR为77bp。编码143个氨基酸,具有典型的几丁质结合结构域。而在st突变中存在58bp的缺失及6处单碱基替换,并有移码产生。突变后的Bm-st的几丁质结构域被完全破坏,功能上存在潜在缺失性。3.mln候选基因的表达模式及其在野生型与突变中的表达差异分析mln突变的候选基因Bm-iAANAT基因的时期表达分析从4龄第3天幼虫开始到羽化结束。RT-PCR分析显示Bm-iAANAT的表达水平在该发育时期具有波动性。在4龄第3天(蜕皮间期)时表达正常,在第4次蜕皮阶段开始时(0-16小时)停止表达,蜕皮阶段后(24小时后)表达重新开始。在5龄起蚕阶段的大部分时间表达量相对较高,在刚上蔟时没有检测到表达。在上蔟末期(W2.5天),与20E的滴度呈负相关,Bm-iAANAT的表达量显著上升(32)。最高表达量出现在化蛹后的第7天(P7)与第8天(P8)。组织表达模式的结果显示,Bm-iAANAT基因在幼虫头部,丝腺和体壁中表达丰度高。尤以头部的表达量为最高。Bm-iAANAT基因的表达量在其它组织中十分低或几乎检测不到。Bm-iAANAT基因在家蚕不同骨化程度部位的表达模式显示：Bm-iAANAT基因在高度骨化的组织(头,胸足和肛板)中的表达量比在低度骨化组织(表皮)中高。也就是说,Bm-iAANAT的高表达量与mln突变型蚕体中的黑色部位有关,而与非黑色部位无关。5龄第4天,化蛹第2天和羽化时期大造-mln与大造中Bm-iAANAT的半定量结果显示：mln中的两种转录产物的表达量比大造中正常转录产物的表达量要低；定量PCR结果表明,在野生型家蚕体中正常Bm-iAANAT的表达量为mln突变体中两种转录产物表达量之和的10-20倍。另外,在能够表现出mln与大造表型差异的部位(头,胸足和肛板)中,Bm-iAANAT的表达量仍然是大造高于mln突变体。4.st候选基因的表达模式,在野生型与突变中的表达差异以及蜕皮激素对该候选基因表达量的影响st突变的候选基因Bm-st基因在4龄及5龄幼虫食桑期均维持较高的表达量；在4龄刚眠时几乎不表达；且该基因自蛹2天后表达量明显下调。主要表现为幼虫及蛹前期表达。组织表达模式显示Bmm-st在幼虫的头部,体壁,脂肪体,气管高表达,其他组织表达量非常弱或几乎检测不到。Bm-st基因的时空表达模式与st突变只在幼虫期出现表型,且出现表型的部位为表皮相吻合。利用20E处理家蚕后,我们发现,处理3小时后,处理组Bm-st基因的表达量已经低于对照组(半定量及定量结果),处理24小时后,处理组已经发育为老熟幼虫,Bm-st基因的表达量明显低于对照组(半定量及定量结果),这表明蜕皮激素对Bm-st基因的表达具有抑制作用。5.黑色素代谢关键基因在mln与野生型中的差异分析及Bm-iAANAT基因的功能研究我们详细比较了黑色素代谢途径中的关键基因在已着色的野生型与mln突变头部,胸足,肛板中的差异,结果显示,在这些部位中Pale,Ddc和Yellow3个基因的表达量在大造中高于在大造-mln中。但是,除头部以外,ebony基因的表达量在大造中低于在大造-mln中。根据Ddc基因的在突变中的表达模式,我们推测mln中因Bm-iAANAT基因功能缺失而累积的大量多巴胺压抑Ddc基因的表达,并通过野生型与突变中多巴胺的定量分析证实了推测。Bm-iAANAT基因的RNAi结果显示,干涉组中约有20%的个体体色明显黑化,且其Bm-iAANAT基因的表达量明显低于对照组,表明Bm-iAANATT基因的确参与多巴胺的消耗及家蚕的着色。本论文的研究第一次报道了AANAT类基因在昆虫体色模式的形成中扮演着重要角色。6.儿茶酚胺类物质与黑色素基因的相互作用对mln突变鞣化部位着色方式及物理性质的影响我们详细比较了不同发育阶段野生型与mln突变体中黑色素代谢基因的表达及儿茶酚胺类物质的含量,并比较了它们在野生型与突变体中的差异。结果表明,突变多巴胺、NBAD与(?))dc,ebony,black及tan基因的相互调控与AANAT基因的缺失,使得mln突变在不同发育阶段按照既定的模式着色。我们在mln突变中注射β-丙氨酸后,改变mln中固有的色素代谢模式,成功反转了mln的黑化表型,并进行了相关色素物质前提及基因的检测。扫描电镜对野生型与mln突变体成虫背板截面的观察结果显示,mln的背板明显出现分层状结构,这与mln中缺乏交联物质NADA有密切关系。野生型与突变体成虫翅膀的机械性能显示,mln翅的弹性模量明显高于野生型,而阻尼性则明显低于野生型,由于是利用近等位基因系及进行分析,因此野生型与突变体翅膀机械性能的不同主要是由于两者间不同的儿茶酚胺代谢所造成的。7.野生型与st突变表皮中几丁质的测定、Bm-st基因的RNAi及原核表达研究st突变与野生型幼虫盛食期表皮几丁质(N-乙酰葡萄糖胺)含量的测定结果显示：st突变中葡萄糖胺的含量为385.95±55.04119μg/mg,野生型中的含量为525.28±38.25767μg/mg。st突变中葡萄糖胺的含量的确是低于野生型。这可能与Bm-st基因的功能缺失或不足有关。我们根据Bm-st的表达模式选定幼虫4龄减食期为注射时期,注射后5龄刚蜕皮的时候观察表型。结果显示,干涉组约有41.1%的个体无法完成蜕皮,且这些个体体壁明显紧绷,手触有坚硬感,与st突变的表型较类似；极端个体表皮异常,影响体形甚至全身渗血黑化。分子检测的结果显示,干涉组有表型的个体Bm-st基因的表达量显著低于对照组。为了研究野生型与突变中正常与异常的Bm-st蛋白与几丁质的结合情况,我们分别构建了含有野生型与突变型Bm-st基因CDS序列的原核表达载体pET32a-WT与pET32a-st。通过诱导表达获得了野生型与突变体的重组型蛋白,为后续的结合实验及野生型与突变中Bm-st天然蛋白表达的检测奠定了基础。更多还原

【Abstract】 Silkworm is an important agricultural economic insect, its moderate body scale, relative short life cycle, high rate of reproduction coupled with complete genomic data, molecular linkage map, hereditary variation map and abundant mutant materials accumulated over the century make it a model organism for Lepidoptera research, various mutation types exist in silkworm, including body color, appendages, body shape, metamorphosis, amino acid metabolism, uric acid metabolism, egg shape, egg color, lethality, etc. More than450Mendelian mutations were documented, among them, about300were mapped on silkworm linkage groups. Such ample resources play an ever increasing role in sericulture, fundamental biological research of silkworm as a model, using silkworm as a bioreactor and basic research of biology. Two kinds of mutations occupy larger proportions in silkworm mutants, body color mutation and body shape mutation. In lepidoptera insects, body color and body shape hold significance to their survival and reproduction. Study the mechanism of body color and body shape of silkworm can not only help researchers better understand metabolism and regulatory pathways of Lepidoptera pigmentation and development, but also provide clues for silkworm functional gene usage (such as breeding of practical strain on silkworm and Lepidoptera pest control). So, we selected melanism (mln) and stony (st) mutants as our study material, they are the representative mutation for melanism and larval body shape, respectively. By positional cloning, we found the candidate genes for these two mutations and carried out corresponding functional verifications. Our main findings are as follows: 1. Molecular mapping of mln and st locus and candidate gene screeningUsing SSR markers, we carried out preliminary mapping for mln and st loci, and constructed molecular marker linkage maps for mln and st. Marker S1807was tightly linked to mln locus. Although the nearest marker, S0809, had a distance of49.6cM from the st locus, the relative positions and distances of other marker on the linkage map were consistent with their physical positions and distances on the genome, so the result can be used for further analysis of st locus. According to the distance between the mutation locus and its nearest molecular marker and the correspondence between the molecular linkage map and the physical map, we calculated the physical position of the candidate genes, and screened the genes within the regions by bioinformatics analysis. For mln, we focused on the gene BGIBMGA008538. Ortholog to this gene in the fruitfly AANAT1can catalyze monoamines like dopamine. Dopamine is one of the precursors of melanin. Tissue microarray analysis showed that this gene was highly expressed in larva head, and head is the region where the mln phenotype is most obvious. Temporal microarray data showed that this gene was highly expressed during late stages of pupation and moth stage, and mln mutants were conspicuously melanized at these stages. If the function of the product of gene BGIBMGA008538was lost, it could cause dopamine to over accumulate, which in turn lead to melanization. Moreover, genotyping of marker P3C (polymorphic marker based on the candidate gene’s sequence) among mapping group showed that no recombination existed between the candidate gene and the mln locus. Sequencing of P3C showed that in the mln mutant, a deletion was found on the third exon of BGIBMGA0085’38. Based on the evidences above, we conclude that BGIBMGA008538is the gene responsible for the mln mutation. For the st mutant, by combining genetic distance with physical distance, we calculated that the nearest marker--S0809had a distance of14.88M from the st locus. According the relative positions of the markers on the molecular linkage map and the length of scaffold and gap on the physical map of8th chromosome, we speculate that the st locus is on nscaf2827. By further analysis of st phenotype, we propose that the st mutation is caused by cuticle protein abnormality. Microarray analysis of three predicted cuticle genes on nscaf2827in association with genotyping and RT-PCR analysis showed that the gene G2expressed specifically during larval stage, and no recombination existed between this gene and st locus. Obvious differences were observed between cDNA amplifications of wild-type and st mutant. From the findings above, we propose G2is the gene responsible for the st mutation.2. Cloning of mln and st candidate gene and sequence analysis between wild-type and mutantWe cloned full-length cDNA of mln and st candidate gene and genomic sequences that exhibit polymorphism. Sequence comparison between wild-type and mutant showed that in the wild-type Dazao, a1460bp fragment was amplified, while in mln two fragments were amplified, measured1407bp and1253bp, respectively. Combine these three transcripts and the sequence of marker P3C mentioned previously, we found that in the mln mutant a deletion of96bp existed (67bp on exon), also was found was a29bp insertion in one of the introns. Bm-iAANAT in Dazao has5exons and encode a product of261amino acids, which possesses intact acetyl-transferase domain. While in the mutant, Dazao-mln, apart from the first2bp, all of the4th exon is lost in the type-1transcript, the resulting frameshift lead to a premature stop codon TAG. In the second abnormal transcript type-2, the last67bp of the4th exon are lost, and an insertion exists in one of the introns. The termination codon of type-2transcript is located at the3’UTR of the of the normal transcript. So, comparing with the product in wild-type, both type-1and type-2transcript encode potentially abnormal proteins, and both lost the acetyl-transferase domain. The candidate gene we select for st Bm-st is547bp long, consists of4exons. Its5’UTR is38bp, and3’UTR is77bp. Bm-st encodes143amino acids, has a typical chitin binding domain. In the st mutant, a58bp deletion and6nucleotide substitutions was found in Bm-st, resulting in a frameshift. The chitin binding domain of the mutated gene is completely disrupted, potentially loses its function. 3. Expression profile analysis of mln candidate gene and investigation of its differential expression between wild-type and mutantTemporal expression profile analysis of Bm-iAANAT was carried out from day3of4th instar to ecdysis. RT-PCR analysis showed that the expression of Bm-iAANAT gene fluctuated during this period. Bm-iAANAT expressed normally at day3of4th instar, and stopped to express at the beginning of4th molt(0-16hours). After molting (24hours later), Bm-iAANAT resumed to express. Bm-iAANAT was highly expressed at the beginning of5th instar, while its expression was not detected at the onset of wandering stage. At the end of wandering stage(2.5day of wandering), expression of Bm-iAANAT is highly elevated(32), while negatively correlated with20E titer. Highest expression of Bm-iAANAT was observed7days after pupation (P7) and8days after pupation (P8). Spacial expression profile showed that Bm-iAANAT was highly expressed in larva head, silkgland and cuticle, with head at the highest level. Expression of Bm-iAANAT was hardly detected in other tissues. Expression analysis of Bm-iAANAT at differently sclerotized regions showed that it expressed higher in highly sclerotized regions (head, thoracic legs and anal plate) than in less sclerotized region (cuticle). That’s to say Bm-iAANATs expression is correlated with melanized regions of mln mutant but not its unmelanized region. Semiquantitative analysis of Bm-iAANAT in Dazao and Dazao-mln at day4of5th instar, day2of pupation and ecdysis showed that the two types of transcripts in mln were expressed lower than their normal counterpart in Dazao; qRT-PCR showed that the transcript level of normal Bm-iAANAT in Dazao was10-20times higher than the two abnormal transcripts in mln combined. Moreover, at the regions where mln mutants manifested the most evident phenotypic differences from Dazao, Bm-iAANAT expressed higher in Dazao than in mln.4. Expression profile analysis of st candidate gene, its differential expression in wild-type and mutant and influence exerted by20EThe candidate gene for st mutation Bm-st was highly expressed during mulberry feeding period at4th and5th instar; but was undetectable at4th molt and was evidently down-regulated from P2. Its expression was restricted to larval and early pupation stage. Spatial expression profile showed that Bm-st was highly expressed in larva head, cuticle, fat body and trachea, and was hard to detect in other tissues. The expression pattern of Bm-st corresponds well with st’s phenotype. In the20E induction experiment, we found that expression level of Bm-st was lower in induced group than in control3hours after commence of experimentation(according to results of semiquantitative and quantitative analyses). After24hours, the induced group already developed into prematured larve, and their Bm-st expression was evidently lower than that of the control’s (according to results of semiquantitative and quantitative analyses). This demonstrates that20E has a repression effect on Bm-st.5. Analyses of key melanin metabolism genes in wild-type and mutant and research on the function of Bm-iAANATWe analyzed the differential expression of key melanin metabolism genes in pigmented regions of wild-type and mln mutant, including:head, thoracic legs and anal plate. Results showed that, expression levels of Pale, Ddc and Yellow were higher in Dazao than in Dazao-mln. However, apart from in head, expression of ebony was lower in Dazao than in Dazao-mln. According to the expression profile of Ddc in mln, we speculate that the over-accumulated dopamine resulted from the abnormal Bm-iAANAT repressed the expression of Ddc, and this was verified by quantitation of dopamine in Dazao and Dazao-mln. In the RNAi experiment, knock down of Bm-iAANAT led to20%of the injected group to melanize, and their Bm-iAANAT expression was evidently lower than in the control group, showing that Bm-iAANAT did participate in consumption of dopamine and pigmentation of silkworm. This is the first report of the important role AANAT gene plays in insect body color pattern determination.6. Influences of the mutual regulation of catecholamines and melanin metabolism genes on color patterns and physical properties of mln sclerotized regionsWe analyzed differences in melanin gene expression level and catecholamine’s content between Dazao and mln at various developmental stages. Result showed that mutual regulation of dopamine, NBAD, Ddc, ebony, black and tan coupled with AANATs function loss made mln mutant to color at its distinctive pattern at different developmental stages. After injection of β-alanine, we successful changed the pigment metabolism pathway in mln, and rescued its melanism phenotype. We also performed pigment precursor and related gene investigation. Scanning electron microscope analysis of cross-section of adult dorsal plate from Dazao and mln showed that stratification existed in mln’s dorsal plate, and this was likely caused by lack of cross-linking agent NBAD in mln. Analysis of mechanical properties of adult wings from Dazao and mln showed that, the elastic modulus of mln was higher than Dazao, while damping of mln was lower than Dazao. Because we used near-isogenic systems to carry out the experiments, so the physical property differences were due to the catecholamine metabolism differences in wild-type and mln mutant.7. Quantitation of chitin between the wild-type and st mutant, RNAi and prokaryotic expression of Bm-stAnalysis of chitin (Glucosamine) content in cuticle of wild-type and st mutant at mulberry feeding stage showed that content of Glucosamine in st mutant was385.95±55.04119μg/mg, and in the wild-type was525.28±38.25767μg/mg. The content of Glucosamine in st mutant was lower than that in wild-type, this might be caused by dysfunction of Bm-st. Based on the expression pattern of Bm-st, we carried out RNAi on4th instar at decline-feeding period, phenotype documentation was performed right after4th molt. Result showed that about41%individuals in the injected group were unable to complete molting, and the cuticles of these silkworms were tight, and felt hard by hand-touching. These resembled the st phenotype. Some extreme individuals had abnormal cuticle and body shape, and might even bleed and melanize. Molecular investigation showed that the Bm-st level in the individuals that showed st-like phenotype was evidently lower than in the control group. To find out the chitin binding abilities of the Bm-st products in wild-type and st mutant, two prokaryotic vectors:pET32a-WT and pET32a-st were constructed, containing the CDS of the wild-type Bm-st and mutant Bm-st, respectively. After induced expression, the recombination protein of wild-type and mutant were obtained, provided experimental foundations for follow-up binding research.更多还原