家蚕淡墨突变体的基因定位克隆及功能研究

发布时间：2018-06-08 22:27

本文选题：家蚕 + 淡墨突变　；参考：《西南大学》2016年博士论文

【摘要】：地球上已被描述和记录的物种大约有200万种,其中昆虫约占53%。昆虫以其强大的适应能力,遍布各地。家蚕(Bombyx mori)是重要的经济昆虫,在大约5000年前由中国野桑蚕(Bombyx mandarina)驯化而来,也是实验遗传学的经典材料。近年来,随着家蚕基础研究的系统化发展和越来越多的生物学关注,家蚕被认为是一种新兴的模式动物,在基础研究、生物反应器、鳞翅目害虫防治及医学模型等研究领域具有重要意义。1941年日本学者首次报道了一种自然发生的家蚕突变体,淡墨(black dilute,bd),其幼虫表皮显著黑化,雌蛾不妊,表现为隐性的孟德尔遗传,被定位于家蚕经典连锁图谱的第9连锁群22.9cM处,该突变体现被保存于日本和中国。昆虫的体色和斑纹是其最为显著的形态特征之一,其着色机制研究具有重要的生态学意义,并已有良好的研究基础;同时,着色在种群中极易分化,是进化生物学研究的优秀模型。生殖发育是昆虫繁衍和扩张的重要基础,有性的繁殖方式能带给生物更多的演化机会,研究其生殖相关的分子机制能够推动对遗传分化发育的理解。家蚕淡墨(bd)突变兼具显著的着色模式改变和生殖障碍表型,对其突变基因及其分子作用的解析既有利于昆虫着色机制研究,同时也为鳞翅目害虫防控提供潜在的靶标。本研究利用分子定位锁定淡墨突变位点在基因组上的范围,结合基因功能注释、基因芯片分析、基因表达差异分析、克隆测序分析等方法确定其候选基因,利用RNAi、过表达和基因敲除等手段鉴定候选基因的功能。并比较分析了淡墨突变与野生型幼虫体壁的转录组,寻找Bmbd基因的可能下游靶基因,综合分析淡墨突变发生的分子机制。本论文主要的结果有:1.bd突变体表型特征分析比较野生型品系大造、淡墨纯合体(bd/bd)、淡墨杂合体(+/bd)和可育淡墨(bdf/bdf)幼虫的着色模式。淡墨纯合体幼虫表皮显著黑化,斑纹处颜色加深;bdf幼虫着色模式与bd类似,只是颜色较浅。蛹期和蛾期bd体色与对照并没有明显的差别。淡墨纯合体成虫翅膀皱缩,不能顺利伸展,活动能力偏低,雄蛾交配能力弱,交配后的雌蛾产卵不能正常着色形似未受精卵。淡墨杂合体(+/bd)和bdf成虫翅膀正常,行为协调,雄蛾能够独立寻找雌蛾交配,雌蛾产卵能正常着色并发育。解剖未交配的淡墨雌蛾,观察内部生殖器,发现其卵巢各结构完整,但整体偏小,卵不整齐。经过电镜扫描发现,淡墨卵的花瓣体结构和卵孔异常。经过孤雌生殖实验,发现淡墨突变体的卵存在发育缺陷。2.bd位点的分子定位因为淡墨(bd)雌表现为不育性,我们选择其等位点突变体可育淡墨(bdf)作为突变亲本,大造n作为正常亲本,两者杂交产生f1。用f1的雌回交bdf的雄配制连锁群体,用f1的雄回交bdf的雌配置定位群体。选取属于第9连锁群的ssr分子标记引物,并根据基因组数据设计相应的引物,在亲本基因组间进行多态筛选,利用多态标记对bd位点进行连锁分析,采用连锁的标记在1162个个体构成的重组分析群体中对淡墨位点进行定位分析。将淡墨位点锁定在家蚕第9染色体大约600kb的区域内,该区域存在11个预测基因。3.候选基因筛查分析将11个预测基因的编码序列与ncbi的非冗余蛋白库(nr)进行比对,对其进行功能注释。利用家蚕基因芯片数据分析11个预测基因在家蚕5龄3天各组织和4龄到5龄幼虫的时空表达模式。结合基因功能、表达模式与突变性状进行关联分析,通过半定量pcr比较大造、淡墨杂合体以及淡墨纯合体中定位区域内的预测基因在转录水平的差异。发现can8基因在淡墨纯合体中几乎不表达,杂合体中表达水平大约为野生型的一半。而其他预测基因在三者之间无明显的差异。所以,我们将can8作为重点候选基因进行进一步研究。4.bd突变候选基因的克隆与序列分析我们在野生型大造和bdf中克隆了can8基因的cdna序列,发现can8基因与nscaf3045上相邻的预测基因can9为一个基因,且该基因至今没有明确的功能注释,将其命名为bmbd,其编码btb-zf蛋白。通过序列分析,检测到该基因存在两种不同的开放阅读框,其差异为羧基端锌指功能域不同,可能结合不同的dna序列。通过序列比对,比较了bdf与野生型中cdna的序列,发现仅存在几个单碱基的突变,其预测蛋白质的高级结构并没有明显的差异。通过基因组dna克隆发现,在bd中bmbd基因从第二内含子到转录上游存在大约160kb的缺失,并插入一段3.6kb的序列,该缺失和插入造成bmbd基因完全沉默,同时疑似产生了一个新基因,编码钾离子依赖的钠/钙离子通道蛋白,将其命名为bmnckx4-like基因,其功能有待研究。通过blast程序,在昆虫中对bmbd基因进行同源搜索,并进行系统发生分析,发现该基因在许多物种中都存在同源基因,在鳞翅目中非常保守。5.野生型中bmbd基因的表达模式分析在野生型大造4龄刚眠的各组织的rna模板中进行荧光定量检测bmbd基因的表达情况,发现其在各组织中均有不同程度的表达,在头、表皮、生殖腺和丝腺等有较高的表达,在血液中表达水平较低。同时,在大造品系中调查了bmbd从蚁蚕到蛾一天的时期表达模式,发现其呈波动表达,在每龄刚眠有上调表达,在食桑期有中度的表达。这一表达模式,与家蚕幼虫蜕皮及新表皮着色具有一致性。6.bmbd基因的rnai设计并合成了bmbd基因的3条sirna。取野生型nis品系幼虫,在4龄2天,bmbd基因上调前一天,将3条sirna混合注入其血淋巴,并及时进行电穿孔诱导。之后,用桑叶在25℃,75%的相对湿度小心饲养。在5龄一天,观察幼虫皮肤表型,发现存在大约47%的注射幼虫在电击正极一侧出现表皮黑化现象。进行分子检测发现,黑化皮肤部分的bmbd基因表达量被显著降低。7.bmbd基因的过表达克隆了bmbd基因的两种orf框,并将其重组入含有绿色荧光蛋白的表达载体中,基因与绿色荧光蛋白由a3启动子启动。将载体与piggybac助质粒混合使其终浓度为1μg/μl,经腹部气门注入到淡墨2龄幼虫的血淋巴中,并及时进行电穿孔诱导。在5龄时观察幼虫表皮,发现有部分幼虫在电击正极一侧出现白色斑块,在荧光显微镜下观察白色区域存在绿色荧光蛋白的表达。结合干涉实验结果,初步说明bmbd基因参与调控家蚕的体壁着色。8.bmbd基因的敲除我们设计并合成了bmbd基因的特异grna,并合成cas9核酸内切酶的rna,进行混合。选野生型n4品系的卵,在产卵1~4小时期间,显微注射大约10nl的混合rna,并用生物粘合剂封住针口。在25℃,相对湿度85%的条件下进行催青孵化。在其后代中,观察表型。结果显示,在子代群体中存在体壁偏黑表型的个体。9.rna-seq通过功能分析,验证了bmbd基因,作为一个转录因子蛋白,其表达能够抑制着色,缺乏时幼虫显著黑化。为了研究其调控体色机制,根据该基因在刚眠时高表达,我们取了大造、淡墨纯合体(bd/bd)和淡墨杂合体(+/bd)4龄刚眠幼虫的体壁,进行RNA测序。分析在该时期转录的基因,并比较三个转录组数据的差异。在三个品系中存在31个共同差异基因,包括候选基因Bmbd,还有15个表皮蛋白(cuticular protein)基因,在黑化淡墨突变中这15个表皮蛋白基因显著上调表达。表皮蛋白是表皮的主要组分之一,在家蚕中鉴定了200多个表皮蛋白基因。在柑橘凤蝶幼虫体壁中发现,表皮蛋白存在斑纹特异性,暗示色素在掺入表皮中时与表皮蛋白可能发生特异结合。本研究发现的15个在黑化表皮中高表达的表皮蛋白基因可能是家蚕幼虫黑化所必须的,推测表皮蛋白基因参与表皮着色,这为昆虫着色提供了新的研究内容。本研究定位克隆了家蚕淡墨突变体的负责基因Bmbd,验证了它抑制黑化的功能,并通过转录组比较分析、荧光定量分析,推测该基因可抑制漆酶基因laccase2,以及影响表皮蛋白基因的表达,对着色模式进行调控。同时,淡墨突变存在严重的生殖缺陷,我们发现在家蚕淡墨突变体中,与果蝇ovo直系同源的基因BGIBMGA000988转录水平显著下调,ovo是卵发育关键基因,推测该基因可能与淡墨雌不育有关。本文通过家蚕bd突变体的研究,揭示了一个与鳞翅目幼虫着色模式和生殖发育相关的新基因。鳞翅目昆虫中普遍存在其保守的同源基因,结合对幼虫体色的调控和对生殖发育的影响,家蚕淡墨可望作为鳞翅目害虫防控研究的潜在模型,有待进一步挖掘其利用价值。
[Abstract]:There are about 2 million species of species that have been described and recorded on the earth, of which insects account for about 53%. insects with their strong adaptability and spread all over the world. The silkworm (Bombyx mori) is an important economic insect. It was domesticated by the Chinese silkworm (Bombyx Mandarina) about 5000 years ago. It is also a classic material for experimental genetics. In recent years, with the silkworm, with the silkworm The systematic development of basic research and more and more biological attention, silkworm is considered to be a new model animal. In the field of basic research, bioreactor, lepidoptera pest control and medical model, it is important for Japanese scholars to report a natural mutant of silkworm, light ink (black DIL) for the first time in.1941. Ute, BD), the epidermis of the larvae is significantly blackened and the female moth is not pregnant. It is shown as a recessive Mendel inheritance. It is located in the ninth chain group of the classic linkage map of the silkworm, 22.9cM, which is preserved in Japan and China. The body color and markings of insects are one of the most significant morphological features, and the study of its coloring mechanism has important ecology. At the same time, coloring is very easy to differentiate in the population. It is an excellent model for the study of evolutionary biology. Reproductive development is an important basis for the propagation and expansion of insects. Sexual reproduction can bring more opportunities for evolution, and the study of its reproductive related molecular mechanism can promote the development of genetic differentiation. It is understood that the mutation of the silkworm (BD) mutation and the reproductive disorder phenotype are significant. The analysis of the mutant gene and its molecular role is beneficial to the study of the insect coloring mechanism and the potential target for the control of Lepidoptera pests. The candidate genes were identified by gene functional annotation, gene chip analysis, gene expression difference analysis, cloned sequencing analysis and other methods. RNAi, overexpression and gene knockout were used to identify the function of the candidate genes. The transcriptional groups of the light ink and wild type larva were compared and analyzed to find the possible downstream target genes of the Bmbd gene. A comprehensive analysis of the molecular mechanism of the occurrence of light ink mutation. The main results of this paper are: the analysis of the phenotypic characteristics of 1.bd mutants compared with the wild type, the light ink homozygous (bd/bd), the light ink complex (+/bd) and the fertile light ink (bdf/bdf) larvae. The epidermis of the light ink homozygous larvae is significantly blackened, the color of the markings is deepened, and the BDF larva is a larva. The color pattern is similar to BD, but the color is shallow. The BD body color of the pupal stage and the moth stage is not significantly different from that of the control. The wings of the light ink homozygote can not extend smoothly, the ability of the activity is low, the male moth mating ability is weak, and the oviposition of the female moth after mating can not be similar to the unfertilized egg. The light ink complex (+/bd) and the wings of the BDF adult are normal. The male moth could find the female moth independently, and the female moth could be coloured and developed normally. The female moth could be coloured and developed normally. The unmated female moth was dissected and the internal genitals were observed. The whole ovary was intact, but the whole egg was small and the egg was not neat. The structure of the petal body and the ovum in the light egg were found to be abnormal. After the parthenogenesis experiment, the female moth was found to have been found to have been found by the parthenogenesis experiment. The molecular location of the developmental defect.2.bd loci of the light ink mutants is due to the male infertility of the weak ink (BD) female, and we select the allele mutants to breed the light ink (BDF) as the mutant parent and make the n as the normal parent. The hybridization produces the male confecting group of the female BDF of the f1. with the F1, and the female configuration of the male return of F1 to the male of the male. The SSR molecular marker primers belonging to the ninth linkage group were selected and the corresponding primers were designed according to the genome data. Polymorphic screening was carried out in the parent genome. The linkage analysis of the BD loci was carried out by polymorphic markers. The linkage markers were used to locate the light loci in the recombinant analysis group composed of 1162 individuals. The light ink loci are locked in the region of about 600KB of the ninth chromosome of the silkworm. There are 11 candidate genes in this region for screening and analysis of the.3. candidate genes. The coding sequences of the 11 predicted genes are compared with the non redundant protein library of NCBI (NR) for functional annotation. 11 prediction genes are analyzed by the silkworm gene chip data in the 3 days of the Bombyx mori 5. The spatio-temporal expression patterns of various tissues and 4 to 5 instar larvae, combined with gene function, expression patterns and mutation traits, were compared by semi quantitative PCR, and the difference in the transcriptional level of the predicted genes in the light ink complex and the light ink homozygous region was found. It was found that the can8 gene was almost non expressed in the light ink homozygous. The expression level in the chimeras is about half of the wild type. And there is no significant difference between the other predicted genes between the three. Therefore, we use can8 as a key candidate gene to further study the cloning and sequence analysis of the.4.bd mutation candidate genes. We found the cDNA sequence of the can8 gene in the wild type and BDF, and found the can8 gene. The predictive gene can9 adjacent to nscaf3045 is a gene, and the gene has no clear functional annotation and named bmbd, which encodes the btb-zf protein. Through sequence analysis, the gene has two different open reading frames. The difference is that the functional domain of the carboxyl terminal zinc finger is different and may be combined with different DNA sequences. Sequence alignment, compared with the sequence of cDNA in BDF and wild type, it was found that there were only a few single base mutations, and there was no significant difference in predicting the high structure of the protein. By genomic DNA cloning, the deletion of the bmbd gene from the second intron to the upstream of the transcription was found in the BD, and a sequence of 3.6kb was inserted into the sequence of 3.6kb. The bmbd gene was completely silenced and a new gene was suspected to produce a new gene, which encodes a potassium ion dependent sodium / calcium channel protein, named bmnckx4-like gene, and its function needs to be studied. The bmbd gene is searched by the blast program in insects, and the phylogenetic analysis is carried out to find that the gene is in many cases. Homologous genes are found in the species, and the expression pattern analysis of bmbd gene in the.5. wild type of Lepidoptera is used to detect the expression of bmbd gene in the RNA template of the wild type 4 years old. It is found that the expression of bmbd gene in various tissues is different in the head, the epidermis, the genital gland and the silk gland. The expression level is low in the blood. At the same time, the expression pattern of bmbd from the silkworm to the moth was investigated in the large production line, and it was found to be in fluctuating expression. The expression was up-regulated at every dormancy and moderate expression during the mulberry period. This expression pattern was consistent with the.6.bmbd base of the molt and the new epidermis of the silkworm larvae. RNAi designed and synthesized 3 sirna. of the bmbd gene from the wild type NIS strain of the NIS strain. In 2 days of age 4, the bmbd gene was mixed into its hemolymph and induced by electroporation one day before the bmbd gene was up-regulated. Then, the mulberry leaves were carefully fed at 25 and 75% relative humidity. At 5 days, the larval skin phenotype was observed and found to be about 4. 7% of the injected larvae appeared on the side of the positive electrode. Molecular detection showed that the bmbd gene expression of the blackened part of the skin was significantly reduced by the overexpression of the.7.bmbd gene and the two ORF frames of the bmbd gene were cloned and reassembled into the surface vector containing the green fluorescent protein, and the gene and green fluorescent protein were initiated by A3 The carrier and piggyBac plasmids were mixed to make the final concentration of the piggyBac plasmids, the final concentration was 1 mu g/ mu, and the abdominal valve was injected into the blood of the 2 instar larvae of the light ink, and the electroporation was induced in time. At the age of 5, the larval epidermis was observed, and some of the white spots appeared on the side of the electric shock. The white area was observed under the fluorescence microscope. The expression of green fluorescent protein. Combined with the results of interference experiment, we preliminarily show that bmbd gene participates in the knockout of the coloring.8.bmbd gene of the silkworm, we designed and synthesized the specific gRNA of the bmbd gene, and synthesized the RNA of the cas9 endonuclease, and mixed it. The eggs of the wild type N4 strain were selected for the microinjection of about 10N during the ovum 1~4 hours. L was mixed with RNA and sealed the needle mouth with a biological adhesive. The incubation was carried out at 25 degrees centigrade and relative humidity of 85%. The phenotype was observed in the offspring. The results showed that the individual.9.rna-seq in the progeny of the progeny showed the bmbd gene, which could be suppressed as a transcription factor protein, and the expression could be suppressed as a transcription factor protein. In order to study the color mechanism of the larvae, in order to study its regulation of body color mechanism, according to the high expression of the gene at the time of dormancy, we took the body wall of 4 instar larvae of light ink homozygote (bd/bd) and light heterozygous complex (+/bd), and sequenced RNA. The transcriptional genes at this period were analyzed and the differences between the three transcriptional groups were compared. Three products were compared. There are 31 common difference genes in the system, including the candidate gene Bmbd and 15 epidermal protein (cuticular protein) genes. The 15 epidermal protein genes are up to up expression in the blackening and light ink mutation. The epidermal protein is one of the main components of the epidermis, and more than 200 epidermal protein genes are identified in the silkworm. It is found that the epidermal protein is speckle specific, suggesting that the pigment may have a specific binding to the epidermal protein when mixed with the epidermis. The 15 epidermal protein genes, which are highly expressed in the blackened epidermis, may be necessary for the blackening of the silkworm larvae. It is suggested that the epidermal protein gene is involved in the coloring of the epidermis, which provides a new color for the insect coloring. In this study, the responsible gene Bmbd of the silkworm mutants was cloned, and the function of inhibiting the blackening was verified. Through the comparison and analysis of the transcriptional group and the fluorescence quantitative analysis, it was suggested that the gene could inhibit the laccase gene laccase2, and influence the expression of the epidermal protein gene and control the color pattern. Meanwhile, the light ink mutation was found. In the presence of severe reproductive defects, we found that in the light mutants of the silkworm, the transcriptional level of the gene BGIBMGA000988 directly homologous to the Drosophila ovo is significantly down, and ovo is the key gene for the development of the egg. This gene may be related to the light female sterility. In this paper, a coloring pattern with the Lepidoptera larva was revealed through the study of the BD mutant of the silkworm. New genes related to reproductive development. There are common conserved homologous genes in Lepidoptera. In combination with the control of the body color of the larvae and the effect on reproductive development, the silkworm can be regarded as a potential model for the control and control of Lepidoptera pests, which need to be further excavated.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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