本文選題：家蠶 + 淡墨突變��； 參考：《西南大學》2016年博士論文

【摘要】：地球上已被描述和記錄的物種大約有200萬種,其中昆蟲約占53%。昆蟲以其強大的適應(yīng)能力,遍布各地。家蠶(Bombyx mori)是重要的經(jīng)濟昆蟲,在大約5000年前由中國野桑蠶(Bombyx mandarina)馴化而來,也是實驗遺傳學的經(jīng)典材料。近年來,隨著家蠶基礎(chǔ)研究的系統(tǒng)化發(fā)展和越來越多的生物學關(guān)注,家蠶被認為是一種新興的模式動物,在基礎(chǔ)研究、生物反應(yīng)器、鱗翅目害蟲防治及醫(yī)學模型等研究領(lǐng)域具有重要意義。1941年日本學者首次報道了一種自然發(fā)生的家蠶突變體,淡墨(black dilute,bd),其幼蟲表皮顯著黑化,雌蛾不妊,表現(xiàn)為隱性的孟德爾遺傳,被定位于家蠶經(jīng)典連鎖圖譜的第9連鎖群22.9cM處,該突變體現(xiàn)被保存于日本和中國。昆蟲的體色和斑紋是其最為顯著的形態(tài)特征之一,其著色機制研究具有重要的生態(tài)學意義,并已有良好的研究基礎(chǔ);同時,著色在種群中極易分化,是進化生物學研究的優(yōu)秀模型。生殖發(fā)育是昆蟲繁衍和擴張的重要基礎(chǔ),有性的繁殖方式能帶給生物更多的演化機會,研究其生殖相關(guān)的分子機制能夠推動對遺傳分化發(fā)育的理解。家蠶淡墨(bd)突變兼具顯著的著色模式改變和生殖障礙表型,對其突變基因及其分子作用的解析既有利于昆蟲著色機制研究,同時也為鱗翅目害蟲防控提供潛在的靶標。本研究利用分子定位鎖定淡墨突變位點在基因組上的范圍,結(jié)合基因功能注釋、基因芯片分析、基因表達差異分析、克隆測序分析等方法確定其候選基因,利用RNAi、過表達和基因敲除等手段鑒定候選基因的功能。并比較分析了淡墨突變與野生型幼蟲體壁的轉(zhuǎn)錄組,尋找Bmbd基因的可能下游靶基因,綜合分析淡墨突變發(fā)生的分子機制。本論文主要的結(jié)果有:1.bd突變體表型特征分析比較野生型品系大造、淡墨純合體(bd/bd)、淡墨雜合體(+/bd)和可育淡墨(bdf/bdf)幼蟲的著色模式。淡墨純合體幼蟲表皮顯著黑化,斑紋處顏色加深;bdf幼蟲著色模式與bd類似,只是顏色較淺。蛹期和蛾期bd體色與對照并沒有明顯的差別。淡墨純合體成蟲翅膀皺縮,不能順利伸展,活動能力偏低,雄蛾交配能力弱,交配后的雌蛾產(chǎn)卵不能正常著色形似未受精卵。淡墨雜合體(+/bd)和bdf成蟲翅膀正常,行為協(xié)調(diào),雄蛾能夠獨立尋找雌蛾交配,雌蛾產(chǎn)卵能正常著色并發(fā)育。解剖未交配的淡墨雌蛾,觀察內(nèi)部生殖器,發(fā)現(xiàn)其卵巢各結(jié)構(gòu)完整,但整體偏小,卵不整齊。經(jīng)過電鏡掃描發(fā)現(xiàn),淡墨卵的花瓣體結(jié)構(gòu)和卵孔異常。經(jīng)過孤雌生殖實驗,發(fā)現(xiàn)淡墨突變體的卵存在發(fā)育缺陷。2.bd位點的分子定位因為淡墨(bd)雌表現(xiàn)為不育性,我們選擇其等位點突變體可育淡墨(bdf)作為突變親本,大造n作為正常親本,兩者雜交產(chǎn)生f1。用f1的雌回交bdf的雄配制連鎖群體,用f1的雄回交bdf的雌配置定位群體。選取屬于第9連鎖群的ssr分子標記引物,并根據(jù)基因組數(shù)據(jù)設(shè)計相應(yīng)的引物,在親本基因組間進行多態(tài)篩選,利用多態(tài)標記對bd位點進行連鎖分析,采用連鎖的標記在1162個個體構(gòu)成的重組分析群體中對淡墨位點進行定位分析。將淡墨位點鎖定在家蠶第9染色體大約600kb的區(qū)域內(nèi),該區(qū)域存在11個預測基因。3.候選基因篩查分析將11個預測基因的編碼序列與ncbi的非冗余蛋白庫(nr)進行比對,對其進行功能注釋。利用家蠶基因芯片數(shù)據(jù)分析11個預測基因在家蠶5齡3天各組織和4齡到5齡幼蟲的時空表達模式。結(jié)合基因功能、表達模式與突變性狀進行關(guān)聯(lián)分析,通過半定量pcr比較大造、淡墨雜合體以及淡墨純合體中定位區(qū)域內(nèi)的預測基因在轉(zhuǎn)錄水平的差異。發(fā)現(xiàn)can8基因在淡墨純合體中幾乎不表達,雜合體中表達水平大約為野生型的一半。而其他預測基因在三者之間無明顯的差異。所以,我們將can8作為重點候選基因進行進一步研究。4.bd突變候選基因的克隆與序列分析我們在野生型大造和bdf中克隆了can8基因的cdna序列,發(fā)現(xiàn)can8基因與nscaf3045上相鄰的預測基因can9為一個基因,且該基因至今沒有明確的功能注釋,將其命名為bmbd,其編碼btb-zf蛋白。通過序列分析,檢測到該基因存在兩種不同的開放閱讀框,其差異為羧基端鋅指功能域不同,可能結(jié)合不同的dna序列。通過序列比對,比較了bdf與野生型中cdna的序列,發(fā)現(xiàn)僅存在幾個單堿基的突變,其預測蛋白質(zhì)的高級結(jié)構(gòu)并沒有明顯的差異。通過基因組dna克隆發(fā)現(xiàn),在bd中bmbd基因從第二內(nèi)含子到轉(zhuǎn)錄上游存在大約160kb的缺失,并插入一段3.6kb的序列,該缺失和插入造成bmbd基因完全沉默,同時疑似產(chǎn)生了一個新基因,編碼鉀離子依賴的鈉/鈣離子通道蛋白,將其命名為bmnckx4-like基因,其功能有待研究。通過blast程序,在昆蟲中對bmbd基因進行同源搜索,并進行系統(tǒng)發(fā)生分析,發(fā)現(xiàn)該基因在許多物種中都存在同源基因,在鱗翅目中非常保守。5.野生型中bmbd基因的表達模式分析在野生型大造4齡剛眠的各組織的rna模板中進行熒光定量檢測bmbd基因的表達情況,發(fā)現(xiàn)其在各組織中均有不同程度的表達,在頭、表皮、生殖腺和絲腺等有較高的表達,在血液中表達水平較低。同時,在大造品系中調(diào)查了bmbd從蟻蠶到蛾一天的時期表達模式,發(fā)現(xiàn)其呈波動表達,在每齡剛眠有上調(diào)表達,在食桑期有中度的表達。這一表達模式,與家蠶幼蟲蛻皮及新表皮著色具有一致性。6.bmbd基因的rnai設(shè)計并合成了bmbd基因的3條sirna。取野生型nis品系幼蟲,在4齡2天,bmbd基因上調(diào)前一天,將3條sirna混合注入其血淋巴,并及時進行電穿孔誘導。之后,用桑葉在25℃,75%的相對濕度小心飼養(yǎng)。在5齡一天,觀察幼蟲皮膚表型,發(fā)現(xiàn)存在大約47%的注射幼蟲在電擊正極一側(cè)出現(xiàn)表皮黑化現(xiàn)象。進行分子檢測發(fā)現(xiàn),黑化皮膚部分的bmbd基因表達量被顯著降低。7.bmbd基因的過表達克隆了bmbd基因的兩種orf框,并將其重組入含有綠色熒光蛋白的表達載體中,基因與綠色熒光蛋白由a3啟動子啟動。將載體與piggybac助質(zhì)�；旌鲜蛊浣K濃度為1μg/μl,經(jīng)腹部氣門注入到淡墨2齡幼蟲的血淋巴中,并及時進行電穿孔誘導。在5齡時觀察幼蟲表皮,發(fā)現(xiàn)有部分幼蟲在電擊正極一側(cè)出現(xiàn)白色斑塊,在熒光顯微鏡下觀察白色區(qū)域存在綠色熒光蛋白的表達。結(jié)合干涉實驗結(jié)果,初步說明bmbd基因參與調(diào)控家蠶的體壁著色。8.bmbd基因的敲除我們設(shè)計并合成了bmbd基因的特異grna,并合成cas9核酸內(nèi)切酶的rna,進行混合。選野生型n4品系的卵,在產(chǎn)卵1~4小時期間,顯微注射大約10nl的混合rna,并用生物粘合劑封住針口。在25℃,相對濕度85%的條件下進行催青孵化。在其后代中,觀察表型。結(jié)果顯示,在子代群體中存在體壁偏黑表型的個體。9.rna-seq通過功能分析,驗證了bmbd基因,作為一個轉(zhuǎn)錄因子蛋白,其表達能夠抑制著色,缺乏時幼蟲顯著黑化。為了研究其調(diào)控體色機制,根據(jù)該基因在剛眠時高表達,我們?nèi)×舜笤�、淡墨純合體(bd/bd)和淡墨雜合體(+/bd)4齡剛眠幼蟲的體壁,進行RNA測序。分析在該時期轉(zhuǎn)錄的基因,并比較三個轉(zhuǎn)錄組數(shù)據(jù)的差異。在三個品系中存在31個共同差異基因,包括候選基因Bmbd,還有15個表皮蛋白(cuticular protein)基因,在黑化淡墨突變中這15個表皮蛋白基因顯著上調(diào)表達。表皮蛋白是表皮的主要組分之一,在家蠶中鑒定了200多個表皮蛋白基因。在柑橘鳳蝶幼蟲體壁中發(fā)現(xiàn),表皮蛋白存在斑紋特異性,暗示色素在摻入表皮中時與表皮蛋白可能發(fā)生特異結(jié)合。本研究發(fā)現(xiàn)的15個在黑化表皮中高表達的表皮蛋白基因可能是家蠶幼蟲黑化所必須的,推測表皮蛋白基因參與表皮著色,這為昆蟲著色提供了新的研究內(nèi)容。本研究定位克隆了家蠶淡墨突變體的負責基因Bmbd,驗證了它抑制黑化的功能,并通過轉(zhuǎn)錄組比較分析、熒光定量分析,推測該基因可抑制漆酶基因laccase2,以及影響表皮蛋白基因的表達,對著色模式進行調(diào)控。同時,淡墨突變存在嚴重的生殖缺陷,我們發(fā)現(xiàn)在家蠶淡墨突變體中,與果蠅ovo直系同源的基因BGIBMGA000988轉(zhuǎn)錄水平顯著下調(diào),ovo是卵發(fā)育關(guān)鍵基因,推測該基因可能與淡墨雌不育有關(guān)。本文通過家蠶bd突變體的研究,揭示了一個與鱗翅目幼蟲著色模式和生殖發(fā)育相關(guān)的新基因。鱗翅目昆蟲中普遍存在其保守的同源基因,結(jié)合對幼蟲體色的調(diào)控和對生殖發(fā)育的影響,家蠶淡墨可望作為鱗翅目害蟲防控研究的潛在模型,有待進一步挖掘其利用價值。
[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.
【學位授予單位】：西南大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q78

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