家蠶GATA結(jié)合蛋白家族鑒定及BmGATA6-like基因功能研究
發(fā)布時(shí)間：2018-07-16 09:03
【摘要】：家蠶作為重要的無脊椎動(dòng)物模型,在其變態(tài)發(fā)育過程中組織形態(tài)會(huì)發(fā)生巨大變化,因此是研究細(xì)胞增殖、分化、凋亡及自噬等生理現(xiàn)象理想的材料。GATA結(jié)合蛋白因子是從低等的線蟲到高等的哺乳動(dòng)物發(fā)育過程中都需要的非常重要的調(diào)控因子,參與個(gè)體胚胎發(fā)育、組織器官發(fā)生、個(gè)體衰老等重要的生命活動(dòng)。本研究以家蠶作為研究對(duì)象,利用家蠶基因組數(shù)據(jù)庫獲得家蠶GATA結(jié)合蛋白序列信息,并重點(diǎn)對(duì)BmGATA6-like基因的功能進(jìn)行分析及研究,將有利于闡明家蠶變態(tài)發(fā)育的分子調(diào)控機(jī)理,促進(jìn)對(duì)昆蟲變態(tài)發(fā)育的全面認(rèn)識(shí),不僅可以為害蟲的生物防治提供線索,甚至還能為人類的退行性疾病提供數(shù)據(jù)參考。主要研究結(jié)果如下:1.家蠶GATA結(jié)合蛋白家族基因的鑒定通過NCBI及家蠶全基因組數(shù)據(jù)庫,分析獲得5個(gè)家蠶GATA結(jié)合蛋白家族基因,分別為BmGATA-A-like,BmGATA-C-like,BmBCF I,BmGATA6-like和BmGATA-X。同其它物種中的GATA結(jié)合蛋白類似,它們都具有保守的GATA鋅指結(jié)構(gòu)域,其中BmGATA-A-like和BmGATA-X只含有一個(gè)GATA鋅指結(jié)構(gòu)域,BmGATA-C-like,BmBCF I和BmGATA6-like都含有兩個(gè)典型的GATA鋅指結(jié)構(gòu)域。5齡3天家蠶各組織芯片數(shù)據(jù)顯示家蠶GATA結(jié)合蛋白的表達(dá)量較低,且各成員間的表達(dá)情況存在較大差異。保守結(jié)構(gòu)域的進(jìn)化分析顯示家蠶GATA結(jié)合蛋白家族成員主要與昆蟲的GATA結(jié)合蛋白聚為一類,其中BmGATA-A-like與果蠅的Pannier聚為一類,BmBCF I與赤擬谷盜的GATAa及果蠅的Serpent聚為一類,BmGATA6-like與鱗翅目昆蟲的GATA結(jié)合蛋白聚為一類,BmGATA-X與意大利蜂的GATA4-like最為保守,而BmGATA-C-like則單獨(dú)聚為一類;保守結(jié)構(gòu)域的物種間多序列比對(duì)顯示GATA鋅指結(jié)構(gòu)域序列同源性非常高。2.家蠶Bm GATA6-like基因的克隆與表達(dá)分析通過家蠶基因組數(shù)據(jù)庫預(yù)測(cè)序列信息及ncbiblast序列,克隆得到家蠶bmgata6-like基因全長cdna序列(為了簡(jiǎn)潔,后文中將以bmgata6代替稱呼bmgata6-like)。該基因全長為4011bp,含有10個(gè)外顯子和9個(gè)內(nèi)含子;其orf框長1974bp,編碼657個(gè)氨基酸,預(yù)測(cè)的蛋白分子量為70.8kda,等電點(diǎn)pi為6.13。bmgata6蛋白含有兩個(gè)典型的gata鋅指蛋白結(jié)構(gòu)域,分別位于第464~516位氨基酸基序和524~576位氨基酸基序;在線軟件預(yù)測(cè)其不含信號(hào)肽。利用實(shí)時(shí)熒光定量pcr技術(shù)檢測(cè)其在家蠶胚胎、5齡3天各組織中的相對(duì)表達(dá)水平,結(jié)果顯示bmgata6基因在家蠶胚胎發(fā)育各個(gè)時(shí)期均有表達(dá),在胚胎發(fā)育的后期持續(xù)高表達(dá),在胚胎發(fā)育第6天及孵化出的蟻蠶中表達(dá)量最高;5齡3天各組織中,bmgata6在各個(gè)組織中均有不同程度的表達(dá),但是在中腸中的表達(dá)量顯著高于其它組織。檢測(cè)bmgata6在表達(dá)較高的中腸組織和表達(dá)較低的血液不同時(shí)期的表達(dá)情況,結(jié)果顯示中腸組織中bmgata6持續(xù)表達(dá),并且在眠期及變態(tài)發(fā)育前期顯著高表達(dá);血細(xì)胞中bmgata6的表達(dá)模式與中腸組織中的表達(dá)模式極其相似,同樣是在眠期及變態(tài)發(fā)育時(shí)期顯著高表達(dá)。免疫熒光檢測(cè)bmgata6在中腸組織和血細(xì)胞中的表達(dá)定位情況,結(jié)果顯示bmgata6在家蠶中腸所有類型的細(xì)胞中均有高量表達(dá),且只集中表達(dá)于中腸組織細(xì)胞的細(xì)胞核中;l5d3家蠶血細(xì)胞中,檢測(cè)到bmgata6在顆粒細(xì)胞中有微量的表達(dá),且只在顆粒細(xì)胞的細(xì)胞質(zhì)中表達(dá),漿細(xì)胞中不能檢測(cè)到bmgata6信號(hào)。這些結(jié)果暗示著bmgata6基因在家蠶生長及變態(tài)發(fā)育過程中扮演重要角色。3.家蠶bmgata6-like基因誘導(dǎo)細(xì)胞凋亡機(jī)制研究構(gòu)建bmgata6過表達(dá)載體,通過細(xì)胞轉(zhuǎn)染的方法在家蠶胚胎細(xì)胞系bme中過表達(dá)bmgata6基因。westernblotting結(jié)果顯示轉(zhuǎn)染后24hbmgata6蛋白就開始表達(dá),且隨著時(shí)間的延長其蛋白含量也逐漸增多。熒光顯微鏡觀察結(jié)果顯示從轉(zhuǎn)染后48h開始,細(xì)胞逐漸出現(xiàn)細(xì)胞碎裂的現(xiàn)象,轉(zhuǎn)染后72h及96h,其周圍細(xì)胞碎片明顯增多。分別用dapi及tunel染色進(jìn)行細(xì)胞凋亡檢測(cè),dapi染色結(jié)果顯示過表達(dá)bmgata6的細(xì)胞核結(jié)構(gòu)松散,染色質(zhì)呈不規(guī)則形狀分布;tunel染色后計(jì)數(shù)結(jié)果顯示tunel的著色率明顯高于對(duì)照組。熒光定量pcr檢測(cè)轉(zhuǎn)染過表達(dá)bmgata6基因后bme細(xì)胞中家蠶凋亡相關(guān)基因的表達(dá)量變化,結(jié)果顯示相關(guān)基因表達(dá)量被顯著上調(diào);凋亡執(zhí)行蛋白caspase3/7活性檢測(cè)顯示其活性也被明顯上調(diào)。以上實(shí)驗(yàn)說明bmgata6可以誘導(dǎo)家蠶細(xì)胞發(fā)生凋亡。利用bmgata6抗體進(jìn)行免疫沉淀,并將得到的產(chǎn)物進(jìn)行蛋白質(zhì)質(zhì)譜分析鑒定,篩選得到可能與bmgata6互作的蛋白數(shù)量為11個(gè),根據(jù)分子量大小及功能等因素最后確定可以促進(jìn)細(xì)胞發(fā)生凋亡的parp為目的蛋白;免疫熒光共定位實(shí)驗(yàn)驗(yàn)證PARP與BmGATA6蛋白共同在細(xì)胞核中表達(dá);進(jìn)一步免疫共沉淀實(shí)驗(yàn)驗(yàn)證BmGATA6可以與PARP蛋白相互作用。這些結(jié)果暗示BmGATA6誘發(fā)的細(xì)胞凋亡可能與PARP蛋白的相互作用相關(guān)。4.FOG蛋白抑制Bm GATA6誘發(fā)細(xì)胞凋亡通過比較在家蠶胚胎細(xì)胞系BmE和家蠶卵巢細(xì)胞系BmNs中分別過表達(dá)BmGATA6的現(xiàn)象,發(fā)現(xiàn)不表達(dá)家蠶FOG因子BmUsh的BmE細(xì)胞系相較于高量表達(dá)BmUsh的BmNs細(xì)胞系,BmGATA6過表達(dá)誘發(fā)的凋亡現(xiàn)象更為快速且更為明顯。同時(shí)在BmE細(xì)胞系中過表達(dá)BmGATA6和BmUsh,經(jīng)顯微鏡觀察結(jié)果顯示由BmGATA6誘發(fā)的細(xì)胞凋亡明顯得到了抑制;Tunel染色后,統(tǒng)計(jì)結(jié)果顯示與單獨(dú)過表達(dá)BmGATA6基因相比,共同過表達(dá)BmGATA6和BmUsh基因的Tunel信號(hào)數(shù)量明顯下降,且Caspase3/7的活性也明顯降低。結(jié)果說明家蠶的FOG因子BmUsh可以抑制由過表達(dá)BmGATA6誘發(fā)的細(xì)胞凋亡。通過原核表達(dá)及蛋白純化,獲得具有活性的BmGATA6重組蛋白,利用Far-western證明BmGATA6可以與BmUsh蛋白相互作用,且其作用區(qū)域在BmGATA6的N端;進(jìn)一步免疫共沉淀實(shí)驗(yàn)也驗(yàn)證了家蠶FOG因子BmUsh蛋白可以在與BmGATA6共同過表達(dá)的情況下與PARP蛋白結(jié)合,但是單獨(dú)過表達(dá)BmUsh時(shí)不能與PARP結(jié)合。說明BmUsh通過與BmGATA6結(jié)合間接與PARP相互作用。熒光定量PCR檢測(cè)家蠶中腸組織和血細(xì)胞中BmUsh和BmGATA6的時(shí)期表達(dá)變化,進(jìn)行比較發(fā)現(xiàn)中腸組織中BmGATA6表達(dá)量較高,血細(xì)胞中BmUsh表達(dá)量較高,但無論是在中腸組織還是血細(xì)胞中,兩者的表達(dá)模式極為相似,都在眠期及變態(tài)發(fā)育時(shí)期高量表達(dá)。由此結(jié)果推測(cè)家蠶FOG因子BmUsh與BmGATA6在眠期和變態(tài)時(shí)期通過相互作用,共同調(diào)控家蠶的正常生長及變態(tài)發(fā)育。5.家蠶Bm GATA6基因的功能綜合上述結(jié)論與分析,我們推測(cè)家蠶BmGATA6在家蠶生長及變態(tài)發(fā)育過程中,被某些生長因子或者激素等誘導(dǎo)上調(diào)表達(dá),在組織細(xì)胞發(fā)生程序性死亡過程中,通過與PARP蛋白的相互作用或者其它目前還未知的調(diào)控機(jī)制共同誘導(dǎo)家蠶組織細(xì)胞發(fā)生凋亡,當(dāng)?shù)蛲龀潭冗_(dá)到預(yù)期水平后,FOG因子BmUsh再通過與其直接作用抑制由其誘導(dǎo)的凋亡,共同維護(hù)家蠶生長及變態(tài)發(fā)育過程的順利進(jìn)行。
[Abstract]:Silkworm, as an important model of invertebrate, has a great change during its abnormal development, so it is an ideal material to study the physiological phenomena such as cell proliferation, differentiation, apoptosis and autophagy, the.GATA binding protein factor is a very important tune from low nematode to higher mammals. In this study, the silkworm GATA binding protein sequence information was obtained by the silkworm genome database, and the function of the BmGATA6-like gene was analyzed and studied. It will be beneficial to clarify the metamorphosis of the silkworm. The mechanism of molecular regulation to promote the comprehensive understanding of insect metamorphosis can not only provide clues for the biological control of insect pests, but also provide data reference for human degenerative diseases. The main results are as follows: the identification of the gene of the GATA binding protein family of the 1. silkworms was analyzed by NCBI and the whole genome database of the silkworm, and 5 families were obtained. The GATA binding protein family of the silkworm, BmGATA-A-like, BmGATA-C-like, BmBCF I, BmGATA6-like and BmGATA-X. are similar to the GATA binding proteins in other species. They all have a conservative GATA zinc finger domain, and BmGATA-A-like and BmGATA-X contain only one domain of GATA zinc finger. Two typical GATA zinc finger domain.5 age 3 days old silkworm microarray data show that the expression of GATA binding protein in silkworm is low, and there is a great difference in the expression among the members. The evolutionary analysis of the conserved domain shows that the members of the GATA binding protein family of the silkworm mainly gather with the GATA binding protein of insects, of which, BmGATA-A-l Ike is clustered with the Pannier of Drosophila melanogaster, BmBCF I and GATAa and Serpent of Drosophila melanogaster are clustered into a class. GATA binding proteins of BmGATA6-like and Lepidoptera are clustered into a class. BmGATA-X and the GATA4-like of the Italy bee are most conservative, while BmGATA-C-like is a single class; the multiple sequence alignment between species in the conservative domain shows GATA. The cloning and expression analysis of the Bm GATA6-like gene of the silkworm, silkworm,.2. is highly homologous, and the whole length cDNA sequence of the Bombyx mori bmgata6-like gene is cloned by the sequence information of the silkworm genome database and the sequence of ncbiblast. (in order to be succinct, the bmgata6 substitution will be called bmgata6-like). The whole length of the gene is 4011BP, It contains 10 exons and 9 introns; the ORF frame is long 1974bp, encodes 657 amino acids, the predicted protein molecular weight is 70.8kda, and the isoelectric point Pi is 6.13.bmgata6 protein containing two typical GATA zinc finger protein domains, which are located in the 464~516 amino acid sequence and 524~576 bit amino acid sequence respectively, and the online software predicts its non signal peptide. Real time fluorescence quantitative PCR was used to detect the relative expression level in the tissues of the silkworm embryos, 5 years and 3 days. The results showed that the bmgata6 gene was expressed in every period of the silkworm embryo development. The expression was high in the later stage of the embryo development, and the highest expression in the embryonic development and hatched silkworms in the sixth days of embryo development; and in the 3 days of 5 instar, bmgat The expression of A6 was significantly higher in the various tissues, but the expression in the midgut was significantly higher than that in other tissues. The expression of bmgata6 was expressed in the middle intestinal tissue and expressed in different periods of the lower blood. The results showed that the expression of bmgata6 in the midgut tissue was continuous, and was highly expressed in the period of dormancy and in the early stage of metamorphosis. The expression pattern of bmgata6 in the blood cells was very similar to the expression pattern in the midgut tissue. It was also highly expressed in the period of dormancy and metamorphosis. The expression of bmgata6 in the midgut tissues and blood cells by immunofluorescence showed that bmgata6 was highly expressed in the types of cells in the middle intestine of the silkworm, and only set in the middle intestine of the silkworm. It is expressed in the nucleus of the cell of the midgut tissue; in the l5d3 silkworm blood cells, the expression of bmgata6 in granulosa cells is detected, and only in the cytoplasm of the granulosa cells, and the bmgata6 signal can not be detected in the plasma cells. These results suggest that the bmgata6 gene plays an important role in the growth and metamorphosis of the silkworm,.3. The mechanism of bmgata6-like gene induced apoptosis of silkworm was constructed and the bmgata6 overexpression vector was constructed. The expression of bmgata6 gene was expressed in the BME of the Silkworm Embryo Cell Line BME by cell transfection. The expression of 24hbmgata6 protein began to express after transfection, and the protein content gradually increased with the time of transfection. The results of microscopic observation showed that the cells began to break up gradually from 48h after transfection. After transfection, the cell fragments of 72h and 96h were significantly increased. The cell apoptosis was detected by DAPI and TUNEL staining. The results of DAPI staining showed that the nuclear structure of the expressed bmgata6 was loose and the chromatin was irregular. After TUNEL, TUNEL was stained. The count results showed that the coloring rate of TUNEL was significantly higher than that of the control group. The expression of apoptosis related genes in the silkworm of BME cells transfected with bmgata6 gene was detected by fluorescence quantitative PCR. The results showed that the expression of related genes was significantly up-regulated, and the activity of caspase3/7 activity of apoptotic executive protein showed that its activity was also obviously up-regulated. It is suggested that bmgata6 can induce apoptosis of silkworm cells. Immunoprecipitation is carried out by using bmgata6 antibody, and the obtained products are identified by protein mass spectrometry. The number of proteins that may be interacted with bmgata6 is 11, and the PARP of cell apoptosis can be promoted according to the molecular weight and function. Protein; immunofluorescence co localization experiment verifies that PARP and BmGATA6 protein are expressed together in the nucleus, and further immunoprecipitation experiments prove that BmGATA6 can interact with PARP protein. These results suggest that the apoptosis induced by BmGATA6 may be associated with the interaction of PARP protein and the inhibition of Bm GATA6 induced apoptosis by.4.FOG protein. The phenomenon of BmGATA6 in the silkworm embryo cell line BmE and the silkworm ovarian cell line BmNs was compared. It was found that the BmE cell line that did not express the FOG factor BmUsh of the silkworm was compared to the BmNs cell line with the high expression of BmUsh, and the apoptosis induced by BmGATA6 overexpression was more rapid and more obvious. Meanwhile, BmGATA6 and Bm were overexpressed in the BmE cell line. Ush, the microscopically observed results showed that the apoptosis induced by BmGATA6 was obviously inhibited. After Tunel staining, the statistical results showed that the number of Tunel signals that expressed the BmGATA6 and BmUsh genes in the common overexpressed BmGATA6 gene decreased obviously, and the activity of Caspase3/7 decreased obviously. The results showed that the FOG factor BmUs of the silkworm was BmUs. H can inhibit the apoptosis induced by overexpression of BmGATA6. Through prokaryotic expression and protein purification, the reactive BmGATA6 recombinant protein is obtained. Far-western shows that BmGATA6 can interact with BmUsh protein and its action region is at the N end of BmGATA6; further immunoprecipitation test also verifies that the FOG factor BmUsh protein of silkworm is available. Combined with PARP protein in the case of co expression with BmGATA6, but it could not be associated with PARP when BmUsh was overexpressed alone. It indicated that BmUsh interacted indirectly with PARP by combining with BmGATA6. The fluorescence quantitative PCR detected the changes in the period table of BmUsh and BmGATA6 in the midgut and blood cells of the silkworm, and was compared to the BmGA in the midgut tissue. The expression of TA6 is high and the expression of BmUsh in the blood cells is high, but both in the middle and the blood cells, the expression patterns are very similar, both in the hibernation and metamorphosis period of high expression. Thus, it is concluded that the FOG factor BmUsh and BmGATA6 can regulate the normal growth of silkworm in the dormant and metamorphic periods. The conclusion and analysis of the functions of the Bm GATA6 gene of the long and abnormal development of.5. silkworm (silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, and silkworm, GATA6) are synthesized by the above conclusion and analysis. The unknown regulation mechanism co induced the apoptosis of silkworm tissue cells. When the degree of apoptosis reached the expected level, FOG factor BmUsh could inhibit the apoptosis induced by its direct action, so as to maintain the smooth progress of the growth and metamorphosis of silkworm.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q966
本文編號(hào)：2125904
[Abstract]:Silkworm, as an important model of invertebrate, has a great change during its abnormal development, so it is an ideal material to study the physiological phenomena such as cell proliferation, differentiation, apoptosis and autophagy, the.GATA binding protein factor is a very important tune from low nematode to higher mammals. In this study, the silkworm GATA binding protein sequence information was obtained by the silkworm genome database, and the function of the BmGATA6-like gene was analyzed and studied. It will be beneficial to clarify the metamorphosis of the silkworm. The mechanism of molecular regulation to promote the comprehensive understanding of insect metamorphosis can not only provide clues for the biological control of insect pests, but also provide data reference for human degenerative diseases. The main results are as follows: the identification of the gene of the GATA binding protein family of the 1. silkworms was analyzed by NCBI and the whole genome database of the silkworm, and 5 families were obtained. The GATA binding protein family of the silkworm, BmGATA-A-like, BmGATA-C-like, BmBCF I, BmGATA6-like and BmGATA-X. are similar to the GATA binding proteins in other species. They all have a conservative GATA zinc finger domain, and BmGATA-A-like and BmGATA-X contain only one domain of GATA zinc finger. Two typical GATA zinc finger domain.5 age 3 days old silkworm microarray data show that the expression of GATA binding protein in silkworm is low, and there is a great difference in the expression among the members. The evolutionary analysis of the conserved domain shows that the members of the GATA binding protein family of the silkworm mainly gather with the GATA binding protein of insects, of which, BmGATA-A-l Ike is clustered with the Pannier of Drosophila melanogaster, BmBCF I and GATAa and Serpent of Drosophila melanogaster are clustered into a class. GATA binding proteins of BmGATA6-like and Lepidoptera are clustered into a class. BmGATA-X and the GATA4-like of the Italy bee are most conservative, while BmGATA-C-like is a single class; the multiple sequence alignment between species in the conservative domain shows GATA. The cloning and expression analysis of the Bm GATA6-like gene of the silkworm, silkworm,.2. is highly homologous, and the whole length cDNA sequence of the Bombyx mori bmgata6-like gene is cloned by the sequence information of the silkworm genome database and the sequence of ncbiblast. (in order to be succinct, the bmgata6 substitution will be called bmgata6-like). The whole length of the gene is 4011BP, It contains 10 exons and 9 introns; the ORF frame is long 1974bp, encodes 657 amino acids, the predicted protein molecular weight is 70.8kda, and the isoelectric point Pi is 6.13.bmgata6 protein containing two typical GATA zinc finger protein domains, which are located in the 464~516 amino acid sequence and 524~576 bit amino acid sequence respectively, and the online software predicts its non signal peptide. Real time fluorescence quantitative PCR was used to detect the relative expression level in the tissues of the silkworm embryos, 5 years and 3 days. The results showed that the bmgata6 gene was expressed in every period of the silkworm embryo development. The expression was high in the later stage of the embryo development, and the highest expression in the embryonic development and hatched silkworms in the sixth days of embryo development; and in the 3 days of 5 instar, bmgat The expression of A6 was significantly higher in the various tissues, but the expression in the midgut was significantly higher than that in other tissues. The expression of bmgata6 was expressed in the middle intestinal tissue and expressed in different periods of the lower blood. The results showed that the expression of bmgata6 in the midgut tissue was continuous, and was highly expressed in the period of dormancy and in the early stage of metamorphosis. The expression pattern of bmgata6 in the blood cells was very similar to the expression pattern in the midgut tissue. It was also highly expressed in the period of dormancy and metamorphosis. The expression of bmgata6 in the midgut tissues and blood cells by immunofluorescence showed that bmgata6 was highly expressed in the types of cells in the middle intestine of the silkworm, and only set in the middle intestine of the silkworm. It is expressed in the nucleus of the cell of the midgut tissue; in the l5d3 silkworm blood cells, the expression of bmgata6 in granulosa cells is detected, and only in the cytoplasm of the granulosa cells, and the bmgata6 signal can not be detected in the plasma cells. These results suggest that the bmgata6 gene plays an important role in the growth and metamorphosis of the silkworm,.3. The mechanism of bmgata6-like gene induced apoptosis of silkworm was constructed and the bmgata6 overexpression vector was constructed. The expression of bmgata6 gene was expressed in the BME of the Silkworm Embryo Cell Line BME by cell transfection. The expression of 24hbmgata6 protein began to express after transfection, and the protein content gradually increased with the time of transfection. The results of microscopic observation showed that the cells began to break up gradually from 48h after transfection. After transfection, the cell fragments of 72h and 96h were significantly increased. The cell apoptosis was detected by DAPI and TUNEL staining. The results of DAPI staining showed that the nuclear structure of the expressed bmgata6 was loose and the chromatin was irregular. After TUNEL, TUNEL was stained. The count results showed that the coloring rate of TUNEL was significantly higher than that of the control group. The expression of apoptosis related genes in the silkworm of BME cells transfected with bmgata6 gene was detected by fluorescence quantitative PCR. The results showed that the expression of related genes was significantly up-regulated, and the activity of caspase3/7 activity of apoptotic executive protein showed that its activity was also obviously up-regulated. It is suggested that bmgata6 can induce apoptosis of silkworm cells. Immunoprecipitation is carried out by using bmgata6 antibody, and the obtained products are identified by protein mass spectrometry. The number of proteins that may be interacted with bmgata6 is 11, and the PARP of cell apoptosis can be promoted according to the molecular weight and function. Protein; immunofluorescence co localization experiment verifies that PARP and BmGATA6 protein are expressed together in the nucleus, and further immunoprecipitation experiments prove that BmGATA6 can interact with PARP protein. These results suggest that the apoptosis induced by BmGATA6 may be associated with the interaction of PARP protein and the inhibition of Bm GATA6 induced apoptosis by.4.FOG protein. The phenomenon of BmGATA6 in the silkworm embryo cell line BmE and the silkworm ovarian cell line BmNs was compared. It was found that the BmE cell line that did not express the FOG factor BmUsh of the silkworm was compared to the BmNs cell line with the high expression of BmUsh, and the apoptosis induced by BmGATA6 overexpression was more rapid and more obvious. Meanwhile, BmGATA6 and Bm were overexpressed in the BmE cell line. Ush, the microscopically observed results showed that the apoptosis induced by BmGATA6 was obviously inhibited. After Tunel staining, the statistical results showed that the number of Tunel signals that expressed the BmGATA6 and BmUsh genes in the common overexpressed BmGATA6 gene decreased obviously, and the activity of Caspase3/7 decreased obviously. The results showed that the FOG factor BmUs of the silkworm was BmUs. H can inhibit the apoptosis induced by overexpression of BmGATA6. Through prokaryotic expression and protein purification, the reactive BmGATA6 recombinant protein is obtained. Far-western shows that BmGATA6 can interact with BmUsh protein and its action region is at the N end of BmGATA6; further immunoprecipitation test also verifies that the FOG factor BmUsh protein of silkworm is available. Combined with PARP protein in the case of co expression with BmGATA6, but it could not be associated with PARP when BmUsh was overexpressed alone. It indicated that BmUsh interacted indirectly with PARP by combining with BmGATA6. The fluorescence quantitative PCR detected the changes in the period table of BmUsh and BmGATA6 in the midgut and blood cells of the silkworm, and was compared to the BmGA in the midgut tissue. The expression of TA6 is high and the expression of BmUsh in the blood cells is high, but both in the middle and the blood cells, the expression patterns are very similar, both in the hibernation and metamorphosis period of high expression. Thus, it is concluded that the FOG factor BmUsh and BmGATA6 can regulate the normal growth of silkworm in the dormant and metamorphic periods. The conclusion and analysis of the functions of the Bm GATA6 gene of the long and abnormal development of.5. silkworm (silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, and silkworm, GATA6) are synthesized by the above conclusion and analysis. The unknown regulation mechanism co induced the apoptosis of silkworm tissue cells. When the degree of apoptosis reached the expected level, FOG factor BmUsh could inhibit the apoptosis induced by its direct action, so as to maintain the smooth progress of the growth and metamorphosis of silkworm.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q966
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