家蚕GATA结合蛋白家族鉴定及BmGATA6-like基因功能研究
发布时间：2018-07-16 09:03
【摘要】：家蚕作为重要的无脊椎动物模型,在其变态发育过程中组织形态会发生巨大变化,因此是研究细胞增殖、分化、凋亡及自噬等生理现象理想的材料。GATA结合蛋白因子是从低等的线虫到高等的哺乳动物发育过程中都需要的非常重要的调控因子,参与个体胚胎发育、组织器官发生、个体衰老等重要的生命活动。本研究以家蚕作为研究对象,利用家蚕基因组数据库获得家蚕GATA结合蛋白序列信息,并重点对BmGATA6-like基因的功能进行分析及研究,将有利于阐明家蚕变态发育的分子调控机理,促进对昆虫变态发育的全面认识,不仅可以为害虫的生物防治提供线索,甚至还能为人类的退行性疾病提供数据参考。主要研究结果如下:1.家蚕GATA结合蛋白家族基因的鉴定通过NCBI及家蚕全基因组数据库,分析获得5个家蚕GATA结合蛋白家族基因,分别为BmGATA-A-like,BmGATA-C-like,BmBCF I,BmGATA6-like和BmGATA-X。同其它物种中的GATA结合蛋白类似,它们都具有保守的GATA锌指结构域,其中BmGATA-A-like和BmGATA-X只含有一个GATA锌指结构域,BmGATA-C-like,BmBCF I和BmGATA6-like都含有两个典型的GATA锌指结构域。5龄3天家蚕各组织芯片数据显示家蚕GATA结合蛋白的表达量较低,且各成员间的表达情况存在较大差异。保守结构域的进化分析显示家蚕GATA结合蛋白家族成员主要与昆虫的GATA结合蛋白聚为一类,其中BmGATA-A-like与果蝇的Pannier聚为一类,BmBCF I与赤拟谷盗的GATAa及果蝇的Serpent聚为一类,BmGATA6-like与鳞翅目昆虫的GATA结合蛋白聚为一类,BmGATA-X与意大利蜂的GATA4-like最为保守,而BmGATA-C-like则单独聚为一类;保守结构域的物种间多序列比对显示GATA锌指结构域序列同源性非常高。2.家蚕Bm GATA6-like基因的克隆与表达分析通过家蚕基因组数据库预测序列信息及ncbiblast序列,克隆得到家蚕bmgata6-like基因全长cdna序列(为了简洁,后文中将以bmgata6代替称呼bmgata6-like)。该基因全长为4011bp,含有10个外显子和9个内含子;其orf框长1974bp,编码657个氨基酸,预测的蛋白分子量为70.8kda,等电点pi为6.13。bmgata6蛋白含有两个典型的gata锌指蛋白结构域,分别位于第464~516位氨基酸基序和524~576位氨基酸基序;在线软件预测其不含信号肽。利用实时荧光定量pcr技术检测其在家蚕胚胎、5龄3天各组织中的相对表达水平,结果显示bmgata6基因在家蚕胚胎发育各个时期均有表达,在胚胎发育的后期持续高表达,在胚胎发育第6天及孵化出的蚁蚕中表达量最高;5龄3天各组织中,bmgata6在各个组织中均有不同程度的表达,但是在中肠中的表达量显著高于其它组织。检测bmgata6在表达较高的中肠组织和表达较低的血液不同时期的表达情况,结果显示中肠组织中bmgata6持续表达,并且在眠期及变态发育前期显著高表达;血细胞中bmgata6的表达模式与中肠组织中的表达模式极其相似,同样是在眠期及变态发育时期显著高表达。免疫荧光检测bmgata6在中肠组织和血细胞中的表达定位情况,结果显示bmgata6在家蚕中肠所有类型的细胞中均有高量表达,且只集中表达于中肠组织细胞的细胞核中;l5d3家蚕血细胞中,检测到bmgata6在颗粒细胞中有微量的表达,且只在颗粒细胞的细胞质中表达,浆细胞中不能检测到bmgata6信号。这些结果暗示着bmgata6基因在家蚕生长及变态发育过程中扮演重要角色。3.家蚕bmgata6-like基因诱导细胞凋亡机制研究构建bmgata6过表达载体,通过细胞转染的方法在家蚕胚胎细胞系bme中过表达bmgata6基因。westernblotting结果显示转染后24hbmgata6蛋白就开始表达,且随着时间的延长其蛋白含量也逐渐增多。荧光显微镜观察结果显示从转染后48h开始,细胞逐渐出现细胞碎裂的现象,转染后72h及96h,其周围细胞碎片明显增多。分别用dapi及tunel染色进行细胞凋亡检测,dapi染色结果显示过表达bmgata6的细胞核结构松散,染色质呈不规则形状分布;tunel染色后计数结果显示tunel的着色率明显高于对照组。荧光定量pcr检测转染过表达bmgata6基因后bme细胞中家蚕凋亡相关基因的表达量变化,结果显示相关基因表达量被显著上调;凋亡执行蛋白caspase3/7活性检测显示其活性也被明显上调。以上实验说明bmgata6可以诱导家蚕细胞发生凋亡。利用bmgata6抗体进行免疫沉淀,并将得到的产物进行蛋白质质谱分析鉴定,筛选得到可能与bmgata6互作的蛋白数量为11个,根据分子量大小及功能等因素最后确定可以促进细胞发生凋亡的parp为目的蛋白;免疫荧光共定位实验验证PARP与BmGATA6蛋白共同在细胞核中表达;进一步免疫共沉淀实验验证BmGATA6可以与PARP蛋白相互作用。这些结果暗示BmGATA6诱发的细胞凋亡可能与PARP蛋白的相互作用相关。4.FOG蛋白抑制Bm GATA6诱发细胞凋亡通过比较在家蚕胚胎细胞系BmE和家蚕卵巢细胞系BmNs中分别过表达BmGATA6的现象,发现不表达家蚕FOG因子BmUsh的BmE细胞系相较于高量表达BmUsh的BmNs细胞系,BmGATA6过表达诱发的凋亡现象更为快速且更为明显。同时在BmE细胞系中过表达BmGATA6和BmUsh,经显微镜观察结果显示由BmGATA6诱发的细胞凋亡明显得到了抑制;Tunel染色后,统计结果显示与单独过表达BmGATA6基因相比,共同过表达BmGATA6和BmUsh基因的Tunel信号数量明显下降,且Caspase3/7的活性也明显降低。结果说明家蚕的FOG因子BmUsh可以抑制由过表达BmGATA6诱发的细胞凋亡。通过原核表达及蛋白纯化,获得具有活性的BmGATA6重组蛋白,利用Far-western证明BmGATA6可以与BmUsh蛋白相互作用,且其作用区域在BmGATA6的N端;进一步免疫共沉淀实验也验证了家蚕FOG因子BmUsh蛋白可以在与BmGATA6共同过表达的情况下与PARP蛋白结合,但是单独过表达BmUsh时不能与PARP结合。说明BmUsh通过与BmGATA6结合间接与PARP相互作用。荧光定量PCR检测家蚕中肠组织和血细胞中BmUsh和BmGATA6的时期表达变化,进行比较发现中肠组织中BmGATA6表达量较高,血细胞中BmUsh表达量较高,但无论是在中肠组织还是血细胞中,两者的表达模式极为相似,都在眠期及变态发育时期高量表达。由此结果推测家蚕FOG因子BmUsh与BmGATA6在眠期和变态时期通过相互作用,共同调控家蚕的正常生长及变态发育。5.家蚕Bm GATA6基因的功能综合上述结论与分析,我们推测家蚕BmGATA6在家蚕生长及变态发育过程中,被某些生长因子或者激素等诱导上调表达,在组织细胞发生程序性死亡过程中,通过与PARP蛋白的相互作用或者其它目前还未知的调控机制共同诱导家蚕组织细胞发生凋亡,当凋亡程度达到预期水平后,FOG因子BmUsh再通过与其直接作用抑制由其诱导的凋亡,共同维护家蚕生长及变态发育过程的顺利进行。
[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.
【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】：Q966
本文编号：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.
【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】：Q966
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