家蚕微孢子虫葡萄糖-6-磷酸脱氢酶基因(G6PDH)的克隆及表达特征分析
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摘要
葡萄糖-6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase,G6PDH)是戊糖磷酸途径的第一个关键酶,催化葡萄糖-6-磷酸形成6-磷酸葡萄糖酸-δ-内酯。家蚕微孢子虫有较为完整的戊糖磷酸途径,该途径除提供糖酵解的中间代谢物外还可形成还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)从而抵抗生物体内的氧化刺激。为了解G6PDH基因在微孢子虫生长发育过程中的作用,根据家蚕微孢子虫G6PDH基因序列设计特异引物克隆了该基因,并对其序列特征进行生物信息学分析,进一步采用荧光定量PCR分析G6PDH基因在家蚕感染不同时间点的中肠组织中的转录水平。结果表明,克隆的家蚕微孢子虫G6PDH基因编码区全长1 305 bp,编码434个氨基酸,预测的G6PDH蛋白无信号肽,分子质量为50. 8 kD,等电点为5. 22,没有跨膜结构域,存在磷酸化位点,不存在N-糖基化位点及O-糖基化位点;家蚕微孢子虫感染家蚕后,从2 h起G6PDH基因表达量开始升高,至12 h时达到最大值,在24～72 h虽然有逐渐降低的趋势,但趋势并不明显,96 h后开始显著降低,至144 h表达量达到最低,而后在168 h时表达量又有所回升。推测G6PDH基因参与家蚕微孢子虫发芽、增殖以及成熟等整个生命周期。
Glucose-6-phosphate dehydrogenase( G6 PDH) is the first key enzyme in the pentose phosphate pathway and catalyzes glucose-6-phosphate to form 6-phosphoglucono-δ-lactone. There is a relatively complete pentose phosphate pathway in Nosema bombycis. This pathway can also produce reduced nicotinamide adenine dinucleotide phosphate( NADPH) and provide glycolysis intermediates to resist oxidation stimulation in vivo. In order to understand the role of G6 PDH in the growth and development of Nosema bombycis,we cloned the G6 PDH gene of Nosema bombycis by using specific primers according to its sequence retrieved from GenBank. Then we performed sequence analysis by bioinformatics tools and expression profile analysis by real-time fluorescent quantitative PCR. The results showed that the cloned fulllength coding sequence of G6 PDH gene of Nosema bombycis is 1 304 bp and encodes 434 amino acids. The predicted G6 PDH protein has no signal peptide,the molecular weight is 50. 8 kD,and the isoelectric point is 5. 22. There is no transmembrane domain,no O-glycosylation site and N-glycosylation site but there are phosphorylation sites. The results of real-time fluorescent quantitative PCR showed that the G6 PDH gene starts to express from 2 h after silkworm infected with Nosema bombycis,and reaches the maximum at 12 h. Although there is a trend of decreasing gradually within 24-72 h,the trend is not obvious. After96 h,it starts to decrease significantly. The expression level reaches the minimum at 144 h,and then the expres-sion level of G6 PDH gene increases at 168 h. It is speculated that the G6 PDH gene may be involved in spore germination,proliferation and maturation of the whole life cycle.
Glucose-6-phosphate dehydrogenase( G6 PDH) is the first key enzyme in the pentose phosphate pathway and catalyzes glucose-6-phosphate to form 6-phosphoglucono-δ-lactone. There is a relatively complete pentose phosphate pathway in Nosema bombycis. This pathway can also produce reduced nicotinamide adenine dinucleotide phosphate( NADPH) and provide glycolysis intermediates to resist oxidation stimulation in vivo. In order to understand the role of G6 PDH in the growth and development of Nosema bombycis,we cloned the G6 PDH gene of Nosema bombycis by using specific primers according to its sequence retrieved from GenBank. Then we performed sequence analysis by bioinformatics tools and expression profile analysis by real-time fluorescent quantitative PCR. The results showed that the cloned fulllength coding sequence of G6 PDH gene of Nosema bombycis is 1 304 bp and encodes 434 amino acids. The predicted G6 PDH protein has no signal peptide,the molecular weight is 50. 8 kD,and the isoelectric point is 5. 22. There is no transmembrane domain,no O-glycosylation site and N-glycosylation site but there are phosphorylation sites. The results of real-time fluorescent quantitative PCR showed that the G6 PDH gene starts to express from 2 h after silkworm infected with Nosema bombycis,and reaches the maximum at 12 h. Although there is a trend of decreasing gradually within 24-72 h,the trend is not obvious. After96 h,it starts to decrease significantly. The expression level reaches the minimum at 144 h,and then the expres-sion level of G6 PDH gene increases at 168 h. It is speculated that the G6 PDH gene may be involved in spore germination,proliferation and maturation of the whole life cycle.
引文
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[4] PAN G Q,XU J S,LI T,et al.Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation[J/OL].BMC Genomics,2013,14:186[2018-05-01]. https://www. ncbi. nlm. nih. gov/pubmed/23496955.DOI:10.1186/1471-2164-14-186
[5] WU Z,LI Y,PAN G,et al.A complete Sec61 complex in Nosema bombycis and its comparative genomics analyses[J].J Eukaryot Microbiol,2007,54(4):379-380
[6]邱璐瑶,平静,李文道,等.3种微孢子虫在我国西南部分地区腹泻患者中的流行情况调查[J].中国真菌学杂志,2017,12(5):257-261
[7] GUO R,CAO G,LU Y,et al.Exogenous gene can be integrated into Nosema bombycis genome by mediating with a non-transposon vector[J].Parasitol Res,2016,115(8):3093-3098
[8] SIRONMANI T A.Biochemical characterization of the microsporidian Nosema bombycis spore proteins[J]. World J Microbiol Biotechnol,1999,15(2):239-248
[9] SIRONMANI T A.Preliminary genomic characterization of microsporidian Nosema bombycis[J].World J Microbiol Biotechnol,2000,16(6):533-540
[10] WANG Y,DANG X,LUO B,et al.Characterization of a novel otubain-like protease with deubiquitination activity from Nosema bombycis(Microsporidia)[J]. Parasitol Res,2015,114(10):3759-3766
[11]张志芳,沈中元,何家禄.微孢子虫研究进展[J].蚕业科学,2000,26(1):38-44
[12] LI Z,WANG Y,WANG L,et al.Molecular and biochemical responses in the midgut of the silkworm,Bombyx mori,infected with Nosema bombycis[J/OL]. Parasit Vectors,2018,11(1):147[2018-05-01].https://doi.org/10.1186/s13071-018-2755-2
[13] KONG W F,CHEN J Y,HOU Z X,et al.Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits[J].J Plant Physiol,2007,164(7):934-944
[14] YU L J,LAN W Z,CHEN C,et al.Glutathione levels control glucose-6-phosphate dehydrogenase activity during elicitor-induced oxidative stress in cell suspension cultures of Taxus chinensis[J].Plant Sci,2004,167(2):329-335
[15] LANDI S,NURCATO R,DE LILLO A,et al.Glucose-6-phosphate dehydrogenase plays a central role in the response of tomato(Solanum lycopersicum)plants to short and long-term drought[J].Plant Physiol Biochem,2016,105:79-89
[16] BHASKAR,KUMARI N,GOYAL N. Cloning,characterization and sub-cellular localization of gamma subunit of T-complex protein-1(chaperonin)from Leishmania donovani[J].Biochem Biophys Res Commun,2012,429(1/2):70-74
[17]董静菲. G6PD在AngⅡ诱导的HUVEC氧化应激中的作用[D].石家庄:河北医科大学,2014
[18]王旭.拟南芥葡萄糖-6-磷酸脱氢酶(G6PD5)基因的克隆及功能研究[D].哈尔滨:黑龙江大学,2013
[19]伍先托,林果为.葡萄糖6-磷酸脱氢酶研究进展[J].国际输血及血液学杂志,1990,13(2):72-74
[20] CORDEIRO A T,THIEMANN O H. 16-bromoepiandrosterone,an activator of the mammalian immune system,inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture[J]. Bioorg Med Chem,2010,18(13):4762-4768
[21]杨文.TKTL1、G6PD单独或联合阻遏PPP代谢对宫颈癌细胞的影响[D].武汉:华中科技大学,2012
[22] YU L J,LAN W Z,CHEN C,et al.Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in Taxus chinensis cultures[J].Biol Plant,2005,49(2):265-268
[23] ALM H,TORNER H,L HRKE B,et al.Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity[J]. Theriogenology,2005,63(8):2194-2205
[24] BHOJWANI S,ALM H,TORNER H,et al. Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer[J].Theriogenology,2007,67(2):341-345
[25] CARDI M,CHIBANI K,CASTIGLIA D,et al.Overexpression,purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley(Hordeum vulgare cv.Nure)roots[J].Plant Physiol Biochem,2013,73(6):266-273
[26]刘阳,严奉坤,丁苗苗,等.发菜G6PDH基因(GND)克隆及其干旱胁迫下的差异表达分析[J].西北植物学报,2017,37(7):1263-1270
[27]王晓晖,刘晓,高博闻,等.腊梅葡萄糖-6-磷酸脱氢酶(G6PDH1)基因的克隆及表达分析[J].中国中药杂志,2015,40(21):4160-4164
[2]吴正理,李艳红,宋元达.微孢子虫蛋白质及其与宿主互作的研究进展[J].蚕业科学,2009,35(4):921-928
[3] DOLGIKH V V,SELIVERSTOVA E V,NAUMOV A M,et al.Heterologous expression of pyruvate dehydrogenase E1 subunits of the microsporidium Paranosema(Antonospora)locustae and immunolocalization of the mitochondrial protein in amitochondrial cells[J].FEMS Microbiol Lett,2009,293(2):285-291
[4] PAN G Q,XU J S,LI T,et al.Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation[J/OL].BMC Genomics,2013,14:186[2018-05-01]. https://www. ncbi. nlm. nih. gov/pubmed/23496955.DOI:10.1186/1471-2164-14-186
[5] WU Z,LI Y,PAN G,et al.A complete Sec61 complex in Nosema bombycis and its comparative genomics analyses[J].J Eukaryot Microbiol,2007,54(4):379-380
[6]邱璐瑶,平静,李文道,等.3种微孢子虫在我国西南部分地区腹泻患者中的流行情况调查[J].中国真菌学杂志,2017,12(5):257-261
[7] GUO R,CAO G,LU Y,et al.Exogenous gene can be integrated into Nosema bombycis genome by mediating with a non-transposon vector[J].Parasitol Res,2016,115(8):3093-3098
[8] SIRONMANI T A.Biochemical characterization of the microsporidian Nosema bombycis spore proteins[J]. World J Microbiol Biotechnol,1999,15(2):239-248
[9] SIRONMANI T A.Preliminary genomic characterization of microsporidian Nosema bombycis[J].World J Microbiol Biotechnol,2000,16(6):533-540
[10] WANG Y,DANG X,LUO B,et al.Characterization of a novel otubain-like protease with deubiquitination activity from Nosema bombycis(Microsporidia)[J]. Parasitol Res,2015,114(10):3759-3766
[11]张志芳,沈中元,何家禄.微孢子虫研究进展[J].蚕业科学,2000,26(1):38-44
[12] LI Z,WANG Y,WANG L,et al.Molecular and biochemical responses in the midgut of the silkworm,Bombyx mori,infected with Nosema bombycis[J/OL]. Parasit Vectors,2018,11(1):147[2018-05-01].https://doi.org/10.1186/s13071-018-2755-2
[13] KONG W F,CHEN J Y,HOU Z X,et al.Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits[J].J Plant Physiol,2007,164(7):934-944
[14] YU L J,LAN W Z,CHEN C,et al.Glutathione levels control glucose-6-phosphate dehydrogenase activity during elicitor-induced oxidative stress in cell suspension cultures of Taxus chinensis[J].Plant Sci,2004,167(2):329-335
[15] LANDI S,NURCATO R,DE LILLO A,et al.Glucose-6-phosphate dehydrogenase plays a central role in the response of tomato(Solanum lycopersicum)plants to short and long-term drought[J].Plant Physiol Biochem,2016,105:79-89
[16] BHASKAR,KUMARI N,GOYAL N. Cloning,characterization and sub-cellular localization of gamma subunit of T-complex protein-1(chaperonin)from Leishmania donovani[J].Biochem Biophys Res Commun,2012,429(1/2):70-74
[17]董静菲. G6PD在AngⅡ诱导的HUVEC氧化应激中的作用[D].石家庄:河北医科大学,2014
[18]王旭.拟南芥葡萄糖-6-磷酸脱氢酶(G6PD5)基因的克隆及功能研究[D].哈尔滨:黑龙江大学,2013
[19]伍先托,林果为.葡萄糖6-磷酸脱氢酶研究进展[J].国际输血及血液学杂志,1990,13(2):72-74
[20] CORDEIRO A T,THIEMANN O H. 16-bromoepiandrosterone,an activator of the mammalian immune system,inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture[J]. Bioorg Med Chem,2010,18(13):4762-4768
[21]杨文.TKTL1、G6PD单独或联合阻遏PPP代谢对宫颈癌细胞的影响[D].武汉:华中科技大学,2012
[22] YU L J,LAN W Z,CHEN C,et al.Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in Taxus chinensis cultures[J].Biol Plant,2005,49(2):265-268
[23] ALM H,TORNER H,L HRKE B,et al.Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity[J]. Theriogenology,2005,63(8):2194-2205
[24] BHOJWANI S,ALM H,TORNER H,et al. Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer[J].Theriogenology,2007,67(2):341-345
[25] CARDI M,CHIBANI K,CASTIGLIA D,et al.Overexpression,purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley(Hordeum vulgare cv.Nure)roots[J].Plant Physiol Biochem,2013,73(6):266-273
[26]刘阳,严奉坤,丁苗苗,等.发菜G6PDH基因(GND)克隆及其干旱胁迫下的差异表达分析[J].西北植物学报,2017,37(7):1263-1270
[27]王晓晖,刘晓,高博闻,等.腊梅葡萄糖-6-磷酸脱氢酶(G6PDH1)基因的克隆及表达分析[J].中国中药杂志,2015,40(21):4160-4164