一个新的人类睾丸特异基因的cDNA克隆和表达分析

运用“数据库消减杂交”(DigitalDifferentialDisplay)方法筛选人类睾丸特异表达新基因 ,获得了有差异显示的代表新基因的克隆重叠群 ,挑选其中一个克隆重叠群HS .12 9794进行多组织RT PCR ,初步证实该重叠群在人睾丸中有高表达。然后从包含该重叠群的IMAGE克隆出发 ,采用生物信息学的方法快速克隆了一个人类新基因的全长cDNA序列 ,其全长 2 4 30bp ,开放阅读框为 6 76～ 12 4 8bp ,定位于 3p2 1 1,编码由 190个氨基酸组成 ,分子量为 2 0 4 17 8Da ,等电点为 5 2 3的一个偏酸性蛋白质 ,该蛋白与已知蛋白质无明显同源性。克隆实验验证该基因阅读框完全正确 ,半定量RT PCR进一步显示该基因在人不同发育时期的睾丸及精子细胞中有表达 ,推测其可能与精子生成相关 ,暂命名为SRG5 (TestisSpermatogenesisRelatedGene 5 ,SRG5 ) (GenBank登录号 :AY2 2 1117)。SRG5基因的成功克隆表明 ,“数据库消减杂交”与实验验证相结合的技术路线是一种快捷高效的发现更多人类功能新基因的新策略。     

小鼠生精相关基因SRG4的cDNA克隆及在小鼠各发育阶段的表达

从大鼠精子尾部基因Spag4出发,在dbEST数据库中同源搜寻与大鼠Spag4基因编码氨基酸同源性较高的表达序列标签(Expressed Sequence Tag,EST),找到两个在小鼠精母细胞中表达的ESTs,BG101130和BG100990。通过电子杂交得到1155bp的片段,包含了小鼠假想基因AK006225的全部序列,定位于2H1-H2,其开放阅读框为87～1133bp,并被RT-PCR所证实,将该基因命名为小鼠生精相关基因4(Sperrnatogenesis Related Gene4,SRG4,GenBank登录号为AY307077)。SRG4基因推定编码348个氨基酸,有一个coiled-coil区,可能是一个跨膜蛋白。该基因与人类同源基因TSARG4同源性为74％(277／374),与大鼠Spag4同源性为45％(103／224)。多组织RT-PCR和Northern blot结果显示,SRG4在睾丸中特异性表达。RT-PCR结果发现,小鼠出生后2周内,SRG4表达量极低;3周开始时SRG4大量表达,到4∽5周时表达量最高。该结果提示。SRG4基因可能在小鼠精子形成过程中发挥着重要的作用。     

一个人类精子发生相关新基因TSARG7的克隆和初步功能研究

精子发生是一个多基因参与的复杂的生理过程,虽然人们克隆了一些与精子发生相关的基因,但迄今为止,人们对精子发生的分子机制了解有限,因而克隆相关的新基因,并研究其在理论上和实践上的功能仍然十分重要。该文从人类睾丸cDNA文库出发,以小鼠精子发生相关基因mTSARG7基因为电子探针,得到1个与人类的精子发生相关的新基因TSARG7（GenBank登录号为AY513610）,该基因的cDNA全长为2463bp,含有12个外显子和11个内含子,定位在人类8号染色体8p11.21上。TSARG7编码的蛋白质含有456个氨基酸,分子量为56．295kDa,等电点为9．13,为胞浆中非分泌性蛋白,具有磷酸酰基转移酶（Hsc）的结构域,属于酰基转移酶家族的新成员,该家族成员具有脂质合成的功能。TSARG7和mTSARG7,TSARG7和Au041707分别具有97％的同源性。该基因在睾丸中特异表达,亚细胞定位在胞浆中表达。TSARG7 mRNA在13岁的睾丸中开始表达,并且随着精子的发生和性成熟而稳定增加,热应急实验显示该基因的表达与温度相关。综上所述,该文克隆了人的1个新基因,该基因与精子的发生和性成熟相关。     

利用巢式PCR技术和人类基因组草图搜索法快速获得人类睾丸生精细胞凋亡相关基因TSARG2

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 44 5 42 )入手 ,设计了基因特异性引物和载体特异性引物进行巢式PCR扩增 ,结合人类基因组草图搜索法 ,从睾丸cDNA文库中快速分离出人类睾丸凋亡相关基因TSARG2的 5′末端而获得全长cDNA ,GenBank登录号为AY0 40 2 0 4(保密期为 1年 ) ,同时应用生物信息学的方法克隆了该基因在小鼠中的同源基因 ,GenBank登录号为AF395 0 83。TSARG2基因的cDNA全长为 12 33bp ,包含 6个外显子 ,基因组跨越 115kb ,编码由 30 5个氨基酸组成的、分子量为 34 75 1的蛋白质 ,与已知蛋白质无明显同源性。查询最新的人类基因组工作草图 ,该基因定位在染色体 4q33～ 34 .1。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 4 2 )入手 ,利用网上生物信息学克隆了该基因全长 ,GenBank登录号为AF395 0 83。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA ,SRG2基因的cDNA全长为 10 5 8bp ,编码由 2 95个氨基酸组成、分子量为 335 79、等电点为 9.6 4的蛋白质 ,与人类同源基因TSARG2相似性为 78% ,与已知蛋白质无明显同源性。RT PCR结果表明该基因只在睾丸中有高表达。     

人类PKNOX1基因一种新剪接型全长cDNA的克隆与表达分析

为了寻找新的Down’s综合征相关基因,利用生物信息学分析与实验技术相结合的方法,从定位于Down’s综合征关键区内(21q22．3)的EST(GenBank登录号H77399)出发,从人类睾丸组织cDNA文库内克隆到含同源盒结构域转录因子PKNOX1的一种新剪接型全长cDNA,命名为PKNOX1B,GenBank登陆号AYl42115。PKNOX1B基因跨越58．4kb,全长cDNA约2．8kb,有11个外显子和10个内含子,编码405个氨基酸残基的酸性蛋白质,分子量为44．628kDa,等电点6．28。PKNOX1B与PKNOX1的前9个外显子及9个内含子完全相同,由于PKNOX1B在第10与11外显子之间发生了差异剪接,以致其在3’端cDNA序列被截短约2kb,所编码的蛋白质在C端较PKNOX1短30个氨基酸残基。但PKNOX1B保留了与PKNOX1完全相同的同源盒结构域,因而它可能与其他含同源盒结构域基因家族成员一样参与了发育的遗传调控。RT-PCR结果显示PKNOX1B除骨髓组织外在人体组织广泛表达。在睾丸组织中PKNOX1可见5kb,2．9kb,2kb 3种转录本,而在其他组织中仅发现2个较大的转录本,2kb的转录本在睾丸组织呈现特异性的表达,它有可能参与了精子的发生过程。     

睾丸生精细胞凋亡相关基因TSARG1与Mtsarg1的分子克隆及Mtsarg1基因的表达谱分析

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段（GenBank登录号：BE644538）出发,利用生物信息学和实验技术,克隆了小鼠睾丸生精细胞凋亡相关新基因Mtsarg1及相应的人类新基因TSARG1,Gen-Bank登录号分别为AF399971和AY032925。小鼠Mtsargl与人类TSARGl基因在氨基酸水平有55％的一致性和61％相似性,与其他已知蛋白质无明显同源性。小鼠10种组织的RT-PCR分析结果表明,Mtsargl基因在睾丸中高表达,在附睾中呈微弱表达,在其他组织不表达,提示Mtsargl和TSARGl基因在生精细胞凋亡或精子发生中具有潜在的重要作用。     

人锰超氧化物歧化酶cDNA的克隆、测序及表达

用逆转录-聚合酶链反应(RT-PCR)以人肝细胞总RNA为模板, 扩增了人锰超氧化物歧化酶(hMnSOD)的cDNA片段, 将此cDNA克隆到载体pGEM-T中.对重组质粒进行限制酶切分析和序列测定, 确定为含hMnSODcDNA的重组质粒将该hMnSODcDNA重组到表达载体pBV220内, 重组质粒在大肠杆菌DH5-α中表达hMnSOD, 表达产物占菌体总蛋白的14%, 具有持异性SOD酶活性.     

赤眼鳟Mx1基因的cDNA克隆及表达特性

为探究赤眼鳟(Squaliobarbus curriculus)是否存在Mx1 (Myxovirus resistance)基因及参与抗病毒免疫反应, 研究利用RACE技术获得了赤眼鳟Mx1基因(ScMx1)的cDNA全长序列, 并对其进行了生物信息学分析; 采用荧光定量PCR技术, 检测了ScMx1在赤眼鳟健康组织中的表达情况以及感染GCRV后ScMx1和ScIFN-Ⅰ的表达特征。结果表明, ScMx1的cDNA全长为3000 bp, 包含5′非编码区124 bp, 开放阅读框1893 bp, 3′非编码区983 bp, 共编码630个氨基酸。预测的ScMx1蛋白包含GTP酶结合区域、中央核心结构域和GTP酶效应结构域。ScMx1与青鱼Mx1的相似性最高(97%), 与ScMx的相似性仅为50%。ScMx1在所检测的10种组织中均有表达, 其中在脾脏中表达量最高。经GCRV感染开始至168h, ScMx1和ScIFN-Ⅰ在肝脏和体肾中的表达量持续上调; 在脾脏和头肾中于感染后72h达到峰值。相关性分析显示脾脏中ScMx1和ScIFN-Ⅰ的表达水平呈显著相关(r=0.94, P=0.018)。研究发现赤眼鳟存在Mx1基因, 且可能参与了抗GCRV免疫应答反应。     

飞蝗发育相关基因Omb的克隆、原核表达及时空表达分析

【目的】本研究克隆飞蝗Locusta migratoria发育相关的optomotor-blind(Omb)基因,并对其进行序列和表达分析,旨在更好地了解Omb基因在飞蝗翅发育中的作用及为进一步蝗灾的治理和防治提供新的理论依据。【方法】利用RT-RCR扩增飞蝗Omb基因cDNA序列,采用生物信息学软件分析该基因的核苷酸和氨基酸序列,利用MEGA 6. 0构建昆虫纲分子系统进化树;构建重组表达载体pET-30a/LmOmb,转化到大肠杆菌Escherichia coli BL21(DE3)中,SDS-PAGE及Western blot鉴定重组表达蛋白;基于qPCR技术分析Omb基因在飞蝗不同发育时期及成虫不同组织中的表达谱。【结果】克隆获得飞蝗Omb基因部分cDNA序列,命名为LmOmb(GenBank登录号:MG867658),其长792 bp,编码264个氨基酸,在第37-219位氨基酸之间存在一个T-box superfamily保守结构域。同源序列比对分析表明Lm Omb与褐飞虱Nilaparvata lugens Nl Omb氨基酸序列一致性为93%。在IPTG诱导下目标蛋白以6×His标签融合蛋白的形式在宿主菌中得到稳定表达。荧光定量PCR结果显示LmOmb基因在飞蝗不同发育时期均有表达,其中胚胎期的表达量最高,进入若虫期后表达量下降,且各龄若虫之间表达量相对平稳。Lm Omb基因在雌性和雄性成虫的胸部、足、腹部和翅中都有表达,且雌性和雄性之间表达量明显不同。【结论】LmOmb基因可能参与了飞蝗的胚胎发育。研究结果为进一步研究飞蝗LmOmb的功能提供了依据。     

人牛精浆蛋白相关新基因的cDNA克隆、定位和表达

为了研究牛精浆 (bovineseminalplasma ,BSP)蛋白及其相关蛋白在受精及受精卵发育中的重要作用 ,寻找BSP蛋白相关新基因 .采用cDNA末端快速扩增 (RACE)技术 ,克隆了一个BSP蛋白相关基因的cDNA序列 .应用辐射杂种细胞系 (RH)技术进行了基因染色体定位 .通过RT PCR检测了该基因在人体各组织中的表达情况 .并将该基因编码的蛋白进行了原核表达 .新基因的cDNA长度为 10 5 2bp ,其开放阅读框架 (ORF)编码了一个含 2 2 3个氨基酸残基的蛋白质 ,氨基酸序列中含有 4个纤连蛋白Ⅱ结构域 ,与BSP蛋白在结构上具有一定的相似性 ,称其为人BSP相关蛋白 (humanBSP relatedproteins ,HBRP) .该基因定位于染色体 19q13,在大肠杆菌中表达为 5 2kD的融合蛋白 .研究结果提示 ,应用RACE方法克隆了一种新的人类与BSP蛋白相关的基因 ,推测其编码蛋白是与BSP蛋白功能相关的结合蛋白 ,通过基因重组技术大量获得表达蛋白 ,对进一步研究新蛋白的生物学功能具有重要的意义 .     

Molecular Cloning and Preliminary Function Study of a Novel Human Gene, TSARG7, Related to Spermatogenesis

A novel human gene TSARG7 (GenBank accession No. AY513610) was identified from a human testis cDNA library by using the m TSARG7 gene (GenBank accession No. AY489184) as an electronic probe. The gene whose full cDNA length is 2 463 bp containing 12 exons and 11 introns is located in the human chromosome 8p11.21. The predicted protein encoded by this gene contains 456 amino acids with a theoretical molecular weight of 56 295 dalton and isoelectric point of 9.13. It is a new member of the acyltransferase family since its sequence possesses the highly conserved PlsC domain existing in all acyltransferase-like proteins. Two groups, the TSARG7 and mTSARG7, the TSARG7 and Au041707, share 97% identity in the 456 amino acids. Expression of the TSARG7 gene is restricted to the testis. Subcellular localization studies show that the EGFP-tagged TSARG7 protein was localized in the cytoplasm of GC-1 cells. The TSARG7 mRNA expression was initiated in the testis of a 13-year-old boy, and its level increased steadily along with spermatogenesis and sexual maturation of the human. The results of heat stress experiment demonstrate that TSARG7 expression has a relation with temperature. In conclusion, our study suggests that we have cloned a novel human gene and this gene may play an important role in human spermatogenesis and sexual maturation.     

Cloning,Characterization and Primary Function Study of a Novel Gene,Cymg1,Related to Family 2 Cystatins

Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags (ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation. Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags(ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation.