简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因

目的 克隆孢子丝菌未知过氧化氢酶基因,命名为Sscat基因.方法 根据生物信息库中7种已知真菌过氧化氢酶氨基酸序列的高度保守区域设计简并引物,PCR扩增获得部分Sscat基因cDNA片段,随后应用RACE技术分别扩增其3’端和5’端未知序列.结果 Sscat基因cDNA序列全长1746 bp,其中包括5’端121 b...     

金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析

根据中性海藻糖酶NTL基因的同源序列设计引物,PCR扩增出杀蝗专一菌株———金龟子绿僵菌CQMa102NTL基因片段,利用5′_RACE和3′_RACE扩增出NTLcDNA的5′和3′端序列,经拼接得到CQMa102NTL基因cDNA全长。根据其全长cDNA序列,设计引物PCR扩增出CQMa102NTL的完整基因。为了解该基因的上游调控信息,采用PanhandlePolymeraseChainReactionAmplification方法扩增其上游序列。序列分析表明,CQMa102NTL全长DNA3484bp,cDNA全长2385bp,编码737个氨基酸的蛋白,推测蛋白分子量为83.1kD;含有3个内含子,包含一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙附着位点(DTDGNMQITIED);上游序列含有一个压力反应元件(CCCCT);与金龟子绿僵菌广谱性菌株ME1NTL的核苷酸序列和氨基酸序列分别具有93%和99%同源性,由此确定该序列为金龟子绿僵菌中性海藻糖酶基因序列。Southern杂交表明,NTL基因在CQMa102基因组中为单拷贝。Northern杂交表明,NTL基因转录出约2.5kb的mRNA单带,在液体培养条件下,对数生长前期表达水平最高,对数生长后期降到最低,进入稳定生长期后表达水平又有所提高。金龟子绿僵菌CQMa102中性海藻糖酶基因DNA全长和cDNA全长登录GenBank,登录号分别为:AY557613,AY557612。     

栓菌420漆酶同工酶B基因克隆及异源表达

根据铜结合区的保守氨基酸序列设计简并引物,扩增栓菌420(Trametessp.420)基因组,结合长距离反向PCR(LD-IPCR)技术,克隆得到新型漆酶同工酶B基因(lacB),包括结构基因(2255bp)及5′-和3′-非编码序列。lacB含12个内含子,其cDNA序列长1560bp,编码495aa成熟多肽和24aa信号肽。lacB与其它不同来源的真菌漆酶基因具有较高的同源性,而与植物、细菌、昆虫的漆酶基因同源性低于25%。将不含信号序列的lacBcDNA通过质粒pPIC9克隆到表达载体pPIC9K上,电击转化毕赤酵母GS115细胞,经BMM-ABTS平板筛选得到漆酶分泌阳性转化子。     

栓菌420漆酶C基因的克隆、高效表达及重组酶的染料脱色潜能

根据漆酶铜结合保守区氨基酸序列设计简并引物,从新型漆酶合成菌株栓菌420(Trametes sp.420)基因组DNA扩增得到一新的漆酶同工酶基因(lacC)片段,应用长距离反向PCR技术获得其两端侧翼序列。克隆得到的lacC序列长3640bp,包括长2263bp的开放读码框及5′和3′-非编码区。lacC cDNA序列长1560bp,编码一519aa的多肽。推导的LacC蛋白序列内部存在有10个潜在的N-糖基化位点和4个铜原子结合区。将不含自身信号序列的lacC cDNA以pPIC9载体为媒介克隆到表达载体pPIC9K上,转化毕赤酵母(Pichiapastoris)GS115细胞。重组菌在含有0.3mmol/LCuSO4和0.8%丙氨酸的BMM培养基中20℃培养9d,重组漆酶(rLacC)产量达到1.62×104U/L。用发酵粗酶液对终浓度50mg/L的染料进行脱色实验,结果表明,6U/L的rLacC对测试的三甲基类和偶氮类染料具有良好的脱色作用,小分子介体ABTS和HBT能够提高rLacC对染料的脱色效率和脱色速度。     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特 的关键作用,因而也称为微囊藻毒素去毒酶. 从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列. 序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920 bp,其中5′-UTR长74 bp,3′-UTR长174 bp,编码区长672 bp,编码223个氨基酸. 应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878 bp序列. 与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特的关键作用,因而也称为微囊藻毒素去毒酶.从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列.序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920bp,其中5′-UTR长74bp,3′-UTR长174bp,编码区长672bp,编码223个氨基酸.应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878bp序列.与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA

为克隆马铃薯晚疫病抗性相关基因,深入研究马铃薯晚疫病抗性机制,应用SMART LD—PCR技术,以晚疫病菌混合小种诱导48h的水平抗性马铃薯(Solanum tuberosum L．)(R—gene—free)叶片为材料,构建了一个富集晚疫病抗性相关基因的cDNA文库。为提高克隆全长cDNA的效率,将cDNA文库与RACE技术结合,依据本实验室得到的病原诱导表达片段测序结果,在其内部设计两个特异引物,与文库载体臂上的通用引物配对,以文库噬菌体DNA为模板,用高保真PCR分别扩增出cDNA5′端与3′端,从而简便、快捷地得到全长cDNA序列。采用此方法,在马铃薯中克隆了一个受晚疫病菌诱导表达的cDNA,该cDNA长904bp,5′端有29bp的非翻译区,3′端具有完整的polyA尾,包含一个678bp的完整开放阅读框架,编码226个氨基酸(GenBank登录号：AY 185207)。BLAST检索发现其氨基酸序列与烟草一个新的病程相关蛋白基因NtPRp27具有90％的同源性,在马铃薯中尚未发现与之同源的已知基因。Northern杂交结果表明,水杨酸(SA)、茉莉酸(JA)、茉莉酸甲酯(MeJA)、机械伤害和渗透胁迫都能诱导该基因表达。该基因可能是马铃薯一个新的病程相关蛋白基因。     

白皮黄瓜鲨烯合酶基因cDNA克隆及生物信息学分析

为认识葫芦科植物中葫芦素生物合成途径所需鲨烯合酶基因结构特征,克隆白皮黄瓜鲨烯合酶(squalene synthase,SS)基因c DNA并进行生物信息学分析。根据葫芦科植物绞股蓝、罗汉果、红花栝楼等的鲨烯合酶基因cDNA序列,设计白皮黄瓜鲨烯合酶引物,分别采用3'RACE和5'RACE技术扩增鲨烯合酶基因3'端和5'端。获得白皮黄瓜鲨烯合酶基因的两个cDNA克隆,命名为CsSS1和CsSS2,其cDNA序列全长分别为1 627 bp和1 534 bp,都编码417个氨基酸残基,分子质量为47.6 k D。成功克隆得到白皮黄瓜鲨烯合酶基因全长cDNA序列并对其进行序列分析,后续可用于葫芦素生物合成途径所需鲨烯合酶基因正选择位点功能分析。     

鸡含锰超氧化物歧化酶cDNA克隆及序列分析

 为弄清鸡含锰超氧化物歧化酶 (manganese containingsuperoxidedismutase ,MnSOD)的cDNA序列 ,以开展动物锰营养学的深入研究 ,根据已知鸡MnSOD的N端氨基酸序列设计简并引物 ,应用 3′RACE(rapidamplificationofcDNAends)技术 ,扩增克隆了鸡心肌MnSOD 990bp的 3′cDNA片段 .再根据 3′RACE片段测序结果设计引物进行 5′RACE ,结果获取了一个与 3′RACE片段相互重叠的鸡心肌MnSOD 52 1bp的 5′RACE片段 ,并对其进行了克隆测序 .最后根据 3′RACE片段和 5′RACE片段序列信息进行拼接 ,从而获取鸡MnSODcDNA的全序列信息 .研究结果表明 :鸡MnSODcDNA全长为 110 8个核苷酸 ,其中 5′非翻译区 2 5个核苷酸 ,编码区 675个核苷酸 ,3′非翻译区 4 0 8个核苷酸 ,编码一个长 2 2 4个氨基酸残基的蛋白质前体 .其中信号肽长 2 6个氨基酸残基 ,成熟肽长 198个氨基酸残基 ,分子量为 2 2kD .与人、大鼠、线虫、果蝇等真核生物MnSOD氨基酸序列的同源性分别为82 4 %、84 .7%、62 .4 %、59.3% .     

3种鲤科鱼β珠蛋白基因的比较研究

根据 β珠蛋白的N末端和C末端氨基酸序列的保守性设计 2 0bp长的简并引物 ,用RT PCR方法扩增并克隆了鲤、草鱼、鲫的 β珠蛋白基因cDNA。结果显示克隆 3种鱼的 β珠蛋白基因cDNA全长为 4 4 1bp。对它们所编码的氨基酸序列的比较可以看出虽然它们都属于鲤科但氨基酸的组成却有较大的差异。根据所克隆的cDNA分别设计特异引物用PCR方法克隆了 3种鱼的基因组DNA全长 ;从起始密码子到终止密码子的长度分别为鲤 6 6 7bp、草鱼6 2 9bp、鲫 6 6 7bp。 3种鱼中均含有 3个外显子和 2个内含子且内含子的插入位置相同。内含子的碱基序列差异很大。鲫与鲤内含子 1和 2的长度完全相同 ,而与草鱼的内含子长度则相差较大。     

Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli

A genomic clone encoding a thiohydroximate S -glucosyltransferase ( S -GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S -GT gene were present in B. napus . The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli , and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S -GT activity detected from the recombinant protein confirmed that this clone was indeed a S -glucosyltransferase.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

西伯利亚蓼非特异性脂质转移蛋白编码序列的克隆及其盐胁迫下的表达

根据西伯利亚蓼地下茎抑制消减文库(SSH)中获得的非特异性脂质转移蛋白(non-specific lipid transfer protein, nsLTP)EST序列,应用RACE技术克隆了具有Poly A的全长cDNA序列.该序列全长604 bp,其5′非翻译区65 bp,3′非翻译区227 bp,开放阅读框编码103个氨基酸残基;序列分析表明,该基因具有N端信号肽,具有nsLTP家族共有的典型保守区域,属nsLTP家族基因,命名为PsnsLTPs;荧光定量PCR分析表明,PsnsLTPs在西伯利亚蓼叶、茎、地下茎中均有表达.在3%NaHCO3诱导表达下,该基因在地下茎中表达明显受盐胁迫的诱导,推测该基因在抵御盐胁迫时具有重要作用.     

茶树肌动蛋白基因（CsActin1）全长cDNA克隆与生物信息学分析

以茶树（Camellia sinensis）萌动芽为材料,根据茶树萌动芽芽抑制消减杂交文库中分离得到的肌动蛋白（actin）基因的5′-片段设计引物,利用3′-RACE技术克隆了其cDNA全长序列,该基因cDNA全长1 470 bp,命名为CsActin1（GenBank登录号HQ235647）。序列分析表明,CsActin1开放阅读框长1 134 bp,编码377个氨基酸,5′非编码区100 bp,3′非编码区236 bp。推测的蛋白质分子量为41.70 kD,等电点约为5.31,具有肌动蛋白家族的特征信号序列（YVGDEAQs.KRG和WIAKaEYDE）和肌动蛋白相关蛋白的特征信号序列（LLTEApLNPkaNR）。CsActin1与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性在80%以上,氨基酸序列相似性在95%以上。与其它植物肌动蛋白的进化树分析结果表明,茶树肌动蛋白与杨树的两个肌动蛋白间的亲缘关系最为密切。并对推导的蛋白结构进行了分析。     

Isolation and characterization of a novel RING-H2 finger gene induced in response to cold and drought in the interfertile Citrus relative Poncirus trifoliata

Many genes in different organisms encode proteins with really interesting gene (RING) finger domain(s). The RING zinc finger domain is involved in a wide variety of functions in diverse organisms. A cDNA clone showing homology with RING zinc finger genes and nine-fold induction in response to cold was previously identified during a gene expression study in the interfertile Citrus relative Poncirus trifoliata (L.) Raf. In this study, the full-length cDNA of this clone was isolated from 2-day cold-acclimated P. trifoliata by a rapid amplification of cDNA ends method using gene-specific primers. The full-length cDNA was 956 bp containing a complete open reading frame of 474 bp encoding a polypeptide of 158 amino acids. The full-length cDNA showed a high level of homology with genes encoding putative RING zinc finger proteins in plants. The deduced amino acid sequence of this gene contained a signature sequence motif for a RING zinc finger close to the C terminus of the protein. The RING zinc finger domain was significantly similar to previously characterized RING zinc finger proteins from different organisms. Additionally, it had a histidine residue at the fifth co-ordination site, indicating that this gene encodes a RING-H2 finger protein. Northern blot hybridization showed that the expression of the RING finger gene was induced in response to cold in cold-hardy P. trifoliata but not to the same extent in cold-sensitive Citrus grandis L. Osb. (pummelo). However, the gene was induced by drought stress similarly in both the species. To our knowledge, this study presents the first isolation of the full-length sequence of a RING zinc finger gene induced in response to abiotic stress in plants and the initial characterization of this gene in Citrus .     

Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase.

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus (Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.     

耐辐射奇球菌超氧化物歧化酶基因的克隆与序列分析

By using a 453 bp length gene fragment of superoxide dismutase(SOD)as a probe,which was firstly amplified from Deinococcus radiodurans genomic DNA by PCR with degenerate oligonucleotide primers corresponding to the conservative regions of known SODs,a putative SOD gene was identified from the database of D.radiodurans whole genome.Its 636 bp length open reading frame and 5′ and 3′ flanking sequence was determined.The conventional E.coli ribosomal and RNA polymerase binding sites were found upstream from SOD encoding region and an inverted repeat sequence downstream of the termination codon.The deduced 211 amino acid sequence of the structural gene showed a high similarity to other manganese and iron containing SODs in normally conserve regions.