dhaT基因克隆及其与gldABC基因串联表达载体的构建

以肺炎克雷伯氏菌As1.1736的基因组DNA为模板,通过PCR扩增得到了目的基因(dhaT)并将该基因克隆至pMD19-T Simple载体。基因的序列分析表明,dhaT基因全长为1164bp,编码387个氨基酸。将含有自身核糖体结合位点的dhaT基因片段插入到pMD19-T Simple/gldABC质粒的gldABC基因下游,形成重组克隆质粒pMD19-T Simple/gldABC-dhaT,并进一步将gldABC与dhaT串联基因亚克隆到表达载体pET28a(+)上。     

苦荞麦主要过敏蛋白N端基因片段的克隆及序列分析

为了获得苦荞麦主要过敏蛋白全长基因并深入开展苦荞过敏蛋白的研究,本实验在先前获得的苦荞麦主要过敏蛋白C端(TBa)基因序列的基础上,设计不同的引物,以开花20d左右的苦荞麦果实为材料提取总RNA,并以此RNA为模板,通过RT-PCR和5’-RACE等方法,扩增,克隆获得苦荞麦主要过敏蛋白N端(命名为TBb)cDNA序列。序列分析结果表明,该序列由1005bp组成,开放阅读框为960bp,可编码一个由320个氨基酸残基组成的功能蛋白。同源性分析显示,此核苷酸序列与甜荞麦过敏性贮藏蛋白及豆球类蛋白的同源性达到90%。由此核苷酸序列推导出的氨基酸序列与甜荞麦13S贮藏蛋白有84%的同源性,与甜荞麦主要过敏蛋白有76%的同源性。苦荞麦主要过敏蛋白N端基因的获得为该蛋白的重组表达及其生物学活性等研究建立了前期基础。     

菠菜亚硝酸还原酶(NIR)基因的克隆与生物信息分析

利用RT-CR方法克隆菠菜NiR基因,并利用ExPASy分析预测了其编码蛋白。结果表明,该NiR基因(登陆号:X07568)全长为2062bp,1785bp开放阅读框,编码一个包含594个氨基酸残基的多肽链。所推导的氨基酸序列与拟南芥、小麦编码的氨基酸序列具有较高同源性,为85%。     

福寿螺(Ampullaria crossean)mfc 基因的分子克隆和序列分析

目的:从福寿螺胃组织细胞中扩增多功能纤维素酶基因,与pGEM T-easy载体连接,转化大肠杆菌删DH5 a.结果:该基因全长为1 225 bp,上游5'端非翻译区19 bp,3'端非编码区21 bp,开放阅读框(0pen reading frame,ORF)1 185bp,编码395个氨基酸,推测等电点pI为6.57,分子质量45.8k Da.推测的多功能纤维素酶具有糖苷水解酶保守的氨基酸序列,属于糖苷水解酶第10家族.同源性比对结果显示,该基因及推测的氨基酸序列与egx(GenBank Accession No.AAP 31 839)的同源性分别为99.3%和99.8%,在8个位点出现核苷酸差异.在1个位点出现氨基酸差异.通过软件分析并预测,该基因编码的蛋白共存在14个丝氨酸、苏氨酸和酪氨酸磷酸化位点.没有信号肽序列.结论:成功克隆出福寿螺多功能纤维素酶基因.     

迟钝爱德华氏菌FimA基因的克隆及序列分析

以迟钝爱德华氏菌Et、XA、EA分离株的DNA为模板,采用PCR技术,扩增菌毛亚基(FimA)基因的DNA片段,将其克隆到pMD18-T载体上。通过序列测定,分析结果表明:Et、XA、EAFimA基因由534个核苷酸组成,编码177个氨基酸。Et、XA、EA之间FimA基因核苷酸同源性为99%,与其它分离物核苷酸同源性为76%～77%,氨基酸同源性为81%～82%。     

甜荞苯丙氨酸解氨酶基因PAL的克隆及序列分析

利用RT-PCR技术,首次从甜荞(Fagopyrum esculentum)中克隆得到苯丙氨酸解氨酶基因(PAL)的cDNA ORF序列,命名为FePAL。该序列长2169bp,编码722个氨基酸,与其他植物PAL基因同源性较高,为80%～97%,其推导的氨基酸序列含有PAL酶活性中心特征序列GTITASGDLVPLSYIA和多个脱氨基、催化活性位点。系统发育树表明,甜荞PAL基因与苦荞PAL基因聚类关系最近。     

盐藻八氢番茄红素合成酶基因组DNA的克隆及序列分析

八氢番茄红素合成酶(PSY)为盐藻β-胡萝卜素代谢途径中的一个关键酶。通过染色体步移技术,采用抑制性PCR,从盐藻基因组DNA中克隆到完整的PSY基因,并对其序列进行了分析。该PSY基因序列全长3315bp,由5个外显子和4个内含子组成。其编码区的氨基酸序列与巴氏杜氏藻相应序列的一致性最高为74%,不一致区主要集中在5'端。同源性分析表明盐藻与巴氏杜氏藻的亲缘关系最近。     

狗头枣ACC I氧化酶基因的克隆及其序列分析

为利用RNAi技术开展调控乙烯合成延迟狗头枣果实成熟等研究,以狗头枣试管苗叶片的总DNA为模板,根据冬枣ACC I氧化酶基因的序列设计引物,通过PCR方法获得一长1 294 bp片段,序列分析结果表明,该片段具有4个外显子和3个内含,依次在106~201、430~541 bp与877~1 002 bp碱基处。此序列与冬枣ACC I氧化酶基因(EU216549.1)的核苷酸和氨基酸序列同源性均为99.69%,表明成功地克隆了狗头枣ACC I氧化酶基因。     

南方红豆杉GGPP合酶基因的克隆与生物信息学分析

根据GeneBank中公布的GGPPS基因保守区设计特异引物,克隆南方红豆杉GGPPS基因的DNA和cDNA序列,并通过生物信息学方法对其核苷酸序列和蛋白质结构进行分析预测。结果表明,GGPPS基因DNA和cDNA序列都为1 182 bp,DNA序列中无内含子,cDNA序列为一个编码393个氨基酸残基的开放式阅读框;GGPPS的理论相对分子质量为42 590,等电点为5.68。核苷酸同源性分析显示,它与Taxus canadensis(AF081514)同源性为98.8%;推导出的氨基酸序列与Taxus canadensis(AAD16018)同源性达99.0%。利用Protparam、SignalP、ProtScale、SOPMA、Swiss-Modeling、Scan Prosite和MEGA4.0等生物信息学工具分别对其理化性质、信号肽、疏水性、亲水性、二级结构、三级结构和进化树进行分析,为其功能研究和生物合成紫杉醇研究提供分子基础。     

受PVY诱导的烟草NtERD1的基因分离与表达分析

通过抑制差减杂交和cDNA芯片法从受PVY诱导的烟草抑制差减杂交文库中筛选得到一段长为745 bp的基因EST片段,经Blast同源性比对分析,该序列与植物脱水诱导早期应答基因(ERD)在核苷酸和氨基酸水平上高度同源。ORfinder分析表明,该序列包含一个492 bp的开放读码框,编码163个氨基酸。氨基酸Blastp比对显示,该氨基酸序列与其他植物脱水诱导早期应答蛋白具有很高的同源性。所以筛选得到的这个新基因属于烟草脱水诱导早期应答基因,被命名为NtERD1(GenBank登录号为GU144573)。实时荧光定量PCR分析表明,NtERD1在PVY接种早期上调表达,因此NtERD1受PVY侵染诱导,可能对烟草抗病毒具有重要作用。     

gldABC基因与dhaT基因串联表达载体的构建

以肺炎克雷伯氏菌As1．1736的基因组DNA为模板，通过基因扩增仪（PCR）扩增得到了目的基因（g1dABC）并将该基因克隆到pMD19-T Simple载体。通过对该基因的序列分析，甘油脱水酶（g1dABC）结构基因的全长为2816bp。将限制性内切酶处理后的含有自身核糖体结合位点的dhaT基因插入到质粒pMD19-T Simple／g1dABC中g1dABC基因的下游，形成重组克隆质粒pMD19-T Simple／g1dABC-dhaT，并进一步将g1dABC与dhaT串联基因亚克隆到表达载体pET28a（＋）上。     

甘油脱水酶基因(gldABC)与1,3-丙二醇氧化还原酶基因(dhaT)的共表达

将dhaT基因片段以顺反子的形式插入到重组质粒pMD19TS2中gldABC基因的下游,形成重组克隆质粒pMD19TS3,进而将gldABC与dhaT基因以多顺反子的形式亚克隆至pET-28a(+)表达载体上。终浓度为1mmol/L IPTG诱导重组大肠杆菌E.coli/pET-28a(+)/gldABC-dhaT 2h,SDS-PAGE电泳分析结果表明分别在甘油脱水酶三个亚基分子量相对应的61ku、21ku、16ku和1,3-丙二醇氧化还原酶亚基分子量相对应的42 ku的位置上出现了特异性条带。上清液中酶活性分别为甘油脱水酶23.7U/mL,1,3-丙二醇氧化还原酶30.4 U/mL。     

Analysis of the THR4 region on chromosome III of the yeast Saccharomyces cerevisiae

The gene encoding threonine synthase (THR4) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr4 mutant. This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome III inserted in the vector lambdaMG3. The THR4 gene encodes a protein of 514 amino acids (M.W. 58 kDa), which has extensive homologies with E. coli threonine synthase (thrC) and B subtilis threonine synthase. The 5' flanking region of the gene contains three regulatory sequences [TGACT(C)] for the general amino acid control (GCN). About 130 bp downstream of the THR4 gene another large open reading frame (563 amino acids) is found in the opposite orientation. This may imply that this open reading frame, called CTR86, shares a terminator region with THR4. The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site suggests that the gene product may also be involved in amino acid biosynthesis.     

Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22

Pz-peptidase is an endopeptidase that cleaves the synthetic substrate Pz-peptide (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg), which was originally developed for the assay of collagenase. The Pz-peptidase gene of Bacillus licheniformis N22 was cloned and sequenced. The gene consists of 628 amino acids with a motif for zinc-dependent metalloprotease, and shares 42% amino acid identity with the oligoendopeptidase of Lactococcus lactis. This is the first report on the gene structure of a Pz-peptidase.     

中国对虾过敏原基因特征的生物信息学分析

为了研究过敏原基因的基本性质、探讨过敏的机理,对中国对虾过敏原基因的部分序列进行克隆、测序,并分析该基因的有效密码子,碱基组成、密码子的偏好性,以及过敏原蛋白的氨基酸组成等性质。结果表明：中国对虾过敏原的部分基因序列长为1034,共编码264 个氨基酸,密码子的偏好性强,不同海产动物过敏原基因的有效密码子及碱基含量有很大的相似性。从基因序列和氨基酸序列的角度看,中国对虾的过敏原蛋白与其他种类海产甲壳动物的过敏原蛋白具有很强的相似性,这可能是导致海产品过敏原蛋白活性交叉反应的重要原因之一。     

Cloning and sequence analysis of a gene (aly PG) encoding poly(alpha-L-guluronate)lyase from Corynebacterium sp. strain ALY-1

The aly PG gene, coding for a poly alpha-l-guluronate lyase (PG lyase) of Corynebacterium strain ALY-1, was cloned and sequenced. The gene consists of 768 bp encoding a signal peptide of 32 amino acids and a mature protein of 224 amino acids. Two disulfide bond cross-linkages were found to be formed between Cys-4 and Cys-51 and between Cys-200 and Cys-206 in the native PG lyase molecule. The deduced amino acid sequence of the Corynebacterium sp. aly PG gene exhibited 29% homology toward that of the Klebsiella pneumoniae, subsp. aerogenes aly A gene, with two conserved regions (the amino acid sequences from Y-102 to M-110 and from Y-221 to Q-229).     

P. nalgiovense carries a gene which is homologous to the paf gene of P. chrysogenum which codes for an antifungal peptide

Penicillium nalgiovense is related to P. chrysogenum. P. chrysogenum carries the paf gene (Penicillium antifungal peptide) with homology to the afp gene of Aspergillus giganteus. This gene codes for a peptide with antifungal activity. Based on the sequence of the published P. chrysogenum paf gene primers were generated. By the use of these primers a PCR product of the expected length could be isolated from strains of P. nalgiovense. This fragment was sequenced and compared to the sequence of the paf gene of P. chrysogenum. According to the results P. nalgiovense carries a gene (naf = P. nalgiovense antifungal peptide) which is highly homologous to the paf gene of P. chrysogenum. The gene also codes for a preproprotein with the same processing sites as the paf gene, suggesting that the mature product is also a 55 amino acid (aa) peptide. The naf gene has three amino acid exchanges compared to the paf gene, which however do not influence the amino acid sequence of the mature peptide. It also carries two introns at the same positions, however, the sequence differences between the introns are higher than between the coding regions. When P. nalgiovense were grown on plates containing other food-relevant fungi it showed weak antifungal activity.