西伯利亚蓼硫堇基因的克隆和序列分析

根据西伯利亚蓼抑制消减文库(SSH)中获得的硫堇(THI)基因的部分序列,应用RACE技术克隆了具有PolyA的全长cDNA序列。基因全长789bp,5'非翻译区90bp,3'非翻译区276bp,开放阅读框编码140个氨基酸。序列分析表明,该编码蛋白与大多数植物THI蛋白前体高度相似,N端具24个氨基酸的信号肽,中间46个氨基酸为成熟THI部分,C端的70个氨基酸为酸性多肽部分。西伯利亚蓼THI蛋白与丹参等双子叶植物THI蛋白有较高的同源性,具保守的植物THI标签序列C-C-X(5)-R-X(2)-[FY]-X(2)-C。此成熟THI蛋白带正电荷,偏碱性,推定可能具有抗病原微生物活性,为一种新的植物THI蛋白,GenBank登录号为DQ981482。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

丹参硫氧还蛋白基因SmTrxh的克隆和生物信息学分析

对丹参EST数据库进行BLAST同源性比对发现,登录号为CV165156的EST序列与硫氧还蛋白基因（Trx）有很高的同源性。进一步用PCR方法从丹参基因组水平上克隆到长1806bp的DNA序列（登录号为FJ217699）,与cDNA序列比对发现,该基因（SmTrxh）含有2个内含子。生物信息学分析表明,SmTrxh所编码蛋白的分子质量为13．4kDa,理论等电点为5．53,无信号肽,属于定位于细胞质中的稳定类蛋白。该蛋白与其他7种植物中的Trx高度同源,同源性介于68％-74％之间。实时定量PCR检测的结果显示,SmTrxh在丹参中为组成型表达基因,在根、茎和叶中都有表达,主要在根部表达,茎中的表达量最低。     

丹参中病程相关蛋白基因SmSTH-2的生物信息学分析

对丹参cDNA文库的表达序列标签(EST)序列进行BLAST分析显示,其中一条序列与病程相关蛋白基因STH-2有较高的同源性。该序列全长691bp,包含1个长483bp的开放阅读框(ORF),编码160个氨基酸,命名为SmSTH-2。生物信息学分析显示:SmSTH-2所编码蛋白的分子质量为17990Da,等电点为5.15,富含谷氨酸、赖氨酸、甘氨酸、丝氨酸,无信号肽,属于稳定类蛋白。与NCBI注册的其他6种植物来源的病程相关蛋白基因编码的氨基酸序列的同源性在42%～46%之间。实时荧光定量PCR的方法检测丹参不同组织部位中SmSTH-2表达和病原菌对该基因诱导表达的影响的结果表明:SmSTH-2在植物的根、茎、叶中均有不同程度的表达,其表达丰度为根>叶>茎;丹参叶片接种黄瓜细菌性角斑病病原菌后,4d内可诱导该基因的表达量持续增加。用PCR方法从基因组水平克隆到SmSTH-2的DNA序列,测序表明SmSTH-2的编码序列在DNA水平上含有一个71bp的内含子,DNA序列注册号为EF621486.     

丹参非特异性脂质转移蛋白基因（SmLTP1）的克隆及其表达分析

对丹参EST序列进行Blast分析,获得一个新的非特异性脂质转移蛋白基因,命名为SmLTP1（GenBank注册号为EF187461）。该基因cDNA全长593bp,包含一个长为357bp的开放读码框,编码118个氨基酸。生物信息学结构分析表明,该蛋白具有植物nsLTP的典型结构,即4对二硫键,4个a-螺旋,1个可结合和容纳脂肪酸分子的类似口袋状的疏水结构。实时荧光定量PCR分析结果表明,SmLTP1基因在丹参不同组织器官中差异表达,其表达受病原菌和茉莉酸甲酯的诱导,显示SmLTP1基因在植物防御反应中发挥作用。     

二色补血草硫氧还蛋白(Trx)的克隆和表达分析

从二色补血草cDNA文库中分离出1个硫氧还蛋白基因全长cDNA序列。基因全长1138bp,其中,5’非翻译（UTR）区128bp,3＇非翻译区212bp,开放阅读框（ORF）全长798bp,编码265个氨基酸,编码蛋白的分子量为28．58kDa,理论等电点（pI）为9．68。BlastP分析表明二色补血草Trx与拟南芥Trx序列同源性为52％,与葡萄7h序列同源性为76％,从11个物种的氨基酸多序列比对可以看出Trx氨基酸序列保守性较高。实时定量RT-PCR方法检测低温、NaCl和PEG胁迫不同时间后的基因在二色补血草中表达模式的结果表明,NaCl能诱导Trx基因在二色补血草叶中表达,胁迫24h后达到高峰,而聚乙二醇和低温处理则抑制Trx在二色补血草根和叶的表达。     

青杆PwUSP1基因的克隆及表达模式分析

广泛逆境胁迫蛋白（universalstressprotein,USP）在非生物胁迫响应中起重要作用,但在植物中其功能还大部分未知。本研究通过BLAST分析青杆EST文库,得到职zP基因的EST序列,通过RACEPCR方法获取USP基因的末端序列,经过与EST序列拼接得到USP基因的cDNA全长序列,命名为PwUSP1。分析发现PwUSP1全长cDNA为1167bp,编码区为519bp,编码172个氨基酸残基。生物信息学分析显示,PwUSP1编码的蛋白相对分子质量为19．07kDa,理论等电点为6．38,为非跨膜的亲水性蛋白。PwUSP1具有USP家族典型的UspA结构域和ATP结合位点G-（2x）-G-（9x）-G（S／T）。半定量RT-PCR与RT-qPCR分析表明,PwUSP1在青杆的根、茎、针叶、花粉、种子中均有表达,在根和花粉中表达量较高。同时,PwUSP1受干旱和盐胁迫的诱导表达上调,均在处理6h后表达量较高,推测该基因可能在青杆逆境胁迫响应中发挥作用。     

西伯利亚蓼PsGRX基因的克隆及其在NaHCO_3胁迫下的表达

采用cDNA末端快速扩增（RACE）技术从西伯利亚蓼叶cDNA文库中克隆到谷氧还蛋白基因（PsGRX）的完整编码区cDNA序列（GenBank注册号为GU139794）,长度为465bp,编码106个氨基酸。根据与其他植物谷氧还蛋白的氨基酸序列的比对以及系统进化分析的结果,初步确定此基因为谷氧还蛋白基因家族成员。实时定量PCR的结果显示,PsGRX在西伯利亚蓼的叶、茎、地下茎中均有表达,叶中表达量最高,地下茎和茎中较低。在NaHCO3胁迫的过程中,此基因在叶、茎和地下茎中的表达模式也有较明显的差异。     

香蕉水通道蛋白SIP2-1基因的克隆和表达分析

从香蕉中克隆了一个水通道蛋白(AQP)基因MaSIP2-1。序列分析表明,该基因存在一个完整的开放阅读框(ORF)717 bp,编码239个氨基酸。多序列比对和进化树分析表明,MaSIP2-1所编码的蛋白与其他植物中AQP编码的蛋白具有较高的一致性。其中与马来西亚野生香蕉、油棕、麻风树、野茶树的AQP编码的氨基酸序列的同源性较高,分别为98%、74%、65%和63%。器官特异性分析表明,Ma SIP2-1在香蕉的根、茎、叶片、花和果实中均有所表达,其中在茎中表达量较高。通过对其在干旱、高盐、低温、涝害胁迫下的表达结果分析显示,该基因响应干旱、高盐、涝害3种胁迫。     

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.     

弗氏链霉菌丝氨酸蛋白酶基因的克隆及表达

从一株具有极强的降解羽毛能力的弗氏链霉菌菌株（Streptomyces fradiae var.k11）中纯化得到了一种丝氨酸蛋白酶SFP2。经蛋白测序,得到部分氨基酸序列,设计简并引物,PCR扩增得到部分基因序列,通过构建基因文库,获得了包括信号肽序列在内的完整的基因sfp2(EMBL收录号AJ784940),开放阅读框全长924bp,包括114bp的信号肽编码序列和810bp的酶原编码序列, 其中成熟蛋白编码基因长576bp,编码191个氨基酸,理论分子量为19.112kD。酶原编码基因和成熟蛋白编码基因均在大肠杆菌和枯草芽孢杆菌中得到了表达,酶原编码基因表达产物具有正常的生物学活性,证明了克隆基因的生物学功能。     

Cloning and sequence analysis of a snake, Atractaspis engaddensis gene encoding sarafotoxin S6c.

A 469 base pair genomic DNA, which encodes the mature region of a snake cardiotoxic peptide, sarafotoxin S6c, was isolated from the liver of the burrowing asp, Atractaspis engaddensis. The nucleotide sequence encoding the mature peptide region showed a high sequence homology with those of mammalian vasoconstrictor peptides, endothelin family as expected from the high homology of their amino acid sequences. In contrast, both of the upper and lower flanking sequences of sarafotoxin gene and the deduced amino acid sequence of the sarafotoxin precursor were quite different from those of endothelin family. These results suggest that the ancestral gene and biosynthetic pathway of sarafotoxins are different from those of endothelin.     

Signal peptide of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.     

苏云金杆菌毒素调控基因plcR的特性与表达

根据蜡状芽胞杆菌plcR基因和papR基因序列设计特异引物,对6个Bt菌株（WB1、WB7、WB9、HD98、8010、8311）及5个Bc菌株（6A1、6A2、6A3、6A4、6S1）进行了PCR检测.结果显示,3个Bt菌株及4个Bc菌株含有plcR-papR基因.克隆了Bt8010、Bc6A2和6A3的plcR、papR基因,核苷酸序列分析表明,三个菌株的plcR、papR基因与NCBI数据库中的Bt、Bc及Ba相应序列都有很高的相似性.Bt8010的plcR基因编码框由846个核苷酸组成,可编码282个氨基酸;papR基因的编码框由144个核苷酸组成,可编码48个氨基酸.推导的氨基酸序列分析表明,Bt8010 的PapR有21个氨基酸的信号肽序列,PlcR没有信号肽序列.与Bc6A2、6A3和Bc 569相比,Bt8010 的PlcR和PapR在氨基酸序列上与Bc 相应序列存在相对较大的差异.将plcR-papR基因连接到表达载体pHT304中,并转化至大肠杆菌JM109中成功进行了表达,为研究Bt plcR基因的功能奠定了基础.     

基于氨基酸特征序列对人类Rh血型系统的蛋白质结构分析

利用代数学中同态思想和物理中的“粗粒化”思想,以及HP模型,根据a,t,c,g的化学结构分类,提出了DNA序列的特征序列概念（σ-,τ-,σ∩τ-）并推广到蛋白质序列中,从而给出一种数值刻划,将蛋白质序列简化成一个（0,1）序列,基于上述给出特征序列的方法,根据氨基酸分子量与简并度的关系,提出了另外一种DNA序列的特征序列概念（-）并推广到蛋白质序列中,进而给出了另外一种数值刻划,将蛋白质序列简化成一个（0,1,2）序列,通过比较RHD基因和RHCE基因的特征序列的数值刻划图,得出RHD基因和RHCE基因均偏爱使用低分子量且高简并度的氨基酸。     

The nucleotide sequence of the yeast MEL1 gene.

The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein.     

烟夜蛾几丁质酶基因的克隆与表达

运用RT-PCR技术扩增编码烟夜蛾Helicoverpaassulta(Guen啨e)幼虫几丁质酶基因的cDNA片段,将其克隆至pMD18-T载体,获得该基因的成熟蛋白阅读框序列。将该基因重组到表达型质粒pGEX-4T-2中,并转化入原核细胞中表达,序列测定结果表明,烟夜蛾幼虫几丁质酶基因的成熟蛋白阅读框全长1338bp,编码445个氨基酸残基,预测分子量和等电点分别为50.1kDa和9.26;推导的氨基酸序列与其近缘种棉铃虫几丁质酶氨基酸序列的一致性达99%,与其他6种昆虫几丁质酶的氨基酸序列也高度一致(65%~76%),并具有几丁质酶的典型特征。将该基因克隆到原核表达载体pGEX-4T-2上并转化BL21,SDS-PAGE和Western印迹分析表明,经IPTG诱导,76kDa附近没有特异蛋白条带出现,表明烟夜蛾几丁质酶基因不能在原核表达载体pGEX-4T-2中表达。     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.