苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

斜纹夜蛾核多角体病毒几丁质酶基因上游4.0kb的序列分析

本文报道了斜 诳蛾核多角体病毒几丁质酶基因（chiA）上游约4.0kb范围内的序列,它包括了六个读码框（ORF1－6）,其长度分别为156bp、279bp、540bp、369bp、1281bp和228bp,可编码的氨基酸长度分别为51、98、179、122、426和75个,分子量分别为6.15kD、11.46kD、21.70kD、14.69kD 、47.59kD和9.09kD。在ORF1ORF2、ORF3起始密码前分别有一个、二个及一个杆状病毒早期启动子基序CAGT;在ORF4、ORF5起始密码前各有一个及二个杆状病毒晚期启动子基序TAAG。在ORF1、ORF4、ORF5终止密码下游有真核生物mRNA转录poly（A)加尾信号。ORF4为AcMNPV ORF53、BmNPV ORF42、OpMNPV ORF56、LdMNPV ORF54的同源基因。ORF1、ORF2、ORF6与已知的杆状病毒基因沿有同源性,可能为三个新的基因。     

斜纹夜蛾核多角体病毒几丁质酶基因上游4.0kb的序列分析

本文报道了斜纹夜蛾核多角体病毒几丁质酶基因(chiA)上游约4.0 kb范围内的序列,它包括了六个读码框(ORF1～6),其长度分别为156 bp、297 bp、540 bp、369 bp、1281 bp和228 bp,可编码的氨基酸长度分别为51、98、179、122、426和75个,分子量分别为6.15?kD、11.46 kD、21.70 kD、14.69 kD、47.59 kD和9.09 kD。在ORF1、ORF2、ORF3起始密码前分别有一个、二个及一个杆状病毒早期启动子基序CAGT;在ORF4、ORF5起始密码前各有一个及二个杆状病毒晚期启动子基序TAAG。在ORF1、ORF4、ORF5终止密码下游有真核生物mRNA转录poly(A)加尾信号。ORF4为AcMNPVORF53、BmNPVORF42、OpMNPVORF56、LdMNPVORF54的同源基因。ORF1、ORF2、ORF6与已知的杆状病毒基因没有同源性,可能为三个新的基因。     

球孢白僵菌tps2基因的克隆和生物信息学分析

运用SMART RACE RT-PCR技术与DNA步移技术,首次从球孢白僵菌中克隆出完整的海藻糖-6-磷酸磷酸酯酶TPS2的基因编码区序列及上游序列。该基因cDNA全长3219 bp,其中开放阅读框（ORF）2619 bp,编码872个氨基酸。成熟蛋白理论分子量为97.8 kD,理论等电点为6.32。编码框结构基因的全长为2821 bp,有两个长度分别为140 bp和62 bp的内含子。分析表明,上游序列中含有TATA-box、CAAT-box和GC-box,并且也存在GATA元件等启动子顺式调控元件。本文结果将为进一步研究海藻糖在虫生真菌中的生理合成以及抗逆调控机制奠定坚实的基础。     

解淀粉芽孢杆菌BamHI甲基转移酶基因克隆、功能鉴定与序列分析

从解淀粉芽孢杆菌Baillus amyloliquefaciens CICIM B2125中克隆了BamHI甲基转移酶基因(bamHIM),并在大肠杆菌JM109中得到了成功表达.该基因含有1 271 bp的开放阅读框(ORF),编码423个氨基酸,成熟蛋白分子量为49 kD.该基因在自身启动子引导下,表达了具有活性的BamHI甲基转移酶(M.BamHI).该酶可以将BamHI位点的碱基甲基化.氨基酸序列分析表明该酶存在有NADB_Rossmann结构域.     

传染性支气管炎病毒核衣壳蛋白基因克隆及在E.coli中的表达

核衣壳蛋白基因 (N基因 )是传染性支气管炎病毒的重要结构基因 .根据已报道的序列设计引物 ,利用RT PCR技术从病毒RNA中扩增和克隆到了N基因的cDNA ,并测定了核苷酸序列 .克隆的N基因片段ORF全长 12 30bp ,编码 4 0 9个氨基酸 .将该片段序列与其他IBV病毒株比较 ,核苷酸的同一性为 87 0 %～ 98 6 %,氨基酸的同一性为 91 0 %～ 98 1%.将该cDNA亚克隆到pBV2 2 0表达载体 ,转化大肠杆菌DH5α菌株 ,Western印迹检测 ,获得了分子量约 4 5kD表达蛋白     

棉铃虫单核衣壳核多角体病毒囊膜蛋白odv-e66基因及其邻近区域的序列分析

杆状病毒ODV-E66蛋白是包涵体来源病毒(occlusion-derived virus,ODV)囊膜的结构蛋白,ODV囊膜对ODV的稳定性和感染性具有重要作用.本文报道了HaSNPV odv-e66基因及其邻近区域共4 237bp的核苷酸序列及其分析结果.HaSNPV的odv-e66基因编码区全长2 019bp,推测编码一个由672个氨基酸残基组成的,分子量为74.5kD的列比较分显示HaSNPV ODV-E66蛋白具有多个保守区域,包括N末端的强疏水功能区、序列中部的一个可能的核定位信号RKIW,两个Leu-zipper以及5个跨膜区.odv-e66基因上游的两个ORF分别与AcMNPV的orf108和orf109具有同源性,下游的ORF与LsNPV的p13基因具有同源性.     

球孢白僵菌热休克蛋白基因Bbhsp70的cDNA及上游序列克隆与分析

运用SMART RACE RT-PCR技术与DNA步移技术,首次从球孢白僵菌中克隆出完整的热休克蛋白基因Bbhsp70编码区序列及上游序列。该基因cDNA全长2405bp,5′端非翻译区171bp,3′端非翻译区263bp,开放阅读框(ORF)1971bp,编码656个氨基酸。成熟蛋白理论分子量为71.3kDa,理论等电点为4.92。上游序列长度3559bp,其中有305bp序列与cDNA序列重叠。分析表明,上游序列中没有明显的TATA-盒和CAAT-盒,但含有CCAAT-bindingfactor、GC-box等重要的转录因子结合位点,以及热激应答元件(HSE)和GATA元件等启动子顺式调控元件。     

棉铃虫单核衣壳核多角体病毒囊膜蛋白odv—e66基因及其邻近区域的序列分析

杆状病毒ODV－E66蛋白是包涵体来源病毒（occlusion-derived virus,ODV）囊膜的结构蛋白,ODV囊膜对ODV的稳定性和感染性具有重要作用。本文报道了HaSNPV odv-e66基因及其邻近区域共4237bp的核苷酸序列及其分析结果。HaSNPV的odv-e66基因编码区全长2019bp,推测编码一个由672个氨基酸残基组成的,分子量为74.5kD的蛋白质。在起始密码子ATG上游具有杆状病毒晚期转录起始信号ATAAG。与其他杆状病毒ODV－E66的氨基酸序列比较分显示HaSNPV ODV-E66蛋白具有多个保守区域,包括N末端的强疏水功能区、序列中部的一个可能的核定位信号RKIW,两个Leu-xipper以及5个跨膜区。odv-e66基因上游的两个ORF分别与AcMNPV的orf108和orf109具有同源性,下游的ORF与LsNPV的p13基因具有同源性。     

烟曲霉几丁质酶基因的克隆与表达

Chi4 4是烟曲霉 (Aspergillusfumigatus)YJ-407产生的一种胞外几丁质酶。通过用真菌几丁质酶保守氨基酸序列与Chi44的N-端序列检索烟曲霉部分基因组序列数据库 ,获得一个编号为contig555的烟曲霉基因组序列 ,可能包含烟曲霉几丁质酶的基因。根据检索结果用RT-PCR方法从烟曲霉YJ-407中克隆到1.4kb的cDNA片段 ,该cDNA的ORF编码一个395个氨基酸的蛋白 ,分子量为43.6kD。对其推导氨基酸序列分析表明该蛋白与其它真菌来源的几丁质酶同源 ,而且活性中心与人巨噬细胞几丁质酶高度同源。该cDNA已在E .coli与PichiapastorisGS115中获得表达 ,分别获得 43kD和44kD的重组蛋白 ,两种重组蛋白均有几丁质酶活性。与野生酶相比 ,大肠杆菌表达的43kD重组酶及Pichia酵母表达的44kD重组酶稳定性下降 ,说明Chi44的糖基化修饰可稳定酶蛋白.     

Characterization of chitinase genes from an alkaliphilic actinomycete,Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Growth of hyperthermophilic archaeon Pyrococcus furiosus on chitin involves two family 18 chitinases

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA(BD)), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB(BD) [11 kDa]), followed by a catalytic domain (ChiB(cat) [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.     

Cloning,sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5 alpha F'. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2 degrees C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.     

Cloning, expression, and characterization of a chitinase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

The chitinolytic bacterium Aeromonas hydrophila strain SUWA-9, which was isolated from freshwater in Lake Suwa (Nagano Prefecture, Japan), produced several kinds of chitin-degrading enzymes. A gene coding for an endo-type chitinase (chiA) was isolated from SUWA-9. The chiA ORF encodes a polypeptide of 865 amino acid residues with a molecular mass of 91.6 kDa. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified into family 18 of glycosyl hydrolases. chiA was expressed in Escherichia coli and the recombinant chitinase (ChiA) was purified and examined. The enzyme hydrolyzed N-acetylchitooligomers from trimer to pentamer and produced monomer and dimer as a final product. It also reacted toward colloidal chitin and chitosan with a low degree of deacetylation. When cells of SUWA-9 were grown in the presence of colloidal chitin, a 60 kDa-truncated form of ChiA that had lost the C-terminal chitin-binding domain was secreted.     

Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum

This report provides the complete nucleotide sequences of the full-length cDNA encoding squalene synthase (SQS) and its genomic DNA sequence from a triterpene-producing fungus, Ganoderma lucidum. The cDNA of the squalene synthase (SQS) (GenBank Accession Number: DQ494674) was found to contain an open reading frame (ORF) of 1,404 bp encoding a 468-amino-acid polypeptide, whereas the SQS genomic DNA sequence (GenBank Accession Number: DQ494675) consisted of 1,984 bp and contained four exons and three introns. Only one gene copy was present in the G lucidum genome. The deduced amino acid sequence of Ganoderma lucidum squalene synthase (Gl-SQS) exhibited a high homology with other fungal squalene synthase genes and contained six conserved domains. A phylogenetic analysis revealed that G. lucidum SQS belonged to the fungi SQS group, and was more closely related to the SQS of U. maydis than to those of other fungi. A gene expression analysis showed that the expression level was relatively low in mycelia incubated for 12 days, increased after 14 to 20 days of incubation, and reached a relatively high level in the mushroom primordia. Functional complementation of Gl-SQS in a SQS-deficient strain of Saccharomyces cerevisiae confirmed that the cloned cDNA encoded a squalene synthase.     

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5αF′. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.