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。     

枯草杆菌NX-2聚谷氨酸解聚酶的克隆表达及其降解性质研究

利用PCR技术从γ-聚谷氨酸产生菌Bacillus subtilis NX-2的基因组DNA上扩增出聚谷氨酸内切解聚酶基因(ywtD),克隆后测序,对该基因编码区进行了序列分析,比对结果表明本文扩增的ywtD基因编码与文献报道的序列相似性为99.0%,碱基的差异均表现为碱基取代,仅有一个碱基取代导致编码氨基酸的变化,即第349位密码子由GCC变为GTC。将ywtD基因克隆入表达载体pET15b中,该载体转化大肠杆菌Rosetta(DE3),经0.5mmol/L IPTG诱导,外源解聚酶蛋白获得高效表达。利用酶的初提液对聚谷氨酸进行降解,结果显示经72小时解聚酶作用后,γ-PGA分子量从700kDa降到20kDa,此后随作用时间的延长,聚谷氨酸分子量趋于定值。     

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。     

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.     

辣椒脉斑驳病毒文昌分离物基因组测序及分析

辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)近年来严重危害海南黄灯笼辣椒的产量,开展针对该病毒的研究以求达到对其有效防控迫在眉睫.本研究分7段克隆了ChiVMV文昌分离物(ChiVMV Wenchang isolate,ChiVMV-WC)的基因组.分段克隆的测序结果通过拼接,共获得ChiVMV-WC基因组9 717个核苷酸(nt)序列(GenBank登录号:GQ981316).在核苷酸序列的第164位~第9 270位存在一个大的开放阅读框(ORF),编码一个多聚蛋白(分子量为350.44 kD).近年来被证实的马铃薯Y病毒科的一个新的ORF (pretty interesting potyviridae ORF,PIPO)存在于ChiVMV-WC基因组核苷酸序列的第2 892位~第3 110位,编码一个由72个氨基酸聚合而成的多肽(分子量为8.26 kD).ChiVMV-WC与ChiVMV其它分离物的基因组核苷酸序列比较发现该病毒存在着较高的变异率.本研究通过与同属内病毒的同源性分析以及系统进化树分析,确定了ChiVMV-WC在生物进化史上的地位.     

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。     

板栗咖啡酸氧甲基转移酶基因CmCOMT的克隆及原核表达

该研究根据板栗(Castanea mollissima Bl.)cDNA文库分析得到EST序列,采用RT-PCR技术,克隆板栗咖啡酸氧甲基转移酶(caffeic acid O-methyltransferase,COMT)基因全长cDNA(CmCOMT),分析其编码蛋白的相关信息并进行原核表达研究,为板栗木质素合成关键酶基因CmCOMT的生物学功能研究与应用奠定基础。结果表明:(1)CmCOMT基因(GenBank登录号为KU365322)具有一个1 098bp开放阅读框(ORF),共编码365个氨基酸,推测蛋白分子质量为39.684 9kD,理论等电点为5.83,具有植物SAM依赖甲基转移酶的典型特征。(2)CmCOMT核苷酸序列及其编码氨基酸序列与垂枝桦(Betula pendula)和白桦(Betula platyphylla)的相应序列一致性均在90%以上;同源建模表明,CmCOMT的3D模型与苜蓿(Medicago sativa)MsCOMT的蛋白结构相似,推测其可能与MsCOMT具有相似的功能;系统发育树分析显示,CmCOMT与其他植物COMTs具有相同的进化祖先,与桦木科植物物种亲缘关系最近。(3)SDS-PAGE电泳分析表明,CmCOMT蛋白最佳诱导表达条件为0.3mmol/L IPTG在25℃下诱导6h,蛋白分子量约为44kD,其主要以可溶性蛋白的形式存在。     

地中海拟无枝菌酸菌U32中amrC/amkC基因的序列分析及在大肠杆菌中的表达

从力复霉素SV产生菌--地中海拟无枝菌酸菌(Amycolatopsismediterranei)U32的硝酸盐同化基因簇的上游克隆了一个2.6kb的EcoRI-XhoIDNA片段并测定其序列.序列分析表明,该DNA片段编码两个完整的开放阅读框架(ORF),ORF2的起始密码子GTG与ORF1的终止密码子TGA在TG处重叠.ORF1编码一个含224个氨基酸的多肽,它同放线菌中典型的应答调节蛋白包括AfsQ1和MtrA有很高的同源性;ORF2编码一个含472个氨基酸的蛋白,它同包括AfsQ2和MtrB在内的组氨酸激酶同源.ORF1和ORF2有可能构成典型的双组份信号传导系统,分别命名为amrC和amkC.在T7启动子的控制下,完整的amrC和去除子N端一个可能的跨膜区的amkC在大肠杆菌中分别得到了高效表达,表达蛋白的分子量分别为30kD和46kD,与推测蛋白的分子量一致.     

钝齿棒杆菌N-乙酰谷氨酸激酶基因的克隆、序列分析及表达

以钝齿棒杆菌(Corynebacterium crenatum)野生株AS 1.542及产精氨酸突变株971.1的基因组为模板,用PCR方法扩增出N-乙酰谷氨酸激酶基因(argB)片段。核酸序列分析结果表明,该片段全长1505bp,包含一个ORF,推测此ORF区编码一条317个氨基酸的多肽,分子量为33.6kDa。C.crenatum野生株AS 1.542与突变株971.1的argB基因序列比较,发现只在结构区有一个核苷酸的差别但没有引起氨基酸变化。野生株AS 1.542argB基因的编码区核苷酸序列与C.glutamicumATCC 13032、Corynebacterium efficiensYS-314和Escherichia colik12的同源性分别是99.89%、76.62%和37.94%,而氨基酸同源性分别是100%、78.55%和25.25%。在C.crenatum argB基因上游存在启动子区域。经IPTG诱导该基因在棒杆菌中得到有效表达,野生株AS 1.542为宿主的重组子酶活明显提高。突变株971.1为宿主的重组菌酶活提高一倍,精氨酸积累提高约25%。     

油菜抗冷相关基因BnCOR14的克隆及序列分析

以油菜沪油15幼叶为材料,采用RACE技术克隆获得1条新的抗冷相关基因(BnCOR14,GenBank登录号AY456378).该基因全长为564bp,含有1个387bp的开放阅读框(ORF),编码129个氨基酸的多肽,其理论分子量约为14kD.序列分析表明BnCOR14与拟南芥及荠菜等COR蛋白具有较高的相似性,且BnCOR14具有典型的LEA蛋白序列特征,表明BnCOR14可能在油菜抵抗冷胁迫的过程中具有重要的作用.     

NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.     

The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.

The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.     

¹³C-metabolic enrichment of glutamate in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert α-ketoglutarate and glutamate. In yeast, NADP-dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from α-ketoglutarate, while an NAD-dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in acetate/raffinose (YNAceRaf) to examine the role(s) of these enzymes during aerobic metabolism. In YNAceRaf the doubling time of wild type, gdh2Δ, and gdh3Δ cells was comparable at ～4 h. NADP-dependent GDH activity (Gdh1p+Gdh3p) in wild type, gdh2Δ, and gdh3Δ was decreased ～80% and NAD-dependent activity (Gdh2p) in wild type and gdh3Δ was increased ～20-fold in YNAceRaf as compared to glucose. Cells carrying the gdh1Δ allele did not divide in YNAceRaf, yet both the NADP-dependent (Gdh3p) and NAD-dependent (Gdh2p) GDH activity was ～3-fold higher than in glucose. Metabolism of [1,2-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-isotopomers at early time points was noticeably greater in gdh3Δ as compared to wild type and gdh2Δ cells. These in vivo data show that Gdh1p is the primary GDH enzyme and Gdh2p and Gdh3p play evident roles during aerobic glutamate metabolism.     

Glutamate dehydrogenase activity can be transmitted naturally to Lactococcus lactis strains to stimulate amino acid conversion to aroma compounds

Amino acid conversion to aroma compounds by Lactococcus lactis is limited by the low production of alpha-ketoglutarate that is necessary for the first step of conversion. Recently, glutamate dehydrogenase (GDH) activity that catalyzes the reversible glutamate deamination to alpha-ketoglutarate was detected in L. lactis strains isolated from a vegetal source, and the gene responsible for the activity in L. lactis NCDO1867 was identified and characterized. The gene is located on a 70-kb plasmid also encoding cadmium resistance. In this study, gdh gene inactivation and overexpression confirmed the direct impact of GDH activity of L. lactis on amino acid catabolism in a reaction medium at pH 5.5, the pH of cheese. By using cadmium resistance as a selectable marker, the plasmid carrying gdh was naturally transmitted to another L. lactis strain by a mating procedure. The transfer conferred to the host strain GDH activity and the ability to catabolize amino acids in the presence of glutamate in the reaction medium. However, the plasmid appeared unstable in a strain also containing the protease lactose plasmid pLP712, indicating an incompatibility between these two plasmids.     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.     

A new class of glutamate dehydrogenases (GDH). Biochemical and genetic characterization of the first member, the AMP-requiring NAD-specific GDH of Streptomyces clavuligerus

A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an alpha(6) oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mm phosphate buffer, pH 7.0, at 30 degrees C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K(m), 33 mm) and sigmoidal responses to changes in alpha-ketoglutarate (S(0.5) 1.3 mm; n(H) 1.50) and NADH (S(0.5) 20 microm; n(H) 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH(4)(+), by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K(m) for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.     

类产碱假单胞菌谷氨酸脱氢酶的提纯、鉴定及某些特性的初步研究

从类产碱假单胞菌纯化出电泳纯的谷氨酸脱氢酶,用聚丙烯酰胺梯度凝胶电泳和ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为２９０ ｋＤ,亚基分子量为４７ ｋＤ,提示该酶为六聚体．该酶对ＮＡＤＰ（Ｈ）和底物均具有高度专一性,对谷氨酸、α－酮戊二酸及ＮＡＤＰ＋ 的Ｋｍ 值分别为：２８ ｍ ｍ ｏｌ／Ｌ、１．２ｍ ｍ ｏｌ／Ｌ及０．０６３ ｍ ｍ ｏｌ／Ｌ．用Ｈｉｌｌ作图法求得酶对ＮＨ＋４ 和ＮＡＤＰＨ 的［Ｓ］０．５分别为２４ ｍ ｍ ｏｌ／Ｌ和０．０３７ ｍ ｍ ｏｌ／Ｌ．最适反应温度为５０℃,催化氨化反应和脱氨反应的最适ｐＨ 分别为８．０和８．８,在热稳定性方面不及嗜热细菌的谷氨酸脱氢酶稳定．提纯的谷氨酸脱氢酶在低温（４℃）条件下,可在Ｔｒｉｓ－ＨＣｌ缓冲液中贮存半年以上,活力无明显下降,冷冻则可导致纯酶液迅速失活．氮源对菌体谷氨酸脱氢酶水平有显著影响．