谷子叶绿体乙酰辅酶A羧化酶cDNA的克隆和禾本科除草剂靶位点的序列分析

乙酰辅酶A羧化酶是一个生物素羧化酶,它所催化的反应是脂肪酸生物合成中的第一个植物叶绿体中的乙酰辅酶A羧化酶是两类禾本科除草剂的靶蛋白.从抗除草剂拿捕净和感拿捕净的谷子(SetariaitalicaBeauv.)中克隆了两个乙酰辅酶A羧化酶的全长cDNA,分别命名为foxACC-R和foxACC-S,它们推导的蛋白质均编码2 321个氨基酸,然而在第1 780个氨基酸处,foxACC-R编码亮氨酸,而foxACC-S编码异亮氨酸.采用生物信息学方法,我们推断这个cDNA编码的是叶绿体中的乙酰辅酶A羧化酶,并预测了它的功能域和保守区.通过这两个cDNA编码的氨基酸序列与其他乙酰辅酶A羧化酶的序列比较得出结论,亮氨酸/异亮氨酸位点可能是APPs和CHDs两类除草剂作用的关键位点.Southern杂交分析的结果显示,该基因在谷子基因组中只有一个拷贝.     

地中海拟无枝菌酸菌U32中生物素羧基载体蛋白结构基因的克隆、表达及转录

乙酰辅酶A羧化酶(Acetyl CoA Carboxylase EC 6.4.1.2, ACC)催化依赖于ATP的乙酰辅酶A羧化形成丙二酸单酰辅酶A,该反应是脂肪酸生物合成途径中的第一步,也是受到调控的关键一步。根据结核分枝杆菌(M. tuberculosis)和天蓝色链霉菌(S. coelicolor)中ACC-α亚基的氨基酸保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好,设计简并引物以U32基因组DNA为模板扩增出一条约250bp的片段,并以此片段作探针成功地从U32基因组cosmid文库中克隆到相应的ACC-α亚基的编码基因accA。该基因对应的ORF长1797bp,编码一个598个氨基酸的蛋白,推算出的分子量是63,714Da;基因G+C mol%含量为70.1%,符合U32基因结构特征,距起始密码子GTG上游6个碱基处有链霉菌典型的RBS序列AGGAGG,并有生物素羧化酶特征的ATP结合区。利用pET28(b)系统构建表达载体,在E. coli BL21(DE3)中实现了accA的诱导表达,产物大部分以可溶形式存在,并通过Western Blot证明该蛋白上确有共价结合的生物素。Northern Blot分析了各种氮源对accA基因转录水平的不同影响。     

油菜叶绿体乙酰辅酶A羧化酶单交换表达载体的构建

根据已知序列设计引物,通过PCR扩增获得质体定位的乙酰辅酶A羧化酶的4个亚基的基因序列。先将该酶4个亚基的基因进行拼接,然后将这4个拼接好的片段,克隆到pMD18-T载体上,得到质粒pH BM714。再以质粒pHBM714 DNA为模板,用分别带有CpoI和Asc I酶切位点的引物进行PCR扩增,PCR产物在dTTP的保护下经T4 DNA聚合酶处理,与将质粒pHBM720DNA纯化后经CpoI和AscI双酶切后得到的大片段连接,连接产物转化大肠杆菌Xl_(10)-gold,得到正确的重组子命名为pHBM726。此质粒pH BM726,即为带有壮观霉素抗性基因(aadA)筛选标记的质体定位的乙酰辅酶A羧化酶基因油菜叶绿体单交换表达载体;在此载体中壮观霉素抗性基因(aadA)、乙酰辅酶A羧化酶的4个亚基的基因(ACC)和绿色荧光蛋白基因(gfp)共6个基因串联在一起,共用一个启动子序列,一起来进行表达;通过酶切检测、PCR验证和测序验证,均表明该表达载体构建成功。最后此载体在大肠杆菌中表达时,发现重组菌能够在含壮观霉素的培养基上生长,且在可见光下,能看到绿色荧光,表明壮观霉素抗性基因和绿色荧光蛋白基因均在大肠杆菌中成功表达;表达产物通过Western印迹验证表明组成乙酰辅酶A羧化酶的4个亚基的基因在大肠杆菌中成功表达。以上结果表明,该表达载体中串联排列的这6个基因均在大肠杆菌中成功表达。该研究结果可为质体定位的乙酰辅酶A羧化酶转叶绿体的研究奠定基础,为油菜油脂代谢研究提供参考。     

谷子叶绿体乙酰辅酶A羧化酶cDNA的克隆和禾本科除草剂靶位点的序列分析

乙酰辅酶A羧化酶是一个生物素羧化酶,它所催化的反应是脂肪酸生物合成中的第一个关键步骤。禾本科植物叶绿体中的乙酰辅酶A羧化酶是两类禾本科除草剂的靶蛋白。从抗除草剂拿捕净和感拿捕净的谷子(Setaria italicaBeauv.)中克隆了两个乙酰辅酶A羧化酶的全长cDNA,分别命名为foxACC-R和foxACC-S,它们推导的蛋白质均编码2 321个氨基酸,然而在第1 780个氨基酸处,foxACC-R编码亮氨酸,而foxACC-S编码异亮氨酸。采用生物信息学方法,我们推断这个cDNA编码的是叶绿体中的乙酰辅酶A羧化酶,并预测了它的功能域和保守区。通过这两个cDNA编码的氨基酸序列与其他乙酰辅酶A羧化酶的序列比较得出结论,亮氨酸/异亮氨酸位点可能是APPs和CHDs两类除草剂作用的关键位点。Southern 杂交分析的结果显示,该基因在谷子基因组中只有一个拷贝。     

金黄色葡萄球菌肠毒素A Asp227Ala基因的克隆及表达

目的：金黄色葡萄球菌肠毒素A Asp227ala基因的克隆及表达。方法：利用错配PCR方法,从含有金黄色葡萄球菌肠毒素A（Staphylococcal enterotoxn A,SEA)基因的质粒中扩增出约720bp的DNA片段,将其克隆到表达载体7ZTS中,并转化于JM109（DE3）。结果：重组质粒的测序结果表明,它含有702bp（不包括N端72bp的信号肽编码区）,其核苷酸序列与文献报道完全一致,推导的氨基酸序列显示227位的天冬氨酸已突变为丙氨酸。结论：该基因所表达的蛋白为可溶性蛋白,表达量占总蛋白51.5%。表达的蛋白与天然肠毒素A产生的抗体能发生凝集作用,具有与天然SEA类同的抗原活性。     

光皮桦中4-香豆酸辅酶A连接酶基因B14CL的克隆和表达分析

以从光皮桦茎叶组织提取的mRNA为模板,根据其他已克隆到的阔叶类树种中4-香豆酸辅酶A连接酶（4CL）基因的同源序列设计兼并引物,进行RT—PCR扩增,获得部分基因片段,然后结合5’,3’RACE方法从光皮桦中扩增出1个4CL基因的全长cDNA序列,命名为B14CL。该基因cDNA全长为1983bp（GenBank登录号FJ410448）,具有完整的开放阅读框架（69—1697bp）,编码蛋白为542个氨基酸,包含一个AMP结合功能域和一个含有12个氨基酸的功能基序。与其他植物中的4CL进行同源性比对的结果显示,B14CL蛋白与东北白桦的同源性最高,达到了98％。该基因在光皮桦的根和茎中表达量较高,而在花和叶中的表达量低。     

FASN基因的原核表达及生物信息学分析

脂肪酸合成酶(FASN)在生物体内起着重要的作用,主要参与恶性肿瘤的数量调控。本研究旨在构建pET28a-FASN原核表达载体,并表达重组His-FASN蛋白,对该基因进行结构与功能的生物信息学分析。设计FASN基因特异性引物,通过PCR扩增获得的目的基因与原核表达载体pET28a连接,经IPTG诱导表达His-FASN蛋白。获得基因片段大小为1 320 bp,编码440个氨基酸;成功构建至pET28a原核表达载体,通过优化表达,确定在温度为35℃、IPTG浓度0.5 mmol/L、诱导时间为6 h的条件下融合蛋白表达量较高,获得蛋白大小约为53 kD;生物信息学分析结果表明FASN基因编码的蛋白是一个不稳定且具有亲水性的蛋白,不存在信号肽及跨膜区,可成为蛋白激酶磷酸化位点有12个Ser、5个Thr、3个Tyr。此外,从蛋白相互作用网络中发现,相互作用的蛋白包括主要酰基辅酶A合成酶长链家族成员及乙酰辅酶A羧化酶家族成员,为开发抑制剂药物提供了理论依据。     

抗人CD3单链抗体四聚体基因的构建及表达

为构建和表达抗人CD3单链抗体 (scFv) 人p5 3四聚功能域融合基因 ,选用人IgG3上游铰链区作为抗人CD3scFv和人p5 3四聚功能域之间连接的linker .利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC18载体中构建pUC18 IgG3 p5 3克隆载体 .将抗人CD3scFv克隆入pUC18 IgG3 p5 3载体中 ,构建抗人CD3scFv 人p5 3四聚功能域融合基因 .经酶切鉴定及序列测定证实后 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后利用流式细胞仪进行亲和活性测定 .获得了抗人CD3scFv 人p5 3四聚功能域融合基因 ,基因全长 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p5 3四聚功能域基因cDNA序列一致 .表达产物经SDS PAGE和Western印迹实验证实为约 35kD的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合人外周血单个核细胞 (PBMC)细胞 ,亲和力高于scFv ,为进一步临床应用奠定基础     

光皮桦中4-香豆酸辅酶A连接酶基因Bl4CL的克隆和表达分析

以从光皮桦茎叶组织提取的mRNA为模板,根据其他已克隆到的阔叶类树种中4-香豆酸辅酶A连接酶(4CL)基因的同源序列设计兼并引物,进行RT-PCR扩增,获得部分基因片段,然后结合5’,3’RACE方法从光皮桦中扩增出1个4CL基因的全长cDNA序列,命名为Bl4CL。该基因cDNA全长为1983bp(GenBank登录号FJ410448),具有完整的开放阅读框架(69～1697bp),编码蛋白为542个氨基酸,包含一个AMP结合功能域和一个含有12个氨基酸的功能基序。与其他植物中的4CL进行同源性比对的结果显示,Bl4CL蛋白与东北白桦的同源性最高,达到了98%。该基因在光皮桦的根和茎中表达量较高,而在花和叶中的表达量低。     

细纹豆芫菁3-羟甲基戊二酰辅酶A-还原酶基因全长cDNA克隆及生物信息学分析

3-羟甲基戊二酰辅酶A-还原酶（3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR）是甲羟戊酸途径的关键酶。获得芫菁体内HMGR基因信息是确定甲羟戊酸途径与斑蝥素合成相关性的基础。本研究利用RACE技术从细纹豆芫菁Epicauta mannerheimi (Maklin)体内克隆获得HMGR基因全长cDNA序列, 命名为EmHMGR（GenBank登录号为JQ690539）。该基因全长3 118 bp, 其中5′端非翻译区178 bp, 3′端非翻译区414 bp, 开放阅读框2 526 bp, 编码842个氨基酸。推测的蛋白质分子量为92.8 kDa, 理论等电点为6.0, 预测分子式为C₄₁₃₅H₆₆₀₄N1₀₉₈O₁₂₁₆S₅₀, 不稳定系数为43.37, 总亲水性系数为0.091, 为疏水性不稳定蛋白。序列分析发现该基因编码的蛋白与已报道的其他昆虫HMGR的氨基酸序列一致性达50%以上, 而且包含HMGR_Class I保守功能域、 固醇敏感多肽区及HMGR蛋白的其他保守功能位点。系统进化分析发现该基因与叶甲科昆虫HMGR基因的关系最近。本研究首次从芫菁科昆虫体内克隆获得甲羟戊酸途径的关键酶EmHMGR基因, 为后期芫菁体内斑蝥素生物合成途径的研究奠定了基础。     

Overexpression of Acetyl-CoA Carboxylase Gene of <Emphasis Type="Italic">Mucor rouxii</Emphasis> Enhanced Fatty Acid Content in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.     

The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.     

Carboxylase genes of Sulfolobus metallicus

Carbon dioxide limitation of Sulfolobus metallicus resulted in increased cellular concentrations of polypeptides that were predicted to be biotin carboxylase and biotin carboxyl-carrier-protein components of a protein complex. These polypeptides were coeluted from a native polyacrylamide gel and were estimated at 19 and 59 kDa after separation by denaturing gel electrophoresis. Their encoding genes were identified, sequenced and shown to code for polypeptides of 18,580 and 58,235 Da with similarities to biotin carboxyl carrier proteins and biotin carboxylases, respectively. The genes overlapped at the second of two stop codons that terminated the carboxylase gene. A third gene occurred on the opposite strand, 293 bp upstream of the biotin carboxylase gene. Its deduced amino acid sequence was similar to those of carboxyl transferase subunits of carboxylase enzymes, in particular to those of the propionyl-CoA carboxylases. It is proposed that the three described genes could encode the key enzyme complex responsible for carbon dioxide fixation during autotrophic growth of the thermoacidophilic archaea. Received: 24 February 1999 / Accepted: 30 July 1999     

Biotinoyl domain of human acetyl-CoA carboxylase: Structural insights into the carboxyl transfer mechanism

Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity. In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available. To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyzed its characteristics and interaction with the biotin ligase, BirA using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the earlier determined domains from E. coli and P. shermanii. However, the local structures near the biotinylation sites have notable differences that include the geometry of the consensus "Met-Lys-Met" (MKM) motif and the absence of "thumb" structure in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional noncovalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the "swinging arm" motion. The chemical shift perturbation and the cross saturation experiments of the human ACC2 holo-biotinoyl upon the addition of the biotin ligase (BirA) showed the interaction surface near the MKM motif, the two glutamic acids (Glu 926, Glu 953), and the positively charged residues (several lysine and arginine residues). This study provides insight into the mechanism of ACC holoenzyme function and supports the swinging arm model in human ACCs.     

Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase.

The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.     

The Bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl‐CoA carboxylase

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate‐limiting step in fatty acid biosynthesis is catalyzed by a multi‐subunit form of the acetyl‐CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl‐CoA to produce malonyl‐CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the P_II family. Here we show that the GlnB paralogues of the P_II proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC‐BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC‐BCCP‐GlnB interaction and ACC inhibition were relieved by 2‐oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2‐oxoglutarate‐sensitive dissociable regulatory subunit of ACC in Bacteria.     

Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.     

Primary structure of chicken liver acetyl-CoA carboxylase deduced from cDNA sequence

The complete amino acid sequence of acetyl-CoA carboxylase from chicken liver has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by Edman degradation of peptide fragments obtained by digestion of the enzyme polypeptide with Achromobacter proteinase I or staphylococcal serine proteinase. Chicken liver acetyl-CoA carboxylase is predicted to be composed of 2,324 amino acid residues, having a calculated molecular weight of 262,706. The biotin carboxyl carrier protein domain is located in the middle region of the enzyme polypeptide. The amino-terminal portion of the acetyl-CoA carboxylase has been found to exhibit a homologous primary structure to that of carbamyl phosphate synthetase. Localization of possible functional domains including biotin carboxylase subsite in the acetyl-CoA carboxylase polypeptide is discussed.