Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae

The structures of the E277A isomaltase mutant from Saccharomyces cerevisiae in complex with isomaltose or maltose were determined at resolutions of 1.80 and 1.40 Å, respectively. The root mean square deviations between the corresponding main-chain atoms of free isomaltase and the E277Α-isomaltose complex structures and those of free isomaltase and the E277A-maltose complex structures were found to be 0.131 Å and 0.083 Å, respectively. Thus, the amino acid substitution and ligand binding do not affect the overall structure of isomaltase. In the E277A-isomaltose structure, the bound isomaltose was readily identified by electron densities in the active site pocket; however, the reducing end of maltose was not observed in the E277A-maltose structure. The superposition of maltose onto the E277A-maltose structure revealed that the reducing end of maltose cannot bind to the subsite + 1 due to the steric hindrance from Val216 and Gln279. The amino acid sequence comparisons with α-glucosidases showed that a bulky hydrophobic amino acid residue is conserved at the position of Val216 in α-1,6-glucosidic linkage hydrolyzing enzymes. Similarly, a bulky amino acid residue is conserved at the position of Gln279 in α-1,6-glucosidic linkage-only hydrolyzing α-glucosidases. Ala, Gly, or Asn residues were located at the position of α-1,4-glucosidic linkage hydrolyzing α-glucosidases. Two isomaltase mutant enzymes – V216T and Q279A – hydrolyzed maltose. Thus, the amino acid residues at these positions may be largely responsible for determining the substrate specificity of α-glucosidases.     

Short-chain prenyl diphosphate synthase that condenses isopentenyl diphosphate with dimethylallyl diphosphate in ispA null Escherichia coli strain lacking farnesyl diphosphate synthase

A short-chain prenyl diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl diphosphate synthase, ispA, was separated from other prenyl diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate and geranylgeranyl diphosphate.     

Cloning and expression of the N-acetylmuramidase gene from Streptomyces rutgersensis H-46

The N-acetylmuramidase SR1 gene from Streptomyces rutgersensis H-46 was cloned in Escherichia coli JM109 and expressed in E. coli BL21(DE3)pLysS. An open reading frame included the leader peptide region encoding a polypeptide of 65 amino acid residues and the mature SR1 enzyme region encoding a polypeptide of 209 amino acid residues. The overall G + C content of the mature enzyme gene was 67.6%, with 98.1% of G or C in the third position of the codons. The calculated molecular weight of the mature enzyme was 23,057 Da. The amino acid sequence of the mature enzyme showed a significant level of identity with bacteriolytic enzymes from Streptomyces globisporus (50.9% identity), Chalaropsis species (40.2% identity) and Saccharopolyspora erythraea (31.0% identity). The mature enzyme gene cloned into plasmid pET26b carrying a signal peptide, peIB, was expressed in E. coli BL21(DE3)pLysS. The signal peptide region was cleaved during the production of the enzyme. Specific activity of the enzyme purified from the transformant was almost identical to that of the native enzyme. Furthermore, the SR1 enzyme gene cloned with the leader peptide gene into plasmid pET28a was also expressed in E. coli. In this case, a proform-like protein was partially processed; 35 amino acid residues were cleaved but 30 amino acid residues remained. This proform like protein has approximately one-nineteenth the activity of the native enzyme. These results indicated that the native SR1 enzyme was produced in the following manner in the cells of S. rutgersensis H-46. The SR1 enzyme gene was translated to a pre-proform protein followed by the deletion of a signal peptide. Finally, the proform-like protein was processed by deletion of the remaining leader peptide.     

Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae.

The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme by threonine. We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine-induced reduction of the carbon flow into the aspartate pathway, leading to methionine limitation. One of the mutants has an aspartate kinase which is 30-fold more strongly inhibited by threonine than the wild-type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP. The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine-phenylalanine-glycine-glycine (KFGG) stretch present in the N-terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved in substrate binding. Mutagenesis of a HOM3 region centred in the KFGG-coding triplets generated alleles that determine threonine sensitivity or auxotrophy for threonine and methionine, but not a phenotype associated with a feedback-resistant aspartate kinase, indicating that this region is not involved in the allosteric response of the enzyme.     

An active-site mutation causes enhanced reactivity and altered regiospecificity of transglucosylation catalyzed by the Bacillus sp. SAM1606 alpha-glucosidase

Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-O(G)-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were allowed to react with 1.75 M sucrose at 60 degrees C, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-O(F)-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the Bacillus cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.     

Yeast KEX2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway

The KEX2 protease (product of the KEX2 gene) functions late in the secretory pathway of Saccharomyces cerevisiae by cleaving the polypeptide chains of prepro-killer toxin and prepro-alpha-factor at paired basic amino acid residues. The intracellular vesicles containing KEX2 protease sedimented in density gradients to a position distinct from those containing mannosyltransferase I (product of the MNN1 gene), a marker enzyme for the Golgi complex. The recovery of intact compartments containing these enzymes approached 80% after sedimentation. We propose that the KEX2 protease and mannosyltransferase I reside within distinct compartments.     

Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone was isolated and sequenced, within which an ORF of 1425 by encoding a protein of 475 as with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted as sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.     

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.     

Isoleucine 259 and isoleucine 260 residues in Streptococcus gordonii soluble inorganic pyrophosphatase play an important role in enzyme activity

The hinge region of the family II soluble inorganic pyrophosphatase (PPase) found in Streptococcus gordonii DL1 (Challis) has previously been shown to play an important role in opening and closing of the active site between the N- and C-terminal domains of PPase. Amino acid residues isoleucine 259 (I259) and isoleucine 260 (I260) are highly conserved among catalytically active family II PPases and are located very close to the hinge region. Substitution of either I259 or I260 with a hydrophilic acidic amino acid (glutamate or aspartate) resulted in adverse effects on the kinetic properties of the enzyme. The I259/E and I259/D variants were nearly catalytically inactive (k(cat)/K(m) <0.2% of the wild type), whereas both I260/E and I260/D variants showed less than 15% of the catalytic efficiency of the wild type S. gordonii PPase. Conservative substitution of both residues to valine (I259/V, I260/V) showed no significant effect on the catalytic activity. The solvent accessibility data for I259 and I260 and the proximity of these amino acids to the hinge region suggest that occlusion of these residues may stabilise the closed and open conformations of the protein, respectively, thus aiding the catalytic activity of the enzyme.     

Biosynthesis of essential oils in aromatic plants: A review

In aromatic plants species, biosynthesis of essential oils occurs through two complex natural biochemical pathways involving different enzymatic reactions. Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the universal precursors of essential oil biosynthesis and are produced by the cytosolic enzymatic MVA (mevalonic acid) pathway or by plastidic and enzymatic 1-deoxy-d-xylolose-5-phosphate (DXP) pathway, also called the 2-C-methylerythritol-4-phosphate (MEP) pathway. In the particular plant cell part, prenyl diphosphate synthases condense isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) further to form prenyl diphosphates, which are used as substrates for geranyl diphosphate (GPP; C₁₀) or for fernesyl diphosphate (FPP; C₁₅). Essential oils are final terpenoid products and are formed by a huge group of enzymes known as terpene synthases (TPS). Essential oils are important secondary metabolites of plants and have been used not only in different industries but also in ethnobotanical medicines for centuries. Hence, considerable research has been undertaken to understand the essential oil biosynthetic pathways. This review will be a valuable source of information in the field of natural products, as we give detailed insights about biosynthesis of essential oils in plants and thus indicate also new unexplored horizons for further research.     

锌螯合肽的两端排序法定量构效关系

为了研究锌螯合肽结构与生物活性之间的关系,构建定量构效关系（quantitative structure-activity relationship,QSAR）模型。采取两端排序法将56 条不同长度的合成肽规格化,采用18 种氨基酸描述符,利用偏最小二乘法进行分析。发现5 种氨基酸描述符对应的QSAR模型的相关系数达到建模要求,分别为描述符FASGAI、Z、HESH、C和ST,其中描述符FASGAI最优（R2＝0.827 3、Q2＝0.602 2、估计均方根误差＝0.168 6、Q2ext＝0.717 2、预测方根误差＝0.255 8）。对描述符FASGAI所构建的模型进一步分析发现,多肽序列中的氨基酸位置对多肽锌螯合活性的影响力依次为C3＞N3＞C1＞N1＞N2＞C2,同时,多肽各位置上氨基酸残基的立体属性会影响其螯合活性。该模型的成功建立为锌螯合肽定量构效关系的研究提供了探索性思路。     

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.     

半理性设计提高甲酸脱氢酶（CbFDH）活力及热稳定性

甲酸脱氢酶(formate dehydrogenase,FDH）是NADH循环再生的最佳酶之一,广泛应用于食品、医药和化工等行业。但是野生型甲酸脱氢酶普遍存在酶活低、催化效率差等缺点,导致产品转化率较低,影响产品的工业化生产。为了获得具有更佳催化性能的甲酸脱氢酶,作者以博伊丁假丝酵母(Candida boidinii）来源的甲酸脱氢酶为模板,利用HOTSPOT WIZARD v3.1进行三维结构模拟预测,构建了P68G、Q197K两个突变体,比酶活较野生型分别提高了11%和33%。这是由于P68G氨基酸残基侧链的苯环被氢取代,减少了甲酸盐底物进入口袋的空间位阻;而Q197K侧链酰胺基突变为胺丁基增强了酶的柔性。然而这两个突变点对甲酸脱氢酶的热稳定性产生了负面影响,因此在I239位引入半胱氨酸突变与C262构成二硫键以提高其热稳定性,最终获得一株热稳定性显著提高,比酶活较野生型提高31%、较I239C提高45%的突变株CbFDH Q197K/I239C。通过半理性预测蛋白质结构提高了甲酸脱氢酶的活力和热稳定性,为高效构建性能稳定、还原力强的甲酸脱氢酶提供了的理论基础。     

基于定向进化技术提高巨大芽孢杆菌谷氨酸脱羧酶活性

为提高巨大芽孢杆菌谷氨酸脱羧酶活性,通过定向进化技术对其进行酶工程改造。经过二轮易错聚合酶链式反应,从13 000 多个突变株中筛选到突变株A5-3、E2-4、E3-11,相对于野生型,其酶比活力提高了157%、115%、97%,且Kcat/Km都有所增大。其中A5-3氨基酸序列发生了2 个突变（A55D和D451E）。三维模拟结果表明,第55位丙氨酸突变为天冬氨酸很可能为酶促反应提供H＋,从而加快酶促反应效率;突变株E2-4第34位由亮氨酸突变成谷氨酰胺,一定程度上改善了酶的热稳定性;突变株E3-11的第325位由丙氨酸突变成丝氨酸有利于蛋白内部形成更多氢键,增大了该部位的柔性,更有利于氨基酸残基之间发生相互作用。圆二色谱分析表明,突变株与野生型具有相似的三维结构,相比于野生酶,突变酶的α-螺旋减少,无规则卷曲增加,说明突变酶刚性有所下降而柔性增加。利用定向进化技术可以明显改善谷氨酸脱羧酶的酶活性,为其工业化的应用提供实验参考。     

北京棒杆菌天冬氨酸激酶五突变体的构建及酶学性质表征

目的:天冬氨酸激酶（aspartate kinase,AK）是催化天冬氨酸族氨基酸合成的第一个关键别构酶,为了提高其活力,并削弱或解除末端产物赖氨酸（Lys）与苏氨酸（Thr）对AK的协同反馈抑制。方法:在获得的北京棒杆菌四突变体T379N/A380C/G171I/Y198N AK（NCIN AK）的基础上,发现G295位点与抑制剂Thr通过氢键相连且高度保守, 对G295位点进行定点饱和突变,提取质粒,转入大肠杆菌BL21感受态细胞中诱导表达,采用高通量筛选获得酶活力显著提高的突变株,对野生型（Wild type,WT）、五突变体T379N/A380C/G171I/Y198N/G295L AK（NCINL AK）进行酶动力学研究和酶学性质表征。结果:成功构建五突变体NCINL AK,最大反应速率V_max为259 U/（mg·min）。与野生型相比,米氏常数K_m值由3.44 mmol/L减小到0.93 mmol/L,酶与底物结合更加紧密;希尔系数n值由2.73降为1.21,正协同性降低;最适反应温度由25 ℃提高到28 ℃,最适pH由8.0升至8.5,半衰期由4.7 h延长至5.2 h;五突变体NCINL AK较野生型WT AK,不同底物抑制剂浓度在0.2、1.0、5.0、10.0 mmol/L时,抑制作用被不同程度的减弱,甚至Lys抑制作用被完全解除,且在10 mmol/L Thr条件下有激活作用。结论:本实验获得酶活力提高87.20倍的五突变体NCINL AK,酶学性质得到明显改善,在一定抑制剂浓度范围内,基本解除Lys对AK的反馈抑制作用,Thr的反馈抑制得到一定缓解,提高了积累大量天冬氨酸族氨基酸的可能性,为构建高产天冬氨酸族氨基酸菌株提供参考,使甲硫氨酸（Met）等氨基酸的无污染、高效生产成为可能。     

ACTIVITIES OF CERTAIN AMINOTRANSFERASES AND NADP-DEPENDENT GLUTAMIC ACID DEHYDROGENASE IN YEAST DURING FERMENTATION

The activities of alanine (E.C.2.6.I.2.), aspartate (E.C.2.6.I.I) and glycine (E.C. 2.6.I.4) aminotransferases and NADP-dependent glutamic acid dehydrogenase (E.C.I.4.I.4) have been followed in Saccharomyces cerevisiae during fermentation in an all-malt wort. The synthesis of alanine aminotransferase is enough to furnish the yeast with alanine, but glycine aminotransferase activity is inadequate for glycine requirements during the fermentation period when glycine and alanine are not assimilated from the medium. The synthesis of all four enzymes is repressed to various extents during the early stages of fermentation, but de-repression occurs towards the end of the fermentation.     

Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-beta-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70-100 amino acids upstream of the G-X-S-X-G consensus sequence.     

Proteolytic processing of certain CaaX motifs can occur in the absence of the Rce1p and Ste24p CaaX proteases

The CaaX motif directs C‐terminal protein modifications that include isoprenylation, proteolysis and carboxylmethylation. Proteolysis is generally believed to require either Rce1p or Ste24p. While investigating the substrate specificity of these proteases, using the yeast a‐factor mating pheromone as a reporter, we observed Rce1p‐ and Ste24p‐independent mating (RSM) when the CKQQ CaaX motif was used in lieu of the natural a‐factor CVIA motif. Uncharged or negatively charged amino acid substitutions at the a₁ position of the CKQQ motif prevented RSM. Alanine substitutions at the a₂ and X positions enhanced RSM. Random mutagenesis of the CaaX motif provided evidence that RSM occurs with approximately 1% of all possible CaaX motif permutations. Combined mutational and genetic data indicate that RSM‐promoting motifs have a positively charged amino acid at the a₁ position. Two of nine naturally occurring yeast CaaX motifs conforming to this pattern promoted RSM. The activity of the isoprenylcysteine carboxyl methyltransferase Ste14p was required for RSM, indicating that RSM‐promoting CaaX motifs are indeed proteolysed. RSM was enhanced by the overexpression of Axl1p or Ste23p, suggesting a role for these M16A subfamily metalloproteases in this process. We have also determined that an N‐terminal extension of the a‐factor precursor, which is typically removed by the yeast M16A enzymes, is required for optimal RSM. These observations suggest a model that involves targeting of the a‐factor precursor to the peptidosome cavity of M16A enzymes where subsequent interactions between RSM‐promoting CaaX motifs and the active site of the M16A enzyme lead to proteolytic cleavage. Copyright © 2009 John Wiley & Sons, Ltd.     

Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.     

烟草半胱氨酸蛋白酶抑制剂（CPI）基因家族的克隆及组织表达谱分析

运用生物信息学方法,结合RT-PCR和SMART RACE技术从烟草（Nicotiana tabacum）中克隆了4个CPI基因的全长cDNA序列,分别命名为NtCPI1、NtCPI2、NtCPI3和NtCPI4, GenBank登陆号分别为KF057988、KF057989、KF057990和KF057991。基因序列分析表明4个基因分别编码98、98、120和123个氨基酸残基的蛋白质,都具有CPI反应位点的保守基序GG、QXVXQ和A/PW,同时具有植物CPI所特有的LARFAV基序,其中NtCPI3和NtCPI4的N端还包含一段27个氨基酸残基组成的信号肽。实时荧光定量PCR试验表明,4个基因的组织表达谱很广,在根、茎、叶和芽组织中都有表达。这些结果为进一步研究半胱氨酸蛋白酶抑制剂在植物中的生理功能奠定了基础。