Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus

A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30-40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity.     

Purification and partial characterization of recombinant Cu, Zn containing superoxide dismutase of Cordyceps militaris in E.coli

The cDNA of Cu, Zn containing superoxide dismutase from the Cordyceps militaris SH (cm-SOD) was overexpressed in Escherichia coli BL 21 (DE3) using the pET-21a expression vector. The recombinant cell overexpressed the protein corresponding to 35+/-3% of total bacterial protein in cytosol. The purification was performed through three steps: DEAE-FF, CM-52, and G-100. After this purification procedure, a specific activity of 27272.7 U/mg of protein was reached, corresponding to 6.1-fold purification with a yield of 85.0%. The purity was homogeneous by SDS-PAGE analysis and 94.2+/-1.0% by CZE analysis. A subunit molecular mass of the recombinant enzyme was 15704 Da with a Cu and Zn element. In addition, the dimeric and polymeric structures were observed on MALDI-TOF-MS. Isoelectric point value of 7.0 was obtained for the recombinant enzyme that was sensitive to H2O2 and KCN. The recombinant enzyme remained 80+/-2% residual activity at pH 7.8, at 50 degrees C for 4h incubation. The properties: N-terminal amino acid sequence (the first 12 amino acid residues), pI, subunit molecular mass, thermo-stability of the purified recombinant SOD are similar to that of the native Cu, Zn-SOD from C. militaris (N-cm-SOD).     

棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析

[目的]从棉花黄萎病真菌Verticillium dahliae中克隆木聚糖酶基因,并在毕赤酵母中进行异源表达,研究酶学性质.[方法]通过多序列比对设计简并引物,扩增出真菌V. dahliae木聚糖酶基因片段,再采用基因组步行PCR技术获得全长木聚糖酶基因序列.经BLAST比对并结合GT-AG原则分析,该基因含有一个大小为63 bp的内含子,利用DpnI介导的缺失方法对含内含子的全长木聚糖酶基因进行剪接,获得该基因的全长cDNA.将克隆到的cDNA在毕赤酵母GS115进行了表达,重组酶经纯化后进行酶学性质分析.[结果]BLAST比对显示,该cDNA推测的氨基酸序列和已知木聚糖酶的最高一致性为72%.测得该酶最适反应温度为45℃,最适反应pH值为6,在pH5-9维持50%以上的活性,对山毛榉材木聚糖具有最好的水解效果.Mg2 和Ca2 对酶有激活作用,分别提高了33.7%和16.6%,EDTA,β-巯基乙醇和NaN3对酶的活性基本没有影响,Tween-80和DMSO使酶活性提高了28.4%和12.8%.[结论]本文从引起棉花黄萎病的真菌V. dahliae中克隆到的木聚糖酶基因是在GenBank上登录的第一个来自棉花黄萎病真菌的木聚糖酶基因序列.本文所用的克隆方法可以高效的从植物病原真菌和白腐真菌克隆只含一个内含子的11家族的新木聚糖酶基因,避免了摸索原始菌株酶表达诱导条件,检测酶的活性等繁琐的操作.酶学性质分析显示该酶在低聚木糖的制备,面包改良上有潜在的应用价值.     

陆地棉叶绿体铜锌超氧化物歧化酶基因的克隆与表达

以陆地棉‘CRI36＇的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1 043 bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%～74%之间。基因的表达谱分析显示：棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实：该基因主要在苗期表达,以后表达逐渐减少。用pET-21a（＋）构建了原核表达载体,在大肠杆菌BL21（DE3）的表达结果显示：表达后得到一个29.0 kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。     

Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari

A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.     

Up-regulation of lysozyme gene expression during metamorphosis and immune challenge of the cotton bollworm, Helicoverpa armigera

Lysozymes act as crucial bacteriolytic enzymes in insect immune system by hydrolyzing the beta (1-->4) bonds between N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan of prokaryotic cell walls. We have isolated and characterized a Helicoverpa armigera cDNA encoding an insect lysozyme named HaLyz. We amplified a fragment by PCR, using degenerate primers derived from the conservative amino acid sequences for performing 5' and 3' RACE. The full-length cDNA was 661 base pairs. The theoretical pI and molecular weight of the protein were computed to be 9.08 and 15.6 kDa, respectively. Prokaryotic expression of the HaLyz ORF by Escherichia coli confirmed the calculated molecular weight of the protein. The deduced 135 amino acids showed high homology with known lysozymes from other insects, ranging from 47% to 89% by BLASTp search in NCBI. Analyses revealed that this protein has a typical lysozyme C signature among amino acids 93-111, CNVTCAEMLLDDITKASTC. An interesting relation between immunity and larva to pupa metamorphosis in insects was discovered. Real time-PCR showed that HaLyz gene expression was transiently enhanced at the onset of metamorphosis of the cotton bollworm, Helicoverpa armigera. The gene expression was up-regulated after the injection of E. coli or entomopathogenic fungi, Beauveria bassiana, but showed different expression patterns.     

Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate

We have studied the enzymological properties of L-galactose dehydrogenase (l-GalDH), a key enzyme in the biosynthetic pathway of l-ascorbate (AsA) in plants. L-GalDH was purified approximately 560-fold from spinach leaves. The enzyme was a homodimer with a subunit mass of 36 kDa. We also cloned the full-length cDNA of spinach L-GalDH, which contained an open reading frame encoding 322 amino acid residues with a calculated molecular mass of 35,261 Da. The deduced amino acid sequence of the cDNA showed 82, 79 and 75% homology to L-GalDH from kiwifruit, apple and Arabidopsis, respectively. Recombinant enzyme expressed from the cDNA in Escherichia coli showed L-GalDH activity. Southern blot analysis revealed that the spinach L-GalDH gene occurs in a single copy. Northern blot analysis suggests that L-GalDH is expressed in different organs of spinach. The purified native L-GalDH showed high specificity for L-galactose with a Km of 116.2+/-3.2 microM. Interestingly, spinach L-GalDH exhibited reversible inhibition by AsA, the end-product of the biosynthetic pathway. The inhibition kinetics indicated a linear-competitive inhibition with a Ki of 133.2+/-7.2 microM, suggesting feedback regulation in AsA synthesis in the plant.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene

Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana that encode homoserine kinase. The HSK gene is composed of an 1113-bp continuous open reading frame that could produce a 38-kDa protein. The gene product has homology with homoserine kinase from bacteria and fungi. It contains a conserved motif, known as GHMP, found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The amino-terminal 50 amino acids of the HSK protein show features of a transit peptide for localization to plastids. Genomic blot analysis revealed that there is a single locus in A. thaliana to which the HSK cDNA hybridizes. The HSK protein expressed as a His-tagged construct in Escherichia coli shows a specific activity in an l-homoserine-dependent ADP synthesis assay of 3.09 +/- 0.25 micromol min(-1) mg(-1) protein at pH 8.5 and 37 degrees C. The apparent K(m) values are 0.40 mM for l-homoserine and 0.32 mM for Mg-ATP. Other hydroxylated compounds are not used as substrates. The enzyme requires 40 mM K(+) and 3 mM Mg(2+) for activity. It has an unusually high temperature optimum, yet it is very unstable, losing more than 80% of its activity after a single cycle of freeze-thawing. The HSK enzyme shows no significant regulation by amino acids in vitro.     

Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum

Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. The production of hydrolytic enzymes appears to be a key element in the parasitic process. Among the enzymes released by Trichoderma, the aspartic proteases play a major role. A gene (SA76) encoding an aspartic protease was cloned by 3' rapid amplification of cDNA ends from T. harzianum T88. The coding region of the gene is 1,593 bp long, encoding a polypeptide of 530 amino acids with a predicted molecular mass 55 kDa and a pI of 4.5. The catalytic aspartic residues characteristic of aspartic proteases are conserved with an active-site motif (DSG); however, the DSG in the N-terminal lobe is unusual in that Ser replaced Thr. Northern blot analysis indicated that SA76 was induced in response to different fungal cell walls. Aspartic protease SA76 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (10.5 U mL(-1)) 72 h after induction with galactose. The temperature optimum of the enzyme was 45 degrees C and its pH optimum was 3.5. The culture supernatant of the S. cerevisiae strain that expressed the aspartic protease SA76 was able to inhibit the growth of five phytopathogenic fungi. The inhibition of mycelial growth varied between 7% and 38%.