HcNPV半胱氨酸蛋白酶基因的核苷酸序列研究

ＨｃＮＰＶ半胱氨酸蛋白酶基因的核苷酸序列研究贡成良１小林淳宫岛成寿金伟１吴祥甫２＊（日本三重大学分子素材工学科,三重５１４,日本;１浙江农业大学蚕学系,杭州３１００２９;２中国科学院上海生物化学研究所,上海２０００３１）关键词美国白蛾;核型多角体病毒...     

几丁质酶基因及其应用新进展

几丁质酶能降解真菌和昆虫细胞壁的主要成分几丁质而在生物防御中具有重要的作用。近年来随着重组DNA技术的进一步发展和对几丁质酶基因表达与调控机理研究的进一步深入,将几丁质酶基因导入植物增强其抗真菌能力方面的研究取得了较大进展,促进了几丁质酶的产业化应用。     

几丁质酶及其研究进展

本文从几丁质酶的分布、发育调节、可诱导性、分子生物学及抗病基因工程等方面近年来的进展进行了综合论述,并对其进一步的应用提出展望。     

一个新型的棉花几丁质酶基因

在对外源水杨酸处理的低酚品系棉花(Gossypium hirsutum cv.CRI13)(中棉13)幼苗进行RT—PCR差异分析并分离出一个cDNA片段的基础上,采用电子克隆和分子克隆相结合的方法克隆出该片段的全长序列,并对其cDNA和核DNA序列进行分析,结果显示该基因是一个新型的几丁质酶基因,命名为GhChia7.推测的GhChia7氨基酸序列与Ⅰ和Ⅱ型几丁质酶的相同性仅有30％,其结构特征也与已鉴定的Ⅰ-Ⅳ有差异,是一个新型的几丁质酶(Ⅶ型)。Northern杂交结果显示,该基因在棉花幼苗的根中以及棉花纤维中信号较强,且在棉花子叶中受水杨酸诱导表达,用7．5mmol／L水杨酸处理18h后mRNA水平达到最大值。本研究的结果显示GhChia7可能会在棉花抗病防卫反应中发挥重要作用。     

基因枪法向小麦导入几丁质酶基因的研究

利用基因枪法,以菜豆几丁质酶基因转化小麦幼胚愈伤组织。在轰击压力1300psi,轰击距离6cm、100μg金粉/枪和轰击距离9cm、150μg金粉/枪的2种处理条件下,获得4株春小麦东农7742转化植株,转化频率分别为0.36％和0.56％。经PCR和PCR-Southern杂交分析,证实菜豆几丁质酶基因已整合到T0和T1小麦基因组中。采用氨基葡萄糖法测定几丁质酶活力,结果表明,转基因小麦的几丁质酶活力明显高于对照株;转基因植株对白粉病症状减缓,并获得一株赤霉菌接种未扩展的转基因T1植株。     

几丁质酶及其在抗真菌病基因工程中的应用

真菌病是作物减产的主要原因之一。而植物界大量存在具有离体抑制真菌生长增殖能力的蛋白质,相应基因在转基因植株中表达,可使这些植物产生抗真菌能力。几丁质酶就是其中之一,它能催化几丁质（真菌细胞壁的重要成分）水解,从而抑制真菌的生长增殖。随着对其作用机理、生化特性、表达调控的深入研究,几丁质酶基因转化植株显示出很高的抗真菌能力,正日益成为植物真菌病防治的新途径。围绕几丁质酶在抗真菌病基因工程中的应用,本文对几丁质酶的活性底物、分类、生化及诱导表达、协同表达特性,进行了简要、全面的阐述。     

杆状病毒几丁质酶基因结构与功能的研究进展

杆状病毒几丁质酶基因是杆状病毒的非必需基因,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶,该酶N端具信号肽,中部是酶的活性区,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的,同时它还是原组织蛋白酶(pro-V-Cath)的分子伴侣,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

基因丢失或失活在肿瘤发生中的作用研究进展

在癌变原理的研究中,分子肿瘤学领域取得了令人瞩目的成就,发现了与肿瘤发生密切相关的两类基因。第一类是所谓的癌基因(原癌基因),它们是细胞的正常基因,在演化中高度保守,在细胞正常生长和分化过程中有着重要的生理功能。但在外界致癌因素的作用下,通过点突变、DNA重排和基因扩增等方式使这些癌基因激活而导致肿瘤发生。在体外的试验中,用已激活的癌基因去转染特定实验动物细胞,可以使     

大白菜几丁质酶CHB4基因的表达研究

根据本研究组已克隆的大白菜ClassⅣ类几丁质酶基因序列设计引物,通过RT-PCR反应扩增得到该几丁质酶成熟肽基因CHB4,构建原核表达载体pET-CHB4,利用IPTG诱导表达并对诱导表达参数进行优化,SDS-PAGE分析表明,该基因表达的蛋白分子量为28 kD左右,其表达产物主要以包涵体的形式存在,25℃并没有改变表达蛋白的可溶性,而在pH9.5的培养基条件下,诱导表达产物的上清液中却有重组蛋白条带出现。以酵母菌为指示菌做抑菌圈实验,结果显示IPTG浓度为0.6 mmol/L时抑菌效果最明显。几丁质酶的活力测定结果表明,当IPTG浓度为0.6 mmol/L时,几丁质酶活力达到最大值2.8 U/mL。     

斜纹夜蛾核多角体病毒几丁质酶基因的克隆及序列分析

以苜蓿丫纹夜蛾核多角体病毒（Ａｕｔｏｇｒａｐｈａ ｃａｌｉｔｉｒｎｉｃａ ｍｕｌｔｉｎｕｃｌｅｏｃａｐｓｉｄ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＡｃＭＮ－ＰＶ）基因组含几丁质酶基因（ｃｈｉＡ）的ｐｓｔＩ Ｍ片段为探针,通过Ｓｏｕｔｈｅｒｎ杂交将斜纹夜蛾核多角体病毒（Ｓｐｏｄｏｐｔｅｒａ ｌｉｔｕｒａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＳｐｌｔＮＰＶ）的ｃｈｉ     

美国白蛾核型多角体病毒几丁质酶基因核苷酸序列研究

美国白蛾核型多角体病毒编码的几丁质酶Ａ基因定位在ＨｉｎｄⅢ－３．５ｋｂ片段上,ＤＮＡ测序表明,半胱氨酸蛋白酶基因的５’上游反向存在ＣｈｉＡ基因,ＣｈｉＡ基因的ＯＲＦ为１６６２个核苷酸,编码５５３个氨基酸,５‘,３’－非编码区分别具有ＴＡＡＧ基序和ＡＡＴＡＡＡｐｏｌｙ（Ａ＾＋）信号序列。     

半胱氨酸蛋白酶拟肽抑制剂设计新进展

半胱氨酸蛋白酶包括多种酶,这些酶在广泛的生命过程中发挥作用。人类正常的半胱氨酸蛋白酶表达失调,寄生虫、病毒的半胱氨酸蛋白酶表达与多种病理情况相关。对于这类疾病,抑制半胱氨酸蛋白酶是一个可行的药物治疗策略。当前这类药物设计的目标是3种结构不同的半胱氨酸蛋白酶,即木瓜蛋白酶家族、半胱氨酸-天冬氨基特异性蛋白酶家族(caspases)和小核糖核酸病毒科半胱氨酸蛋白酶抑制剂家族。本文综述了近年来有关半胱氨酸蛋白酶抑制剂的设计思路。     

美国白蛾核型多角体病毒超氧化物歧化酶基因的序列分析和表达

根据测序结果 ,HcNPVsod的核苷酸序列与BmNPVsod的完全一致 ,与AcNPVsod的核苷酸序列相比 ,同源性达到 97 2 % ;推测HcNPVsod编码 1 51个氨基酸 ,与BmNPVsod的完全一致 ,与AcNPVsod编码的氨基酸相比 ,有三个氨基酸的差别。按基酸序列分析表明 ,HcNPVSOD蛋白中含有对SOD结构和活性必需的氨基酸残基 ,在HcNPVsod中均是保守的。SOD活性测定表明酶活为 1 47 0 9U/mL菌液     

原位杂交检测斯氏肺吸虫童虫半胱氨酸蛋白酶的基因表达部位

目的 从基因水平研究半胱氨酸蛋白酶基因在斯氏肺吸虫童虫的表达,并进行虫体定位。方法 利用地高辛标记原位杂交技术检测半胱氨酸蛋白酶基因在斯氏肺吸虫童虫的表达并定位。结果 在斯氏肺吸虫童虫的肠管上皮细胞中呈强阳性着色,在虫体皮层细胞中有弱阳性着色。结论 半胱氨酸蛋白酶主要在斯氏肺吸虫童虫的肠管上皮细胞中表达,在虫体皮层细胞中也有弱表达。     

Biochemical analysis of Hyphantria cunea NPV attachment to Spodoptera frugiperda 21 cells

Binding characteristics of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) to Spodoptera frugiperda 21 (Sf21) cells was determined. The cells displayed an affinity of 0.9 × 10¹⁰ M^-1 with about 8900 binding sites per cell. The biochemical nature of HcNPV-binding sites on the cell surface was also partially elucidated. There were 45 to 49% reductions in HcNPV binding following the pretreatment of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, which inhibits N-linked glycosylation and the expression of some membrane proteins on the cell surface, reduced virus binding suggesting a role for glycoprotein(s) in binding. Treatment of cells with wheat germ agglutinin or neuraminidase did not measurably reduce virus binding, indicating that oligosaccharides containing N-acetylglucosamine or sialic acid are not directly involved in HcNPV attachment. The negative effect of methylamine on HcNPV binding seems to be due to the fact that HcNPV entry via an endocytic pathway is blocked by the increased pH of the endosome. Data on energy inhibitors (sodium azide and dinitrophenol) indicates that HcNPV attachment to Sf21 cells may be closely linked to viral entry via receptor-mediated endocytosis. These findings suggest that the binding site moiety has a glycoprotein component, but that direct involvement of oligosacccharides containing N-acetylglucosamine or sialic acid residues in binding is unlikely, and that HcNPV attachment to Sf21 cells might be via receptor-mediated endocytosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

粘质沙雷氏菌几丁质酶chiB基因的克隆与序列分析

采用改进的方法提取粘质沙雷氏菌基因组DNA,通过PCR扩增,得到大小为1 500 bp特异性DNA片段(chiB基因),以pUC18质粒构建了pUC-ch iB克隆载体,转化至感受态细胞E.coliDH5α培养,并筛选出重组质粒。经测序分析,证明克隆片段与文献报道相一致。     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Isolation and Characterization of a Cysteine Protease of Freesia Corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M _r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.