昆虫几丁质酶及其在植物抗虫品种改良中的应用

几丁质是昆虫外壳和围食膜的重要组成成分 ,在适当的时期昆虫分泌适量的几丁质酶降解几丁质以保证昆虫的正常生长。植物几丁质酶能够抵御病原菌的入侵 ,但是对昆虫没有明显的效果 ,而昆虫几丁质酶基因在转基因植物中的组成型表达却对昆虫具有明显的抗性。本文综述了昆虫几丁质酶的特性 ,阐述了昆虫几丁质酶及其在植物抗虫方面应用的研究进展。     

生物信息学在昆虫几丁质酶研究中的应用

昆虫几丁质酶及类似蛋白由多种不同的基因编码而成,它们在分子量大小、空间结构、化学性质和酶学性质等方面存在较大的差异,并在昆虫不同的组织、不同时期的表达量各不相同,对昆虫生长发育过程中几丁质的代谢起着重要的作用.在昆虫几丁质酶的研究过程中,生物信息学在几丁质酶基因克隆、结构分析、同源性比较等方面应用广泛.该文主要对昆虫几丁质酶及生物信息学在昆虫几丁质酶研究中的应用、存在的问题及展望做了全面的综述.     

与宿主昆虫液化相关的杆状病毒基因及其蛋白

昆虫被杆状病毒感染后会发生液化现象,这有利于病毒向周围环境扩散。目前在杆状病毒苜蓿银纹夜蛾核型多角体病毒NPV和GV中,发现与昆虫宿主液化相关的基因有组织蛋白酶基因V-cath基因和几丁质酶基因。V-cath基因表达产物在苜蓿银纹夜蛾多角体病毒(AcMNPV)中能特异性降解昆虫细胞内的肌动蛋白。几丁质酶不仅参与了虫体体表面几丁质的降解,同时还参与V-CATH蛋白前体的加工过程,起分子伴侣的作用。对家蚕核型多角体病毒(BmNPV)的研究表明其FP25K基因表达产物通过影响组织蛋白酶的释放与分泌而参与虫体液化。简要综述了此3种基因及其表达产物的结构、功能与特性,并讨论了它们在生产上的应用前景。     

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。     

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。     

东亚飞蝗中肠几丁质酶基因的克隆、序列分析及组织定位

通过RACE方法,克隆了东亚飞蝗Locusta migratoria manilensis (Meyen)几丁质酶基因 (LmChi)cDNA全序列 (GenBank 登录号：EF092841)。获得的cDNA全长1 604 bp,其中可读框1 452 bp, 编码483个氨基酸。推测其氨基酸序列与18家族昆虫几丁质酶有较高的相似性。与其他几丁质酶一样,东亚飞蝗几丁质酶序列也包含一个信号肽、一个几丁质酶活性位点、一个碳端丝氨酸富集区和一个几丁质结合域。半定量RT-PCR研究表明,LmChi基因只在东亚飞蝗不同发育阶段的中肠组织中表达,而在东亚飞蝗体壁、前肠和后肠均没有发现LmChi基因的转录。     

昆虫几丁质合成及其调控研究前沿

几丁质合成与降解是昆虫最重要的生理过程之一。本文根据国外和作者自己的研究,综述了昆虫几丁质合成及其调控研究进展。昆虫几丁质的生物合成通路始于海藻糖,终止于几丁质,其中共有8个酶参与。目前研究最多的为海藻糖酶和几丁质合成酶。昆虫存在2个海藻糖酶基因和2个几丁质合成酶基因。可溶性海藻糖酶基因对昆虫表皮的几丁质合成影响更大,而膜结合海藻糖酶基因则主要影响中肠的几丁质合成。几丁质合成酶A主要负责表皮和气管几丁质的合成,而几丁质合成酶B则负责中肠围食膜的几丁质合成。目前,昆虫几丁质合成的调控途径主要有两种:利用RNAi技术和几丁质合成抑制剂。     

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。     

HaSNPV几丁质酶基因在大肠杆菌和昆虫细胞中的表达及其产物对Bt杀虫剂的增效作用

为了探索杆状病毒几丁质酶对微生物杀虫剂的增效作用及其利用途径 ,分别在大肠杆菌和昆虫细胞中表达棉铃虫单粒包埋型核型多角体病毒 (HaSNPV)几丁质酶 .用PCR方法扩增出不含N端信号肽编码序列的几丁质酶基因片段 ,并分别克隆至原核表达载体pET2 8a和重组到杆状病毒BactoBac表达系统 ,在大肠杆菌 (E .coli)BL2 1和粉纹夜蛾 (Trichoplusiani)细胞系Tn 5B1 4中分别进行了表达 .在大肠杆菌中表达量约占细菌总蛋白 15 % ,在昆虫细胞中表达量约占细胞总蛋白10 % .将含有几丁质酶的大肠杆菌和昆虫细胞表达产物添加到苏云金杆菌 (Bt)菌液中一起喂食 2龄家蚕 .结果显示 ,HaSNPV几丁质酶基因的 2种表达产物和Bt杀虫剂的混合物使处理的家蚕的致死时间较对照处理均明显缩短 .昆虫细胞和大肠杆菌表达产物与Bt混合物处理的LT50 分别从 93 5h和 95 1h缩短到 5 6 2h及 6 7 2h ,并且供试家蚕的生长速度明显缓慢 .研究结果表明 ,重组的HaSNPV几丁质酶有望作为Bt杀虫剂的增效剂     

甜菜夜蛾几丁质合成酶基因的克隆与表达模式

几丁质合成酶(CHS)是昆虫几丁质生物合成过程中至关重要的酶。但是,迄今有关昆虫CHS的研究工作仍不多见。中山大学昆虫学研究所张文庆教授和他的研究生从鳞翅目昆虫甜菜夜蛾Spodoptera exigua中克隆得到了A类CHS基因(SeCHSA),这是我国克隆获得的第1个昆虫CHS基因(DQ062153)。此     

Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome

MOTIVATION: Multiple chitinases as well as lectins closely related to them have been characterized previously from many insect species and the corresponding genes/cDNAs have been cloned. However, the identification of the entire assortment of genes for chitinase family proteins and their differences in biochemical properties have not been carried out in any individual insect species. The completion of the entire DNA sequence of Drosophila melanogaster (fruit fly) genome and identification of open reading frames presents an opportunity to study the structures and functions of chitinase-like proteins, and also to identify new members of this family in DROSOPHILA: We are, therefore, interested in studying the functional genomics of chitinase-like gene families in insects. METHODS: We searched the Drosophila protein sequences database using fully characterized insect chitinase sequences and BLASTP software, identified all the putative chitinase-like proteins encoded in Drosophila genome, and predicted their structures using domain analysis tools. A phylogenetic analysis of the chitinase-like proteins from Drosophila and several other insect species was carried out. The structures of these chitinases were modeled using homology modeling software. RESULTS: Our analysis revealed the presence of 18 chitinase-like proteins in the Drosophila protein database. Among these are seven novel chitinase-like proteins that contain four signature amino acid sequences of chitinases belonging to family 18 glycosylhydrolases, including both acidic and hydrophobic amino acid residues critical for enzyme activity. All the proteins contain at least one catalytic domain with one having four catalytic domains. Phylogenetic analysis of chitinase-like proteins from Drosophila and other insects revealed an evolutionary relationship among all these proteins, which indicated gene duplication and domain shuffling to generate the observed diversity in the encoded proteins. Homology modeling showed that all the Drosophila chitinase-like proteins contain one or more catalytic domains with a (alpha/beta)8 barrel-like structure. Our results suggest that insects utilize multiple family 18 chitinolytic enzymes and also non-enzymatic chitinase-like proteins for degrading/remodeling/binding to chitin in different insect anatomical extracellular structures, such as the cuticle, peritrophic membrane, trachea and mouth parts during insect development, and possibly for other roles including chitin synthesis. AVAILABILITY: Perl program and supplementary material are available at http://www.ksu.edu/bioinformatics/supplementary.htm     

Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

昆虫几丁质酶及其在害虫防治中的应用

几丁质是昆虫重要的结构性组分,在昆虫生长发育的各个时期都需要一定量的几丁质来维持其代谢平衡.昆虫几丁质酶可以降解昆虫体壁和围食膜中的几丁质,作为一种潜在的生物杀虫剂在害虫防治方面具有广阔的应用前景.随着对昆虫几丁质酶研究的不断深入,目前已克隆到了30余种昆虫几丁质酶,并应用于转基因作物和基因工程微生物中,对害虫具有一定...     

Problems of reproducibility--does geologically ancient DNA survive in amber-preserved insects?

Apparently ancient DNA has been reported from amber-preserved insects many millions of years old. Rigorous attempts to reproduce these DNA sequences from amber- and copal-preserved bees and flies have failed to detect any authentic ancient insect DNA. Lack of reproducibility suggests that DNA does not survive over millions of years even in amber, the most promising of fossil environments.     

A Chitinase Encoding Gene (chit1 Gene) from the Entomopathogen Metarhizium anisopliae: Isolation and Characterization of Genomic and Full-Length cDNA

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome. Received: 13 March 1998 / Accepted: 14 April 1998     

小地老虎几丁质酶基因的克隆与序列分析

利用昆虫几丁质酶对几丁质的调控作用破坏几丁质新陈代谢的平衡来防治害虫, 在生物防治策略中具有很大的发展潜力。从处于预蛹期的小地老虎Agrotls ipsilon (Hufnagel)体中肠内提取总的RNA, 经反转录, 利用cDNA末端快速扩增技术（RACE）获得了几丁质酶基因的cDNA序列。该基因序列已经登录GenBank并获得登录号为EU035316。该序列长度为2 823个碱基, 含有一个1 674个碱基的开放读码框。开放读码框编码558个氨基酸残基, 预测的分子量为62.5 kDa, 等电点5.12。推导得到的氨基酸序列含有2个N-位糖基化位点,20个O-位糖基化位点, 含有2个几丁质酶所具有的保守序列：N-端的催化区和C-端的几丁质结合区。氨基酸序列与其他昆虫, 特别是鳞翅目昆虫的几丁质酶高度同源。     

基因探针及其在昆虫学上的应用

八十年代以来以基因工程技术为主导的分子生物学研究大大丰富了人们对生命过程和本质的认识。基因工程技术在昆虫学研究中日益受到重视。一个新兴的学科─-昆虫分子生物学已经形成。在分子生物学研究中基因探针是必不可少的重要的工具。由于在系统进化上人和哺乳类遗传距离较近,其基因探针具有较大的通用性,所以医学发展起来的人基因探针为哺乳动物研究带来了许多方便。而无脊椎动物的分子生物学研究一向十分薄弱,因此可用于昆虫的基因探针来源困难,研究者常需在实验设计初期对已有的众多基因探针进行预选或自己制备。所以基因探针的选择和使用对昆虫分子生物学研究至为关键。本文就昆虫学常用的基因探针的类型,标记方法,特别是应用等方面选择若干典型实例作一些介绍和评述。