白背飞虱几丁质合成酶1基因的结构及特性研究

几丁质合成酶1(CHS1)是昆虫几丁质合成的关键酶,在昆虫几丁质合成的组织中发挥着重要作用。为探索白背飞虱Sogota furcifera几丁质合成酶1(Sf CHS1)的基因结构特征及其功能,利用转录组测序结合PCR扩增技术,研究了Sf CHS1基因的结构特性,采用定量PCR技术研究Sf CHS1基因的时空表达特性,最后利用显微注射RNAi方法研究Sf CHS1基因的RNAi效果。通过转录组测序获得的Sf CHS1基因开放阅读框为4 719 bp,编码1572个氨基酸,预测蛋白分子量为180.6 k D,预测有6个糖基化位点和16个跨膜螺旋。PCR扩增及测序结果表明Sf CHS1基因存在两个可变剪切,产生两个转录本(CHS1a和CHS1b)。通过系统进化树分析,Sf CHS1和灰飞虱Laodelphgax striatellus及褐飞虱Nilaparvata lugens的CHS1同源性最高,为97%。时间表达特异性分析结果表明,Sf CHS1基因在4龄期第1天,5龄期第1天,成虫第1天即几丁质合成时的表达量最高。组织特异性检测结果表明,Sf CHS1基因主要在白背飞虱的表皮中表达,其次为气管,在肠中也有少量表达。显微注射RNAi的结果表明该方法能显著降低Sf CHS1基因的转录水平,并导致白背飞虱的死亡。研究结果说明,Sf CHS1基因具有两个可变外显子结构,Sf CHS1基因在几丁质合成阶段及几丁质含量高的组织表达量较高,沉默Sf CHS1基因的表达会对白背飞虱产生致死的表型,出现较高的死亡率。     

家蚕几丁质去乙酰化酶BmCDA2的基因表达和免疫定位

几丁质的去乙酰化修饰与昆虫的发育变态密切相关,几丁质去乙酰化酶(chitin deacetylase,CDA)是这个过程中的关键酶。家蚕(Bombyxmori)是鳞翅目昆虫的代表性昆虫,目前对家蚕CDAs的研究较少。为了更好地揭示BmCDAs对家蚕变态发育的作用,本研究采用生物信息学分析、蛋白表达纯化以及免疫荧光定位等方法对表皮中高量表达的BmCDA2进行了研究。结果发现,BmCDA2有两种mRNA拼接形式BmCDA2a和BmCDA2b,分别在幼虫眠期和化蛹期表皮高量表达,两个基因均有几丁质去乙酰化酶催化结构域(catalyticdomain)、几丁质结合结构域(chitinbinding domain)和低密度脂蛋白受体结构域(low density lipoprotein receptor domain);Western blotting结果显示,该蛋白在表皮存在,荧光免疫定位发现BmCDA2蛋白随着幼虫新表皮的生成而逐渐增多,推测BmCDA2可能参与了幼虫新表皮的形成。该结果丰富了家蚕CDAs的生物学功能信息,也为其他昆虫CDA的研究提供一些有价值的参考。     

小菜蛾几丁质酶基因的克隆及表达分析

昆虫几丁质酶参与昆虫蜕皮、消化、防御及免疫等多种生理过程,在昆虫的变态和发育中发挥着重要作用。本研究采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆获得小菜蛾Plutella xylostella几丁质酶基因,命名为PxyChi。该基因开放阅读框长1677 bp,编码558个氨基酸,推测的蛋白分子量为62.03 kDa。氨基酸序列分析表明,小菜蛾几丁质酶第1-19位氨基酸为蛋白的信号肽,且该序列具有典型昆虫几丁质酶的4个保守氨基酸序列。蛋白结构域分析表明,PxyChi属于几丁质酶Group II家族。进化树分析表明:PxyChi与同属鳞翅目二化螟Chilo suppressalis的几丁质酶亲缘关系最近,为75.5%。不同发育时期的荧光定量PCR分析表明,PxyChi在小菜蛾各个发育时期的表达量不同,其中PxyChi在2、3、4龄幼虫、蛹和成虫中的表达量分别是1龄幼虫的28.22、0.76、0.50、84.83和286倍,PxyChi在成虫的表达量最高。暗示PxyChi蛋白可能参与小菜蛾蛹期旧表皮降解和成虫翅的发育等生理过程。本研究可为探索几丁质酶在小菜蛾发育中的功能提供理论依据。     

棉铃虫Ⅰ型和Ⅶ型几丁质酶基因表达规律的比较分析

几丁质酶（chitinase, CHT）在昆虫生长发育过程中主要有降解围食膜、外骨骼、角质层以及肠道内层的几丁质成分,从而参与昆虫的蜕皮和细胞增殖的功能。在本研究中,分析比较棉铃虫I型和Ⅶ型几丁质酶HaCHTⅠ、HaCHTⅦ的结构域及其时空表达特性,对的进一步功能研究提供重要的素材。首先利用生物信息学在线工具SMART对本课题组前期所获得的几丁质酶基因HaCHTⅠ、HaCHTⅦ的结构域进行分析;之后采用qRT-PCR方法研究棉铃虫6龄幼虫不同组织和幼虫不同龄期蜕皮前后的中肠中HaCHTⅠ、HaCHTⅦ的时空表达特性。结果表明：对几丁质酶HaCHTⅠ、HaCHTⅦ结构域进行分析,发现HaCHTⅠ和HaCHTⅦ均具有信号肽序列、催化区域、连接区域,且HaCHTⅠ有一个几丁质结合结构域,而HaCHTⅦ则无;棉铃虫Helicoverpa armigera几丁质酶基因HaCHTⅠ和HaCHTⅦ在6龄幼虫不同组织部位的均有表达,其中HaCHTⅠ在各个部位的表达量没有显著的差异,而HaCHTⅦ在头壳和体壁中表达显著高于其它组织;几丁质酶基因HaCHTⅠ、HaCHTⅦ在棉铃虫幼虫蜕皮前后中肠的表达量变化较大,其中HaCHTⅠ在3、4、5、6龄末期及蛹期时的表达量呈递减的趋势,在4、5、6龄初期时表达量则是先减后增的趋势,而HaCHTⅦ在3、4、5、6龄末期及蛹期时的表达量呈递增的趋势,恰与同一时期HaCHTⅠ的表达量相反,在4、5、6龄初期时HaCHTⅦ的表达量呈递减的趋势。棉铃虫HaCHTⅠ和HaCHTⅦ的结构域和时空表达都存在差异,据此我们测这些差异可能对其功能具有一定的影响。本结果为深入研究几丁质酶基因HaCHTⅠ和HaCHTⅦ的功能奠定了基础。     

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。     

梨小食心虫幼虫中肠中高表达消化酶和解毒酶基因的筛选

【目的】挖掘梨小食心虫Grapholita molesta幼虫中肠中高表达消化酶和解毒酶基因,为今后研究以肠道为靶标的新型农药和转基因作物提供理论依据。【方法】基于梨小食心虫4龄幼虫中肠转录组高通量测序数据的FPKM值,筛选高表达基因,进行GO功能注释和KEGG通路富集分析,并使用BLAST软件进行比对筛选高表达的消化酶和解毒酶基因,利用MEGA对这些高表达的消化酶和解毒酶及其他鳞翅目昆虫的同源蛋白进行系统发育分析。利用qRT-PCR技术对梨小食心虫幼虫不同龄期中肠中的高表达代表性消化酶和解毒酶基因表达量进行定量分析和验证。【结果】在GO数据库中注释了103 677个在梨小食心虫4龄幼虫中肠中高表达基因,包括细胞组分、分子功能和生物学进程三大类功能共41个分支。KEGG通路分析表明,10 846个高表达基因参与了5类生化代谢通路。筛选到具有完整开放阅读框的消化酶基因17个[5个胰蛋白酶(trypsin, TRY)基因、3个氨肽酶(aminopeptidase, APN)基因和9个羧肽酶(carboxypeptidase, CP)基因]和解毒酶基因32个[11个谷胱甘肽S-转移酶(glu...     

褐飞虱表皮蛋白基因NlICP的克隆及功能研究

表皮蛋白与几丁质结合构成抵御外界不良环境的昆虫角质层, 在昆虫的生长发育及蜕皮硬化中具有重要的作用。为了探讨表皮蛋白基因在褐飞虱Nilaparvata lugens生长发育中的功能, 本研究根据褐飞虱转录组测序信息, 对其中1个预测为编码表皮蛋白的Unigene36450序列进行了克隆, 并应用荧光定量PCR和RNA干扰（RNAi）技术分别对该基因的表达规律和功能进行了研究。结果表明： 克隆的 Unigene36450全长cDNA开放阅读框长585 bp, 编码的蛋白含194个氨基酸, 具有典型的表皮蛋白R&R保守结构域, 命名为NlICP。转录水平的时序表达分析发现, NlICP仅在褐飞虱若虫期表达, 且在3龄若虫体内表达量最高, 提示该基因编码的蛋白属于幼虫表皮蛋白。RNA干扰结果显示, 取食dsNlICP的1龄末2龄初（孵化后第3 天）若虫在干扰6 d和8 d时, NlICP基因的表达量分别较取食dsGFP的对照组下降58.8%和45.6%, 差异极显著(P<0.01); 干扰后部分褐飞虱若虫因蜕皮不完全死亡, 干扰5 d的存活率较对照下降26.7%。本研究结果提示, NlICP与褐飞虱若虫的生长发育及蜕皮相关, 可以作为褐飞虱防治的潜在靶标基因。     

甜菜夜蛾几丁质脱乙酰酶SeCDA7的表达与结合活性分析

几丁质脱乙酰酶(Chitin deacetylase,CDA)是昆虫几丁质代谢酶系中的重要组分,是害虫防治的重要靶标。通过RT-PCR技术克隆得到编码甜菜夜蛾几丁质脱乙酰酶secda7基因(Gen Bank登录号为MG604929),该基因长1 431 bp,包含开放阅读框长1 134 bp,SeCDA7蛋白的预测分子量分别为43.156 k D。结构域分析显示,SeCDA7具有一个多聚糖乙酰基转移酶催化区,属于第Ⅴ类CDA蛋白。分别构建了原核和真核重组表达载体,利用大肠杆菌和Bac-to-Bac昆虫杆状病毒表达系统转染Sf9昆虫细胞,成功表达了SeCDA7蛋白,纯化SeCDA7蛋白并分析几丁质结合活性,结果表明SeCDA7蛋白具有几丁质结合活性;荧光定量PCR结果显示secda7基因主要在中肠组织表达。本研究实现了甜菜夜蛾几丁质脱乙酰酶基因secda7的外源表达,并鉴定出SeCDA7蛋白具有几丁质结合活性,为深入探究甜菜夜蛾几丁质脱乙酰酶的生理功能提供了理论依据。     

昆虫几丁质酶基因的分子特性概述

昆虫几丁质酶是分解昆虫体壁和中肠围食膜几丁质的重要酶类。已从烟草天蛾、家蚕等多种昆虫中分离到几丁质酶的cDNA和DNA序列。昆虫几丁质酶基因有着相似的分子特性,这些特性可为构建杀虫工程菌及转几丁质酶基因植物奠定基础。作者结合自己在该领域的工作,着重就昆虫几丁质酶基因结构特点,基因的拷贝数,基因在体内的时空表达以及异源表达及活性测定等多个方面的研究方法和研究进展进行了较为全面地介绍。     

菜粉蝶osiris基因家族鉴定与发育动态

osiris基因家族是昆虫特异性基因,迄今尚未在昆虫纲以外的物种中发现同源基因。本研究利用菜粉蝶转录组数据,鉴定了菜粉蝶16个osiris基因家族成员,分属11个亚家族。通过与菜粉蝶基因组比对,发现菜粉蝶osiris基因均为断裂基因,外显子数量为3-15个;通过结构域分析,发现菜粉蝶Osiris完整编码蛋白含有信号肽和一个未知功能结构域DUF1676,且多数Osiris蛋白含跨膜结构域。系统发育分析表明,osiris基因家族成员与其他昆虫种类相应成员更似直系同源,而非种内基因扩张,再次验证了osiris基因是在昆虫物种分化之前就已形成的多基因家族。发育转录组基因表达分析表明,osiris家族不同成员表达量在不同发育阶段趋势几乎完全一致,多在菜粉蝶1龄幼虫和5龄幼虫高表达,卵期、蛹期与成虫期低表达,预示着osiris基因家族不同成员转录调控机制的相似性与发育的相关性。     

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.     

Cloning and expression analysis of Chitinase genes from Populus canadensis

Plant chitinases play a key role in conferring resistance to environmental stresses, including attack by fungal pathogens. In the present study, we employed rapid amplification of cDNA ends (RACE) to identify five chitinase genes in Populus canadensis Moench. Sequence alignment revealed that these genes belong to five subfamilies of chitinase genes. The full-length cDNAs of these genes ranged in size from 991 to 1358 bp and encoded proteins with mol wts from 29.5 to 40.3 kD. Five genes were grouped into three major clades based on amino acid sequences of encoded proteins. Exon-intron gene structure and protein domain analysis further supported the designation. A three-dimensional structure comparison showed the high similarity between five P. canadensis chitinases and three well-studied chitinases from other species. The expression levels of all five genes were up-regulated during Populus infection with the pathogenic fungus Marssonina brunnea, and four of them were highly induced by salt and drought stresses. Furthermore, such factors as elicitors, wounding, and low temperature also elevated the expression of these chitinase genes to varying extents. We postulated that these chitinase genes may be involved in pathways of the defense against fungal infection and function in response to various abiotic stresses.     

棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱

【目的】昆虫几丁质酶(chitinase, CHT)主要参与蜕皮、围食膜的降解和机体免疫防御等重要生理生长发育过程。本研究旨在对棉铃虫Helicoverpa armigera Ⅳ型(group)几丁质酶基因进行克隆和表达分析,为以该基因作为棉铃虫防控的分子靶标提供理论依据。【方法】采用RT-PCR和RACE技术从棉铃虫中肠中克隆Ⅳ型几丁质酶基因,分别运用DNAMAN和MEGA软件进行多序列比对和构建系统发育树。在大肠杆菌Escherichia coli(DE3)中诱导表达其体外重组蛋白,利用Western blot进一步验证;用Ni-NTA纯化柱纯化重组蛋白,之后研究该蛋白的酶学性质。qPCR分析该基因的在棉铃虫不同发育阶段和6龄幼虫不同组织中的表达谱。【结果】克隆获得棉铃虫几丁质酶基因HaCHT4(GenBank登录号: MH500771),其cDNA长1 624 bp,ORF长1 527 bp,编码509个氨基酸,预测的分子量为55.2 kD。蛋白质序列的N末端具有信号肽,中间序列部分含有一个催化结构域(catalytic domain, CAD), C末端含有一个几丁质结合结构域(chitin binding domain, CBD)。多序列比对显示,HaCHT4具有几丁质酶的保守区域;系统发育分析表明,HaCHT4属于Ⅳ型几丁质酶。重组蛋白His-HaCHT4在大肠杆菌中成功表达。纯化的重组蛋白对胶体几丁质底物具有降解活性,最适温度和pH分别为50℃和7,动力学参数K_m和V_max值分别为1.76±0.35 mg/mL和0.0220±0.0012 μg/mL·s。qPCR分析表明,HaCHT4在1龄和2龄幼虫期的表达量显著高于其他幼虫龄期及预蛹期;主要在中肠和脂肪体中高度表达,体壁和头部中低表达。【结论】结果提示棉铃虫HaCHT4可能参与围食膜中几丁质降解过程。这些结果为深入研究HaCHT4的功能奠定了基础,并为害虫防治提供了有用的信息。     

A gel diffusion assay for visualization and quantification of chitinase activity

Higher plants, bacteria, fungi, insects, and crustaceans all produce chitinases. Chitinase genes in many organisms are currently under investigation. Chitinase activity is usually assayed with radiolabeled or fluorogenic substrates. We developed a simple, inexpensive, nonradioactive gel-diffusion assay for chitinase that can be used to screen large numbers of samples. In this assay, chitinase diffuses from a small circular well cut in an agarose or agar gel containing the substrate glycol chitin, a soluble, modified form of chitin. Chitinase catalyzes the cleavage of glycol chitin as it diffuses through the gel, leaving a dark, unstained circular zone around the well, because the fluorescent dye calcofluor binds only to undigested chitin. Sample activities can be determined from linear regression of logstandard enzyme concentration versus the zone diameter of internal standards on each Petri dish used for a diffusion assay.