稻纵卷叶螟海藻糖酶基因的克隆、分子特性和表达分析

【目的】海藻糖酶(trehalase,Tre)是昆虫体内海藻糖代谢的一个关键酶,包括可溶型(Tre1)和膜结合型(Tre2)两种类型,在昆虫发育和能量调节中具有重要作用。本研究旨在克隆稻纵卷叶螟Cnaphalocrocis medinalis海藻糖酶基因(Cm Tre),解析其在稻纵卷叶螟不同发育阶段和不同组织中的表达模式,分析该基因及酶蛋白的分子特征。【方法】通过稻纵卷叶螟转录组数据结合RACE技术,克隆稻纵卷叶螟Cm Tre的全长c DNA序列,并对该基因进行生物信息学分析;采用实时荧光定量PCR(RT-q PCR)解析Cm Tre在稻纵卷叶螟不同发育阶段及成虫不同组织部位的m RNA表达模式。【结果】获得两种类型的稻纵卷叶螟Cm Tre基因,即可溶型海藻糖酶基因Cm Tre1和膜结合型海藻糖酶基因Cm Tre2。Cm Tre1的全长c DNA长度为2 364 bp,开放阅读框长1 704 bp,编码567个氨基酸;Cm Tre2的全长c DNA长度为2 079 bp,开放阅读框长1 923 bp,编码640个氨基酸。生物信息学分析表明,Cm Tre前端有一个信号肽,Cm Tre1无跨膜结构,Cm Tre2有一个跨膜结构。同源性和聚类分析表明,Cm Tre1和Cm Tre2的氨基酸序列与竹蠹螟Omphisa fuscidentalis海藻糖酶Tre1和Tre2氨基酸序列的一致性最高,分别为74%和79%。同源建模预测结果显示,Cm Tre1的三维分子结构包含19个α螺旋和2个β折叠片;Cm Tre2的三维分子结构含有23个α螺旋,没有β折叠片。RT-q PCR结果显示,Cm Tre在稻纵卷叶螟整个发育历期都有表达,在成虫期表达水平最高,在整个幼虫期都有相对稳定的表达;Cm Tre1在蛹期表达水平最低,Cm Tre2在5龄幼虫时期表达水平最低。Cm Tre在所检测的成虫组织(中肠、体壁、马氏管、头、卵巢、脂肪体、肌肉和精巢)中均有表达;Cm Tre1在中肠和体壁中的表达水平较高,Cm Tre2在肌肉和中肠中的表达水平较高。【结论】本研究克隆了稻纵卷叶螟两种类型的海藻糖酶基因,分析了其基因特征和表达模式。研究结果为阐明海藻糖酶基因的功能进而以海藻糖酶基因为靶标防治害虫奠定了基础。     

亚洲玉米螟几丁质合成酶B(CHSB)启动子序列克隆与分析

昆虫几丁质合成酶是昆虫蜕皮和变态发育过程中几丁质生物合成的关键酶,同时也是环境友好杀虫剂的理想靶标。昆虫几丁质合成酶可分为两类,即A类和B类。其中A类几丁质合成酶(CHSA)主要合成昆虫表皮的几丁质,而B类几丁质合酶(CHSB)在围食膜的形成阶段表达。从亚洲玉米螟3龄幼虫体内提取基因组DNA,利用染色体步移获得了CHSB完整的启动子序列。该DNA片段长度为1484 bp,分析显示该片段为Of CHSB基因5'端侧翼序列,转录起始位点从+1开始,Of CHSB的开放阅读框从+348至+402,分析表明6种类似的转录因子(GATA-1、C/EBPalpha、Oct-1、Dfd、CREB、ER)可能参与Of CHSB的转录调控。转录因子结合位点分别于+116至+127(CREB)、+120至+131(ER)和+292至+302(Oct-1)的区域可能是该启动子的核心顺式元件。     

朱砂叶螨几丁质合成酶基因1全长cDNA的克隆与表达分析

【目的】克隆朱砂叶螨Tetranychus cinnabarinus几丁质合成过程中的关键酶几丁质合成酶基因,并检测该基因在朱砂叶螨生长发育不同阶段的相对表达量。【方法】本研究采用逆转录聚合酶链反应(RT-PCR)以及c DNA末端快速扩增(RACE)技术首次克隆获得朱砂叶螨几丁质合成酶基因1的全长c DNA序列(命名为Tc CHS1,Gen Bank登录号为KM242062),并使用实时荧光定量PCR技术首次检测了Tc CHS1基因在朱砂叶螨生长发育不同阶段的相对表达量。【结果】朱砂叶螨Tc CHS1基因的c DNA全长为4 881 bp,包括198 bp的5'非翻译区(5'-UTR),4 425 bp的开放阅读框(ORF),258 bp的3'非翻译区(3'-UTR),开放阅读框编码1 474个氨基酸,预测其蛋白质分子质量约为168.35 ku,理论等电点为6.26。其包含EDR和QRRRW这2个几丁质合成酶基因的标签序列。氨基酸序列同源性分析结果表明:Tc CHS1与其他昆虫该基因编码蛋白的氨基酸序列相似度在50%左右,与二斑叶螨Tetranychus urticae的氨基酸相似度最高(98%),与西方盲走螨Metaseiulus occidentalis的相似度为55%。分子系统进化的结果也表明Tc CHS1与其他昆虫的CHS1聚在一起,并且和二斑叶螨具有最近的亲缘关系。荧光定量分析表明Tc CHS1基因在朱砂叶螨生长发育的不同阶段(卵、幼螨、第1若螨、第2若螨、雌成螨和雄成螨)均有表达,在卵和雌成螨中的表达量较高,在第2若螨的表达量最低。【结论】Tc CHS1基因可能在朱砂叶螨生长发育过程中具有重要作用。     

小麦条锈菌II类几丁质合成酶基因PstChsII的克隆及表达特征

摘要:【目的】克隆小麦条锈菌几丁质合成酶基因PstChsII,分析其在小麦条锈菌不同发育时期的表达水平。【方法】利用RT-PCR和PCR技术克隆PstChsII的cDNA序列和基因组序列,利用不同的生物信息学软件对序列进行分析,运用实时荧光定量技术分析基因在孢子、芽管以及不同侵染时间的表达水平。【结果】PstChsII基因（Genbank登录号GQ329851）编码区存在15个内含子,开放阅读框长2727 bp,编码908个氨基酸。PstChsII蛋白C端含有7个跨膜螺旋区,N端含多个保守结构域和“QXR     

稻纵卷叶螟氨肽酶N基因的克隆及时空表达分析

【目的】昆虫中肠氨肽酶N(Aminopeptidase N,APN)是Bt杀虫蛋白的重要受体之一,与Bt蛋白的杀虫机制及昆虫对Bt蛋白的抗性密切相关。为阐明稻纵卷叶螟Cnaphalocrocis medinalis(Guenee)APN基因的功能及明确Bt蛋白对稻纵卷叶螟的毒力机制,本研究系统开展了稻纵卷叶螟中肠APN基因的克隆及时空表达分析。【方法】通过简并引物PCR结合RACE技术克隆并获得4条稻纵卷叶螟APN基因的cDNA序列全长,采用实时定量PCR技术研究了APN基因在稻纵卷叶螟不同虫态及幼虫不同组织中的时空表达情况。【结果】经NCBI同源比对分析,认为这4个基因分属于。4PN基因家族的不同类别,分别将其命名为CmAPN1(GenBank登录号:HQ853294)、CmAPN2(GenBank登录号:HQ853295)、CmAPN3(GenBank登录号:KJ143755)、CmAPN4(GenBank登录号:HQ853296)。序列分析表明,CmAPN1-4 cDNA序列全长分别为:3 698、3 478、3 150和3 149 bp,开放阅读框分别为:3 045、2 877、3 045和2 862 bp,分别编码965、958、1 014和952个氨基酸。其推导的氨基酸序列具有鳞翅目昆虫氨肽酶N的典型结构特征,即含有糖基化位点、N-末端信号肽序列、谷氨酸锌化氨肽酶保守结构GAMEN、锌结合位点HEX_2HX_(18)E、C-末端糖基磷脂酰肌醇(GPI)锚信号肽。实时定量研究表明,CmAPNs在幼虫中的表达量显著高于卵、蛹和成虫;在幼虫中,CmAPN7的表达水平明显低于CmAPN22-4,且其在不同龄期中的表达差异显著;CmAPN2-4的表达量随幼虫龄期的增加而增加;CmAPNs在幼虫肠道组织中的表达量显著高于其它组织器官,且CmAPN1和CmAPN2分别在中肠和后肠中呈现高水平表达,CmAPN3在前、中肠内均高水平表达;但CmAPN4在各个组织器官中的表达均保持较低水平。【结论】CmAPNs基因在稻纵卷叶螟的不同虫态和不同组织中呈现了差异显著的时空表达,采用RNA干扰方法进行CmAPNs基因功能研究时,要选择适宜的虫态和虫龄进行干扰。     

蜕皮激素和有效霉素对粘虫几丁质合成酶B基因表达的影响

【目的】克隆粘虫Mythimnaseparata几丁质合成酶B基因的全长cDNA序列,研究该基因的时空表达特性,分析蜕皮激素(20-hydroxy ecdysone, 20 E)和有效霉素(Validamycin)对该基因表达水平的影响。【方法】本试验通过高通量测序法获得粘虫几丁质合成酶B基因的cDNA全长序列,利用RT-qPCR技术分析粘虫几丁质合成酶B基因在不同发育阶段和不同组织的特异性表达及蜕皮激素和有效霉素对其表达的影响。【结果】基因cDNA全长4 617 bp,包含一个完整开放阅读框,编码1个1 538个氨基酸组成的多肽,分子量为175.629 ku,理论等电点为5.96,包含17个跨膜螺旋,4个几丁质合成酶的标签序列CATMWHET,DGD,EDR和QRRRW及1个催化结构域。该基因命名为MsCHSB,GenBank登录号为KY348776。氨基酸序列比对表明,该基因与其他昆虫的几丁质合成酶B基因同源性高于52%,其中与蓓带夜蛾Mamestra configurata和棉铃虫Helicoverpa armigera的几丁质合成酶B基因同源性最高,分别为92%和83%。RT-qPCR技术表明粘虫在不同发育阶段和组织中均有mRNA的特异性表达,其中3龄第1天和中肠中MsCHSB基因相对表达量最高。注射10μg/μL浓度的蜕皮激素6 h和12 h后,表现为对该基因的诱导效应,与对照组差异显著;有效霉素处理后该基因相对表达量均被显著抑制,其中注射20μg/μL浓度的有效霉素48h后,抑制作用最为明显。【结论】本试验得到了一条新的粘虫几丁质合成酶B基因cDNA序列全长。蜕皮激素对MsCHSB基因的表达有一定的诱导作用,有效霉素对MsCHSB基因的表达有一定的抑制作用,该结果为进一步研究昆虫几丁质合成酶B打下了基础。     

梨小食心虫几丁质合成酶2基因的克隆与表达研究

【目的】研究梨小食心虫Grapholitha molesta(Busck)几丁质合成酶2(GmCHS2)基因序列和表达特性,为进一步研究其功能及新型杀虫剂的开发提供理论依据。【方法】利用转录组数据和RACE技术首次获得GmCHS2基因cDNA序列(命名为GmCHS2,GenBank登录号为KY242360),利用其他昆虫同源序列构建系统发育树,利用实时荧光定量PCR技术研究GmCHS2在不同发育阶段和不同组织的表达特性。【结果】GmCHS2基因序列全长4991bp,开放阅读框为4554bp,其中5′端非编码区长267bp,3′端非编码区长170bp,共编码1517个氨基酸,包含了14个跨膜螺旋。系统发育树结果表明该基因属于几丁质合成酶2类基因。GmCHS2基因在试虫不同发育阶段都有表达,预蛹期和成虫期表达量最高;不同组织中,前肠和中肠的表达量最高,其次是后肠,其余组织表达量很少或不表达。【结论】本文研究了GmCHS2基因在梨小食心虫不同发育阶段和组织部位的表达特性,该基因可能在梨小食心虫生长发育过程中具有重要作用。     

金针菇几丁质合成酶基因家族鉴定及其表达分析

【背景】几丁质是真菌细胞壁的重要成分,由几丁质合成酶（chitin synthase,CS）催化合成。几丁质合成酶编码基因在大型食用真菌金针菇中的数量及表达规律尚不明确。【目的】探究几丁质合成酶基因在金针菇中存在的数量及其在子实体不同发育时期的表达规律,为其在大型真菌子实体生长发育过程中的功能研究提供基础。【方法】基于已有的金针菇菌株L11基因组数据,结合NCBI其他真菌CS序列鉴定金针菇中几丁质合成酶编码基因的数量,并对其进行生物信息学分析。进一步根据金针菇F19转录组数据以及实时荧光定量PCR （RT-qPCR）技术分析金针菇CS基因家族的表达规律。【结果】在金针菇单核体菌株L11的基因组中鉴定到9个几丁质合成酶基因,系统发育分析表明它们在子实体发育过程中的表达模式可分为4类（皮尔森相关系数=0.85）。【结论】金针菇CS基因家族表达模式在金针菇不同生长发育时期均存在差异,可能参与了子实体发育不同时期和组织的形态建成。     

稻纵卷叶螟羧酸酯酶基因的鉴定与表达谱分析

【目的】鉴定稻纵卷叶螟Cnaphalocrocis medinalis的羧酸酯酶基因,并检测这些基因在成虫不同组织中的表达模式。【方法】从稻纵卷叶螟转录组中搜索羧酸酯酶基因,使用生物信息学软件对所获得的基因序列进行分析,使用荧光定量PCR检测这些基因在各组织中的相对表达水平。【结果】获得了15个羧酸酯酶基因,分别命名为Cm Car E1~Cm Car E15。其中Cm Car E12缺少3′区域,其余14个序列均含有完整的开放阅读框。除Cm Car E11外,其余14个基因编码的蛋白均具有羧酸酯酶的典型特征,如保守的五肽结构域,催化三联体和氧阴离子穴等。系统进化分析显示15个Cm Car Es被聚在不同的进化支内,Cm Car E13、Cm Car E14和Cm Car E15聚在"胞内催化类"进化支,其余12个Cm Car Es聚在"分泌催化类"进化支。Cm Car E1、Cm Car E2、Cm Car E3、Cm Car E7、Cm Car E8、Cm Car E10和Cm Car E13特异表达于成虫腹部,而Cm Car E9特异表达于雌雄成虫触角。其他基因的表达没有组织特异性。【结论】Cm Car E1、Cm Car E2、Cm Car E3、Cm Car E7、Cm Car E8、Cm Car E10和Cm Car E13编码的酯酶可能参与了内外源化合物的代谢,而Cm Car E9编码的酯酶可能参与了气味分子的降解。     

德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析

【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 Blattella germanica TPS基因,研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列,利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因,分别命名为 BgTPS1 (GenBank登录号：KR050213) 和 BgTPS2 (GenBank登录号：KR050214)。其中,BgTPS1基因cDNA序列全长2 987 bp,开放阅读框 (ORF) 2 502 bp,编码833个氨基酸;BgTPS2基因cDNA序列全长3 212 bp,开放阅读框2 469 bp,编码822个氨基酸。BgTPS1和BgTPS2基因都主要在5龄幼虫脂肪体中表达,且BgTPS2基因的表达量为BgTPS1基因表达量的3.9倍。在两种不同极端温度诱导下,BgTPS1和BgTPS2基因mRNA均上调表达。其中,BgTPS2 的表达量始终显著高于 BgTPS1。在0℃时,BgTPS1和BgTPS2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达,且BgTPS2基因的表达量显著高于BgTPS1基因;低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。     

Light-scattering and infrared-spectrophotometric studies of chitin and chitin derivatives

A sample of chitin isolated from the shell of the crab Scylla serrata had, when in lithium thiocyanate solution, an average, weight-average molecular weight (1) of 1.036 x 10⁶ daltons, an intrinsic dissymmetry (2) of 1.93, and a Z-average radius of gyration (3) of 64.14 nm. Carboxymethylchitin and glycol chitin, in 0.5M sodium chloride, had, respectively, (1) 1.896 and 1.819 x 10⁶ daltons, (2) 3.25 and 4.31. and (3) 143.49 and 251.57 nm. They had similar degrees of polymerization, they underwent dissociation as the concentration of sodium chloride was increased to 2.5M, and the molecular packing of the chains was essentially side-by-side. Chitin in 5.55M lithium thiocyanate and carboxymethylchitin in 2.5M sodium chloride had similar degrees of polymerization. It is concluded that a small but significient number of the amino groups in the chitin molecule are not acetylated.     

Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles

The enzymatic deacetylation of various chitin preparations was investigated using the fungal chitin deacetylase (CDA) isolated from Rhizopus oryzae growth medium. Specific extracellular enzyme activity after solid state fermentation was 10 times higher than that after submerged fermentation. Natural crystalline chitin is a very poor substrate for the enzyme, but showed a five-time better deacetylation after dissolution and reprecipitation. Chitin particles, enzymatically deacetylated for only 1% exhibited a strongly increased binding capacity towards ovalbumin, while maintaining the rigidity and insolubility of chitin in a moderate acidic environment. Because of the unique combination of properties, these CDA treated chitin materials were named "chit-in-osan". Chitinosan was shown to be an attractive matrix for column chromatography because no hydrogel formation was observed, that impaired the flow of eluent. Under the same conditions, partially deacetylated chitosan swelled and blocked the flow in the column.     

TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan

A commercial chitin, regenerated chitin prepared from chitin solutions in 6.8% NaOH and N-acetylated chitosans with degrees of N-acetylation (DNAc) of 77–93% were subjected to oxidization in water with NaClO and catalytic amounts of 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) and NaBr. When regenerated chitin with DNAc of 87% and N-acetylated chitosan with DNAc of 93% were used as starting materials, water-soluble β-1,4-linked poly-N-acetylglucosaminuronic acid (chitouronic acid) Na salts with degrees of polymerization of ca. 300 were obtained quantitatively within 70 min. On the other hand, the original chitin and N-acetylated chitosan with DNAc of 77% did not give water-soluble products, owing to incomplete oxidation. The high crystallinity of the original chitin brought about low reactivity, and the high C2-amino group content of the N-acetylated chitosan with DNAc of 77% led to degradations rather than the selective oxidation at the C6 hydroxyls. The obtained chitouronic acid had low viscosities in water, and clear biodegradability by soil microorganisms.     

Shrimp chitin as substrate for fungal chitin deacetylase

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.     

Studies on chitin. 2. Preparation and properties of chitin membranes

A chitin membrane was prepared by a new procedure involving coagulation of the chitin solution in N,N-dimethyl acetamide, N-methyl 2-pyrrolidone and lithium chloride (DMA-NMP-LiCl) with 2-propanol. The solute permeability, water sorption and mechanical properties were compared with membranes prepared by two previously reported methods (coagulation of a formic acid and dichloroacetic acid (FA-DCA) solution of chitin with 2-propanol; and coagulation of a trichloroacetic acid and dichloroethane (TCA-DCE) solution of chitin with acetone). The permeability coefficients of the three chitin membranes were higher than a regenerated cellulose membrane (Cuprophane®). The membrane prepared from DMA-NMP-LiCl solution had a higher tensile strength (3·3 Mpa) in the wet state than the others. The membrane obtained from TCA-DCE solution absorbed more water (360%) and the membrane prepared from FA-DCA solution was relatively weak (1·8 MPa) in the wet state. It was suggested that 2-propanol was a favourable coagulant for membrane production. In addition, the effect of the origin of chitin on molecular weight and tensile properties of the membranes was studied.     

Separation of C-glycoside flavonoids from Aleurites moluccana using chitin and full N-acetylated chitin

This paper describes a comparative study using different chromatographic supports (fully N-acetylated chitin, chitin and silica gel) to separate the flavonoids swertisin and 2"-O-rhamnosylswertisin from Aleurites moluccana. The results show that the flavonoids have apparently been separated by the hydrogen bond between the stationary phase (chitin and chitin-100) and flavonoids under the conditions studied.