Cloning and Sequencing of a Gene Encoding Aminopeptidase N in the Midgut of Helicoverpa armigera (Hübner)

A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACEtechnique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp inlength, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108.3kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features withaminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18 E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini ishydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accessionnumber is AY181026.     

The changes in digestive enzymes and hormones of gilthead seabream larvae (Sparus aurata, L 1758) fed on Artemia nauplii enriched with free methionine

Variations in digestive enzymes and hormones during the larval development of gilthead seabream (Sparus aurata) fed on live prey (Artemia nauplii) enriched with free methionine were investigated for 16 days (from day 24 to day 40). Prior to initiation of the experiment, newly hatched larvae were transferred from incubators to fiber glass tanks (300 l) with black walls and fed with same diets until day 24. Each experiment was performed in triplicate. In the experimental group, the content of the free methionine in the Artemia nauplii was increased by adding a 5.3 mM free methionine solution to the culture water during a 16-h enrichment period. The larvae of both the control and enriched-methionine groups were sampled four times, with 4-day intervals between samplings, during a 16-day period. The larvae in the control group had a significantly lower growth than those of the methionine group at the end of the study (P < 0.05). The highest trypsin activity and leucine aminopeptidase N/leucine–alanine peptidase ratios were observed in the control group. A significant difference between bombesin activities in the treatment groups was not found at 5th minute after the initiation of feeding (P > 0.05), but they were significant at 15th minute post-initiation of feeding (P < 0.05). A significant difference between the cholecystokinin levels of the treatment groups was found (P < 0.05).     

美国白蛾氨肽酶N的基因克隆表达及与苏云金杆菌3种毒素蛋白的结合特性分析

美国白蛾(Hyphantria cunea)是桑树的重要害虫。为明确苏云金杆菌(Bacillus thuringiensis)杀虫晶体蛋白对该虫的作用机制,利用已知昆虫氨肽酶N基因序列设计引物,以美国白蛾中肠cDNA为模板,利用RACE-PCR技术获得美国白蛾中肠氨肽酶N基因hcapn1全长序列(GenBank登录号:KP053647)。该基因开放阅读框为3 000 bp,编码999个氨基酸,预测蛋白质的分子质量和等电点分别为113.14 kD和4.85。设计去信号肽引物PCR扩增获得hcapn1基因序列,构建原核重组表达载体pET30a-hcapn1,诱导表达的HcAPN1重组蛋白约110 kD。将苏云金杆菌Cry1Ac、Cry2Ab33和Cry9Ea6原毒素经胰蛋白酶消化后与HcAPN1重组蛋白分别进行体外配体印迹试验,发现HcAPN1重组蛋白与Cry9Ea6结合,而不与Cry2Ab33、Cry1Ac发生特异结合,推测HcAPN1可能为Cry9Ea6在美国白蛾中肠的受体蛋白。分别设计引物扩增HcAPN1蛋白2个结构域Asp₃₄～Asp_( 527)和Leu₅₆₃～Gln₉₂₅的编码序列,在大肠埃希菌中表达获得62 kD和48 kD 2种重组蛋白,体外结合试验显示Cry9Ea6与HcAPN1的结合发生在Leu₅₆₃～Gln₉₂₅之间。研究结果为深入理解苏云金杆菌Cry9Ea6蛋白对美国白蛾的杀虫机制提供了基础数据。     

CrylAb敏感和抗性亚洲玉米螟氨肽酶N基因的克隆及序列差异分析

研究亚洲玉米螟(Ostrinia furnacalis)对Cry1Ab产生抗性的分子机理,对今后制定和实施合理的抗性治理策略具有重要的意义。本研究通过PCR方法克隆和测序,鉴定了Cry1Ab敏感-抗性亚洲玉米螟幼虫中肠氨肽酶N(APN)基因家族的一个成员Ofapn3,其cDNA全序列长3591bp,开放阅读框包括3045bp,编码1014个氨基酸。预测蛋白质分子量为115.1kD,等电点(pI)为4.44。其推导的氨基酸序列中具有鳞翅目昆虫氨肽酶典型结构特征,即N-末端具有18个氨基酸的剪切信号肽,谷氨酸锌化氨肽酶保守结构GAMEN,锌结合位点HEXXHX18E,C-末端具有22个氨基酸的糖基磷脂酰肌醇(GPI)锚信号肽。系统分类归为第3支系。与欧洲玉米螟(Ostrinia nubilalis)的Onapn3(GenBank登录号:ABL01483)同源性为96.6%。与Cry1Ab敏感亚洲玉米螟cDNA相比,抗性品系的开放阅读框中有40个碱基发生了点突变,导致氨基酸序列中有10个氨基酸改变。其中Ser735在抗性品系中突变为Pro的现象在抗性棉铃虫APN3的氨基酸突变中也被检测到。鉴定的Cry1Ab敏感和抗...     

梅山猪氨肽酶基因N(pAPN)cDNA克隆、序列分析及重要变异位点的筛选

氨肽酶N(aminopeptidase N,APN)蛋白是猪(Sus scrofa)传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)和猪的流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)等一系列冠状病毒的受体蛋白。为探讨梅山猪APN基因的分子结构特征以及重要的变异位点,本研究通过PCR扩增和测序技术,结合生物信息学分析梅山猪APN基因功能区域和重要变异位点,对其蛋白质结构进行预测和分析。结果表明,梅山猪APN基因cDNA全长2 886 bp(GenBank登录号:KF280271),含有20个外显子,编码961个氨基酸。与GenBank数据库公布的标准序列(登录号:NM_214277.1)相比,梅山猪APN基因编码区变异共引起10处氨基酸突变与2处氨基酸缺失,其中Phe82Asn、Leu107Phe、Leu108 Ile、Ser330Pro、Trp399Arg和Glu465 Gly等6处突变位于APN酶催化活性区域,Gln747His突变、748Tyr和749Ser缺失突变位于APN病毒结合区域。蛋白结构和编码产物功能分析发现,pAPN为不稳定的亲水性蛋白,无信号肽,具有1个跨膜螺旋(跨膜区位于12~34氨基酸,二级结构元件以α螺旋和β折叠为主,编码产物主要参与细胞被膜(cell envelope)、中央中间代谢(central intermediary metabolism)、辅酶因子生物合成(biosynthesis of cofactors)等功能。Gln747His、748Tyr和749Ser缺失突变可能与氨肽酶N结合病毒能力有关。本研究对梅山猪pAPN基因功能的分析及变异位点的筛选,为今后筛选猪抗病毒性腹泻的有效遗传标记提供理论基础。     

目标昆虫对Bt杀虫晶体蛋白抗性的研究概况

综述了多种目标昆虫对Bt杀虫晶体蛋白的抗性机制和遗传特性。     

抗转Bt基因棉棉铃虫幼虫中肠氨肽酶N基因的克隆与序列特征

通过对抗性和敏感棉铃虫中肠氨肽酶的克隆和测序,鉴定了氨肽酶基因家族的2个成员:Haapn3和Haapn4,其cDNA序列具有3053和2862个核苷酸,分别具有3045和2853bp的开放阅读框,编码产生1014和951个氨基酸的蛋白质。其推定氨基酸序列均具有锌结合模体HEXXH和所有锌依赖氨肽酶共有的序列GAMEN。N 末端分别具有18和20个氨基酸的疏水性信号肽,C 末端均具有21个氨基酸的GPI添加信号肽。比对抗性和敏感棉铃虫cDNA的开放阅读框,在Haapn3的开放阅读框中,抗性品系的cDNA具有25个核苷酸的不同,其中9个碱基替代造成氨基酸的变化:苏氨酸189→天冬酰胺、甘氨酸229→天冬氨酸、赖氨酸231→精氨酸、谷氨酸250→丙氨酸、异亮氨酸275→缬氨酸、天冬酰胺281→天冬氨酸、酪氨酸302→组氨酸、缬氨酸648→异亮氨酸、脯氨酸975→谷氨酰胺,其它16个碱基替代为无义突变。在Haapn4的开放阅读框中,有15个点突变,其中8个突变为无义突变,其它7个导致了氨基酸的改变,即赖氨酸4→天冬酰胺、组氨酸162→酪氨酸、脯氨酸185→丝氨酸、谷氨酸297→甘氨酸、苯丙氨酸385→丝氨酸、甲硫氨酸450→异亮氨酸、缬氨酸630→异亮氨酸。本文报道的氨肽酶4个cDNA序列已提交GenBank,AY279536和AY279537分别是抗性和敏感品系的Haapn3,AF511038和AY279534分别是     

Non-destructive Limits to Seed Growth and Leaf Protease Activities in Nodulating and Non-nodulating Soybean Isolines

Leaf senescence leads to a progressive decline in the photosynthetic competence of the leaf. This paper describes some effects of source:sink imbalance on leaf protein catabolism and senescence in soybean. We manipulated pod growth by restricting 100 or 50 % (PR-100 or PR-50, respectively) of young pods at the R4 stage in plastic drinking straws. This effectively reduces final seed mass without interrupting the vascular connections of pods. Nodulating (NOD+) and non-nodulating (NOD−) isolines of the 'Clay' soybean were grown in drainage lysimeters and three pod-restriction (PR) treatments were compared. Pod restriction decreased seed biomass per plant as a result of lower individual seed mass, which was only partially balanced by the increase in seed number. The nitrogen concentration in seeds remained unchanged in NOD+ plants, while it increased with the degree of sink restriction in seeds of NOD− plants. Leaf soluble protein, CO₂ exchange rate and seed nitrogen content were consistently lower in NOD− plants; the leaf protein level remained stable with time in PR-100 plants, decreased for PR-50 and dropped for controls. Endoprotease (HBase) and carboxypeptidase (CPase) activities were significantly lower in leaves from PR-100 plants, while aminopeptidase activity was enhanced, indicating a de novo synthesis of leaf protein. This is consistent with the reported accumulation of vegetative storage proteins (VSPs) in soybean and other legumes after moderate or severe sink reduction. Thus, small modifications of the source:sink ratio such as those obtained by the non-destructive PR technique have an impact on leaf protein catabolism. Nodulating and non-nodulating soybean isolines showed similar responses to PR in terms of leaf senescence initiation and progression, but the rate of the processes appear to be largely influenced by plant N status.     

巴西橡胶树HbEBP1生物学特性、原核表达及氨肽酶活性分析

从巴西橡胶树中筛选到了Hb EBP1基因,该基因序列长度为1 462 bp,其中5′端非编码区长度为141 bp,3′端非编码区长度为133 bp,其c DNA序列具有完整的阅读框,编码395个氨基酸,推导的Hb EBP1分子量为43.596 ku,等电点位为6.45。生物信息学分析结果表明巴西橡胶树Hb EBP1具有3个保守结构域:增值相关蛋白2G4(PA2G4-like)、氨肽酶M24(APP_Met AP)、甲硫氨酸氨肽酶(MAP)结构域。对原核表达的Hb EBP1蛋白进行氨肽酶活性测定,发现体外表达的Hb EBP1蛋白没有Map活性。这些结果表明,橡胶树Hb EBP1基因并不存在氨肽酶活性。     

昆虫中肠Bt毒素受体氨肽酶N的研究进展

氨肽酶N(APN)是昆虫中肠中主要的Bt毒素受体,它与Cry毒素特异结合后,毒素插入细胞膜,在膜上形成孔洞,细胞裂解,最终导致昆虫死亡.APN的变异导致昆虫对Bt敏感性下降甚至产生抗性.就APN的结构特征、分类、APN与Cry毒素的相互作用机制以及APN与昆虫Bt抗性的关系作一综述.