苹果蠹蛾瞬时感受器离子通道基因的克隆及温度胁迫表达分析

瞬时感受器离子通道(transient receptor potential,TRP)是影响昆虫温度感知系统的关键组成。为探讨苹果蠹蛾温度感知和温度适应的机理,本研究以苹果蠹蛾Cydia pomonella为研究对象,通过分子克隆获得TRPA家族中的CpPainless和CpWater_witch基因,进行生物信息学分析,并利用实时荧光定量PCR技术分析靶基因在高低温胁迫后的表达。结果表明,CpPainless基因编码938个氨基酸,N端有8个锚蛋白重复序列。CpWater_witch基因编码980个氨基酸,N端有10个锚蛋白重复序列,二者编码产物均有6个跨膜结构,具瞬时感受器离子通道家族成员结构典型特征。表达分析结果显示,和对照26℃相比,高低温胁迫1 h后,CpPainless在5龄幼虫雌虫体内的表达量显著下调,而5龄幼虫雄虫经低温胁迫1 h后CpPainless表达量显著上调,但高温胁迫1 h后表达差异不显著;CpWater_witch在雌雄虫体内均没有显著变化。研究结果为研究瞬时感受器离子通道在苹果蠹蛾温度感知中的作用奠定基础。     

桃蚜电压门控钠离子通道基因cDNA克隆及其生物信息学分析

克隆获得桃蚜电压门控钠离子通道基因cDNA序列,明确钠离子通道的典型特征,为研究桃蚜抗性分子机理奠定基础。采用实验技术主要有RT-PCR和PCR,克隆桃蚜钠离子通道基因cDNA序列,利用相关软件对其序列进行生物信息学分析。克隆得到两段cDNA序列MpNa_v-1（NCBI登录号：MN124170）和MpNa_v-2（NCBI登录号：MN176136）。MpNa_v-1长度为2945 bp,包括2877 bp的完整开放阅读框,共编码958个氨基酸;MpNa_v-2长度为3546 bp,包括3486 bp的完整开放阅读框,共编码1161个氨基酸。MpNa_v-1和MpNa_v-2共同组成桃蚜的钠离子通道α亚基,MpNa_v-1包含同源结构域Ⅰ和同源结构域Ⅱ,MpNa_v-2包含同源结构域Ⅲ和同源结构域Ⅳ。同源比对发现,桃蚜与豌豆蚜和高粱蚜钠离子通道基因相似度分别高达97.67%和97.65%,所克隆序列包含昆虫钠离子通道α亚基典型特征,具有MFM模块,并含有蚜虫类钠通道特有模块DENS。成功地克隆桃蚜钠离子通道基因,为阐明其对拟除虫菊酯类药剂产生靶标抗性的分子机制奠定基础。     

国槐凝集素基因的克隆及其功能

植物凝集素是一类能高度特异可逆结合到单糖或寡糖上的蛋白,含有至少一个非催化结构域,近年来在农作物抗虫工程中得到了广泛关注。本文从豆科植物国槐中克隆得到了一个新的凝集素基因SjLectin并初步研究了其对小菜蛾的抗性功能。从国槐幼叶中提取总RNA,采用RT-PCR法分离克隆出SjLectin基因的cDNA片段,该基因在GenBank中登录号为KC140286.1。SjLectin基因全长837 bp,编码279个氨基酸组成的蛋白。该蛋白与其他豆科凝集素的同源性均高达66%以上,属于豆科凝集素家族中的一员。在35S启动子控制下,利用根癌农杆菌介导法转化烟草品种W38,获得了该基因的抗卡那霉素植株。通过PCR和RT-PCR法筛选阳性转基因植株,证明SjLectin基因已经转化到烟草基因组中并在转录水平上得到了有效表达。接虫试验证明,阳性植株对小菜蛾的抑制率平均达62.2%。     

禾谷缢管蚜水通道蛋白基因RpAQP1的克隆、分子特性和表达分析

为探究禾谷缢管蚜Rhopalosiphum padi (Linnaeus)水通道蛋白基因RpAQP1的序列特征及其在不同龄期的表达,利用RT-PCR和RACE技术克隆了RpAQP1基因的cDNA全序列,采用生物信息学软件分析了RpAQP1编码蛋白的特性,利用qRT-PCR分析了RpAQP1在不同龄期的表达量。结果显示:禾谷缢管蚜水通道蛋白基因RpAQP1的cDNA全长为1 216 bp,其750 bp的开放阅读框编码250个氨基酸,蛋白分子量为27.36 kD。RpAQP1属于水通道蛋白亚家族DRIP(果蝇内嵌蛋白Drosophila integral protein)的一员,具有6个跨膜区,2个保守的NPA结构单元和1个压缩区域Ar/R;qRT-PCR结果显示,RpAQP1在整个发育历期均有表达,其在2龄若蚜中表达水平最高,是成蚜表达量的1.432倍;在4龄若蚜中表达量最低,为成蚜表达量的0.444倍,显著低于其它龄期,而其余各龄期RpAQP1表达量无显著差异。     

福寿螺18S rRNA和28S rRNA基因片段的克隆与进化分析

为从分子水平上明确入侵我国的福寿螺在分类学上的地位,采用分子克隆和序列比对的方法,对来自菲律宾及我国广东、广西、浙江等不同地理种群福寿螺的18S rRNA基因和28S rRNA基因片段进行扩增、克隆和序列测定,并同瓶螺科、田螺科和环口螺科相关物种进行系统发育分析。结果表明,获得的福寿螺18S rRNA基因和28S rRNA基因片段长度分别为602 bp、325 bp,且不同地理种群间碱基序列无差异。通过邻接法（NJ）和最大简约法（MP）构建的系统树基本一致,证实福寿螺隶属于瓶螺科,与田螺科物种亲缘关系较近,而与环口螺科亲缘关系较远。     

苹果类甜味蛋白编码基因Mdtl2克隆及功能的初步验证

植物类甜味蛋白(Thaumatin like proteins, TLPs)是一类具有抗菌活性和抗非生物胁迫作用的病程相关蛋白。本研究克隆获得富士苹果(Malus domestica cv. Fuji)TLPs家族蛋白Mdtl2编码基因,该基因开放阅读框全长为912 bp,编码303个氨基酸。生物信息学分析显示Mdtl2含有多个保守的半胱氨酸及TLPs家族保守序列片段,且具有TLPs典型的三维结构,属于TLPs家族的糖苷水解酶64家族(GH64-TLP-SF Superfamily)。在烟草中过表达Mdtl2能够显著抑制疫霉(Phytophthora infestans)的侵染。在苹果中过表达Mdtl2显著抑制苹果树腐烂病菌(Valsa mali)的侵染,并且能够诱导胼胝质的积累。上述结果表明,Mdtl2作为糖苷水解酶家族蛋白,能够通过诱导胼胝质的积累,提高植物对病原菌的抗性。     

异色瓢虫乳酸脱氢酶基因的克隆与序列分析

乳酸脱氢酶（1acticdehydrogenase,LDH）是糖酵解途径的终止酶,在整个昆虫的发育阶段长期存在。本试验采用同源克隆和锚定PCR技术,从异色瓢虫Harmoniaaxyridis中克隆到HaxLDH基因的cDNA全序列（GenBank登录号HM362898）,全长1336bp,包含214bp的3’非编码区域和123bp的5,非编码区域,可读框长999bp,编码332个氨基酸。通过软件分析,预测该基因编码蛋白的分子量为36．2kD,理论等电点为8．62;包含1个糖基化位点,无信号肽序列和跨膜结构。同源比对不同昆虫的LDH基因cDNA序列,发现HaxLDH拥有8个非常保守的结构域：DLQHG、TAGV／ARQK／RE／DGES／TRL、LVQRN、GSGTNLD、SCHGW／YI／VI／VGEHGD、VWSG／AVNV／IAGV、AYEV／IIK／RLKGYTS／NWAI／VGLS和LSLP。HaxLDH与赤拟谷盗Triboliumcastaneum的LDH同源性最高,达67％;系统发育分析也表明二者亲缘关系较近。本研究为探讨LDH在昆虫发育和逆境中的作用奠定了基础。     

小麦诱导抗性基因TaWIR1b的克隆及表达分析

全蚀病是小麦上一种重要的土传病害。选育和种植抗病品种是防治小麦全蚀病的根本途径,抗病基因研究是抗病育种的基础性工作。根据基因TaWIR1b(Accession no.M94959.1)的全长序列设计引物扩增‘新农19’的cDNA,获得了完整ORF,编码85个氨基酸残基,比对后发现与TaWIR1b序列同源性达100%。根据获得的TaWIR1b基因全长序列设计定量引物,分析TaWIR1b在全蚀菌胁迫条件下不同互作模式的表达特征。结果表明接种全蚀病菌后抗病小麦品种‘新农19’中TaWIR1b基因被诱导表达,接菌后3d达到峰值143.97,感病品种‘新麦19’中峰值出现在接菌后8d,表达量仅为对照的4.22倍,提示该基因可能参与小麦对全蚀病的抗病过程。     

茶尺蠖脂肪酶合成基因EoL2的克隆和组织表达特征

昆虫脂肪酶可参与脂类消化、吸收和运输,并能介导植物的抗病虫功能。本研究通过茶尺蠖唾液腺转录组数据库发掘到该基因片段,采用RACE技术克隆获得了一条茶尺蠖脂肪酶合成基因序列,命名为Eo L2,并检测了其在茶尺蠖不同生育期及不同组织部位中的相对表达水平。研究结果表明,Eo L2的c DNA序列全长1310 bp,开放阅读框为1041 bp,编码一个包含346个氨基酸的蛋白,推测该蛋白相对分子量为37.96 k D,推测等电点(PI)为8.41,具有脂肪酶典型的GXSXG丝氨酸活性保守序列,其氨基酸序列与家蚕相似度最高,为57%。实时荧光定量PCR结果表明,Eo L2在卵中的表达量显著低于其它虫态;在1龄幼虫期的相对表达水平显著高于2~5龄;在成虫中的表达量显著高于蛹;在血淋巴中的表达量显著高于中肠、表皮、脂肪体和唾液腺。     

昆虫Kr-h1基因的生物信息学分析

Kr-h1基因是研究昆虫变态发育与害虫生物防治的重要靶标分子。本研究解析了昆虫Kr-h1基因结构、保守结构域、二级结构与三级结构,开展了多序列比对、表达模式分析与系统发育树重构。结果表明,Kr-h1基因外显子数量较为保守,多为2个,而内含子长度变异较大。Kr-h1蛋白一般含8个锌指结构,二维与三维结构显示锌指结构保障了蛋白的稳定性。基于全长序列的系统发育分析显示,昆虫Kr-h1为直系同源,亲缘关系与物种进化相吻合,基于锌指结构的系统发育则显示Kr-h1不同模块进化中的差异。菜粉蝶转录组数据显示,该基因表达模式与变态密切相关。本研究丰富了昆虫Kr-h1基因的知识,以期为菜粉蝶、西花蓟马等昆虫变态发育研究与生物防治提供新策略。     

Laboratory and field evaluations of rhodojaponin-III against the imported cabbage worm Pieris rapae (L.) (Lepidoptera: Pieridae)

The activity of rhodojaponin-III (R-III), a grayanoid diterpene compound isolated from Rhododendron molle G. Don flowers, was determined under laboratory and field conditions as an antifeedant, stomach poison, contact toxicant and insect growth inhibitor against Pieris rapae (L.) larvae. The median antifeedant concentration (AFC(50)) values in no-choice leaf disc tests were 1.16 and 15.85 mg L(-1) at 24 h after treatment when tested against third and fifth instars respectively. The median lethal concentration (LC(50)) values in leaf disc tests were 2.84 and 9.53 mg L(-1) at 96 h after treatment against third and fifth instars respectively. R-III showed an almost 30 times higher contact toxicity against third instars than for fifth instars, and the median lethal dose (LD(50)) values for topical application were 1.18 and 34.09 mg kg(-1) at 72 h after treatment respectively. R-III disrupted the development of larvae to pupae or adults with median concentration for inhibiting growth (IC(50)) values of only 1.36 mg L(-1) for third instars and 11.28 mg L(-1) for fifth instars. In field trials, a greater than 80% reduction in the adjusted larval numbers was obtained against P. rapae 14 days after treatment when Rhodo 0.1% EC, a commercial botanical insecticide based on R-III, was applied at both 937.5 and 625 mL ha(-1). These results suggest that further research to develop R-III, and extracts from R. molle, as biorational pesticides or as lead compounds for integrated pest management deserve consideration.     

长春地区大菜粉蝶生物学特性观察

大菜粉蝶Pieris brassicae是十字花科蔬菜上的重要害虫之一。本文通过田间观察结合室内饲养,系统描述了该虫各发育阶段的形态特征,记录了发育历期、生活史及其生活习性。大菜粉蝶在长春地区1年发生3代,以蛹越冬。成虫多产卵于叶背面,每块卵10～207粒。幼虫5个龄期,群集为害,随着幼虫龄期的增长,头壳宽度与体长逐渐增加,各龄期头壳宽度和体长生长模型为：y_W=1.727e^0.557x(R²=0.998)和y_L=0.031x²+0.339x-0.013(R²=0.994)。取食白菜、油菜、甘蓝和萝卜4种寄主的幼虫发育历期存在显著差异,取食白菜发育最快,取食油菜的蛹最重,白菜和油菜更适宜其生长发育。本研究描述了大菜粉蝶各发育阶段的形态特征,明确了其主要生物学特性,为该害虫的识别监测和综合防控提供了理论基础。     

Food preferences and consumption parameters of Cabbage butterfly,Pieris brassicae (Lepidoptera: Pieridae) on different kale genotypes

Journal of Plant Diseases and Protection - Food preferences and consumption parameters of Pieris brassicae larvae were carried out on 17 kale (Brassica oleracea var. acephala) genotypes....     

麦长管蚜电压门控钠离子通道基因克隆及序列分析

克隆获得麦长管蚜电压门控钠离子通道基因cDNA序列,明确其典型特征,为研究麦长管蚜抗性分子机理奠定基础。采用RT-PCR技术,克隆麦长管蚜钠离子通道基因cDNA序列,利用DNASTAR Lasergene 7.10软件对其序列进行分析。克隆得到两条cDNA序列SaNa_v1和SaNa_v2（GenBank登录号分别为MN176137和MN161584）,SaNa_v1序列长3423 bp,共编码1141个氨基酸;SaNa_v2序列长2874 bp,共编码958个氨基酸。同源比对发现,SaNav1与禾谷缢管蚜和桃蚜的第一部分钠离子通道基因相似度分别高达97.81%和97.91%;SaNav2与禾谷缢管蚜和桃蚜的第二部分钠离子通道基因相似性高达97.90%和96.43%。所克隆序列包含4个同源结构域,每个结构域6个跨膜片段（S1～S6）,存在钠通道选择性关键残基“DENS”。本文成功克隆了麦长管蚜中钠离子通道基因,为麦长管蚜钠离子通道抗性机制的研究提供依据。     

Characterization of a nucleopolyhedrovirus variant of the gypsy moth,Lymantria dispar (Lepidoptera: Lymantriidae) in Turkey

The gypsy moth (Lymantria dispar L., Lepidoptera: Lymantriidae) is one of the most serious pest of various forestal, food and industrial crops worldwide. We have characterized a new Lymantria dispar nucleopolyhedrovirus (LdMNPV-T4) variant, which was isolated from dead L. dispar larvae in Turkey. Scanning electron microscope observations showed that the polyhedral occlusion bodies (OBs) of the LdMNPV-T4 were irregularly shaped. Transmission electron microscopy revealed that OBs of LdMNPV-T4 were occupied with several virions in which multiple nucleocapsids packaged by viral envelope. Restriction analysis of the LdMNPV-T4 DNA purified from the viral inclusion bodies yielded BamHI, BglII, EcoRI and HindIII fragments. The mean size estimated for the complete LdMNPV-T4 genome was calculated to be 163.3 kb. Phylogenetic analysis of amplified polh, lef-8 and lef-9 sequences showed its relation to the other NPVs from Lymantria species. Mortality values of the LdMNPV-T4 at four different concentrations against third instar larvae of L. dispar ranged from 45% to 88%. These results suggest that LdMNPV-T4 isolated from Turkey is a promising microbial control agent to be utilized for the biological control of L. dispar.     

Cloning,characterisation and expression profiling of the cDNA encoding the ryanodine receptor in diamondback moth,Plutella xylostella (L.) (Lepidoptera: Plutellidae)

BACKGROUND: The rynodine receptors (RyRs) are the main targets of diamide insecticides such as chlorantraniliprole. To provide the basis for a good understanding of the molecular mechanisms of diamide insecticide resistance, an RyR gene from Plutella xylostella was cloned and characterised in the present paper. RESULTS: A full‐length cDNA sequence of RyR was cloned from P. xylostella through RT‐PCR and rapid amplification of cDNA ends (RACE). The gene (named PxRyR1) is 15 753 bp long, with an open reading frame of 15 354 bp, encoding a predicted RyR of 5117 amino acids. An alternative splicing of the PxRyR1 was also cloned and named PxRyR2. The PxRyR1 shares 77–93% identity with other insect RyRs. Quantitative real‐time PCR analysis showed that the PxRyR was expressed at a high level in second‐instar larvae and adults, at a low level in prepupae and pupae and abundantly in the body wall muscle and head (respectively 6.00 and 3.12 times the expression in the gut). Western blot analysis with anti‐RyR antibodies showed that the RyR was mainly present in the body wall muscle and head, but barely present in the haemocyte and gut. CONCLUSIONS: There are at least two alternative splices of PxRyR expressed in all developmental stages and tissues in P. xylostella at various levels. The results provided the basis for further understanding of the mechanisms of resistance to diamide insecticides in P. xylostella. Copyright © 2012 Society of Chemical Industry     

The effects of Artemisia annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera: Pieridae)

The biological effects of two important medicinal plants, Artemisia annua L. and Achillea millefolium (L.) (viz, mortality, growth, and feeding indices as well as enzyme and non-enzymatic activities) were studied on small white Pieris rapae L a deleterious pest of cruciferous plants under controlled conditions (16:8 h L:D at 25 ± 1 °C and 65 ± 5% RH). The LC₅₀ and LC₂₅ values were 9.387% and 3.645% for A. annua L. and 4.19% and 1.69% for A. millefolium (L.), respectively. At the lowest concentration (0.625%), the deterrency was 29.826% and 44.185% for A. annua L. and A. millefolium (L.), respectively. Feeding indices were variously affected with changes in a number of parameters and an increase in larval and pupal duration. The activity level of alkaline phosphatase increased sharply while alanin and aspartate aminotransferases showed a sharp decrease. For non-enzymatic compounds, the amount of glucose and uric acid increased, but total protein and cholesterol decreased. These results indicate that these two medicinal plants might possess potential secondary metabolites that may be useful for controlling potential insect pests.     

甜菜夜蛾中肠碱性磷酸酶alp2基因的克隆、表达及功能分析

为探究甜菜夜蛾Spodoptera exigua中肠碱性磷酸酶(alkaline phosphatase protein 2,ALP2)是否为Cry1Ac杀虫蛋白的受体,采用同源克隆和RACE技术克隆了编码alp2基因的完整c DNA序列,利用荧光定量PCR比较了甜菜夜蛾幼虫中肠不同龄期ALP2表达量的差异,利用Ligand blot方法检测了中肠ALP2与Cry1Ac杀虫蛋白的结合。结果表明,alp2基因序列全长1 629 bp(Gen Bank序列号为KP420013),编码542个氨基酸,预测在氨基酸序列N端包含1个由21个氨基酸组成的信号肽,在C端存在1个GPI修饰的锚定位点,且在整个氨基酸序列中存在多个糖基化修饰位点。在整个甜菜夜蛾幼虫期均有ALP2表达,但不同龄期的表达量差异显著,1龄幼虫期表达量最低,4龄幼虫期最高。Ligand blot方法检测结果表明原核表达的ALP2片段与活化的Cry1Ac杀虫蛋白可以结合。研究表明,甜菜夜蛾中肠的ALP2可能是Cry1Ac的受体之一。     

滞育七星瓢虫酰基辅酶AΔ11去饱和酶基因的克隆及表达分析

去饱和酶（Fatty acid desaturase,FAD）是生物体不饱和脂肪酸合成中的关键酶,酰基辅酶AΔ11去饱和酶基因是其中重要的一种。本试验基于七星瓢虫Coccinella septempunctata L.转录组数据,利用RT-PCR和RACE技术从滞育七星瓢虫中克隆得到酰基辅酶AΔ11去饱和酶基因全长,并命名为CsFadΔ11（GenBank登录号：MF458996）,该基因全长1447 bp,开放阅读框（ORF）1095 bp,编码364个氨基酸,预测蛋白质分子量为42.99 kD,等电点（pI）为8.87,无信号肽。氨基酸序列分析结果表明,CsFadΔ11有3个组氨酸富集区和6个跨膜结构域,与膜翅目的内华达古白蚁Zootermopsis nevadensis同源性较高。利用实时荧光定量PCR技术研究其时间表达模式,发现CsFadΔ11基因在七星瓢虫初羽化、滞育诱导10 d、滞育诱导20 d、滞育初期、滞育后期、滞育解除期以及正常发育状态下均有所表达,且在滞育早期表达量最高,其次是在滞育解除个体中。其整体表达趋势与相应时期脂积累变化情况基本相符,推测CsFadΔ11基因参与七星瓢虫脂积累调控,并进一步影响七星瓢虫滞育。