小菜蛾碱性磷酸酶基因cDNA片段的克隆及其序列分析

利用DNAman设计简并引物,通过反转录一多聚酶链式反应(RT-PCR)克隆小菜蛾Plutella xylostella(L.)抗性、敏感种群中可能与苏云金芽孢杆菌(Bt)抗性相关的碱性磷酸酶基因。琼脂糖电泳结果显示扩增条带与预期片段长度一致,阳性克隆经测序获得了403bp的基因片断。经blast比较得出所克隆基因属于碱性磷酸酶基因。与敏感种群相比,抗性种群中该基因片段有18个碱基发生变化,有1个氨基酸发生替换(缬氨酸转变为苏氨酸)。研究结果对于进一步研究小菜蛾全基因结构、功能有重要的意义。     

印楝素乳油对小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用。结果表明,其控制作用主要表现在对成虫产卵的显忌避作用。对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害为等方面,印楝素乳油0.005mL/59.0%,使小菜蛾种群趋势指数（I值）降为2.4干扰作用控制指数（IIPC）为0.1079,采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显的控制作用表现在种群发展初期对成虫的忌避作用。     

小菜蛾的耐热性

小菜蛾是世界范围内十字花科蔬菜上的重要害虫.临界高温(critical thermal maxi-mum,CTMax)是昆虫耐热性的常用指标.采用动态加热方法,利用自行组装的装置测定了小菜蛾的临界高温,以此作为其耐热性指标,研究发育阶段、饲养温度、世代、性别和热激对小菜蛾耐热性的影响.结果表明:25℃下饲养的小菜蛾4龄幼虫的CTMax均值为50.31℃,显著高于1龄幼虫(43.03℃)、2龄幼虫(46.39℃)、3龄幼虫(49.67℃)以及雌性成虫(45.76℃)和雄性成虫(47.73℃);不同饲养温度(20、25和30℃)下成虫耐热性无显著差异;30℃下饲养1代、3代及6代的不同世代成虫CTMax也无显著变化;所有处理雌雄成虫的CTMax无显著差异;40℃下45 min热激可使5日龄雄成虫的CTMax值从45.51℃增加到46.49℃.     

小菜蛾在温带地区越冬研究进展

小菜蛾是世界性重要害虫。在热带和亚热带地区冬季的十字花科植物上能正常发育繁殖,可见到各种虫态。但在温带冬季十字花科植物不能生长的地区,小菜蛾的越冬成为一个重要生态学问题。综述了亚洲、北美洲和欧洲小菜蛾越冬的研究进展。小菜蛾在日本的北海道、本州岛的北陆和东北大部分地区不能越冬,越冬北限相当于冬季积雪覆盖时间为60d的区域;在中国,小菜蛾不能在寒冷的东北地区越冬,在长江中下游以南地区冬季可见各虫态,但越冬北限尚不清楚;小菜蛾在冬天气候温和的韩国以及澳大利亚东南部继续发生;北美洲的加拿大西部和安大略地区大量的试验证明小菜蛾不能成功越冬,在美国南部小菜蛾冬季可正常发生,北部小菜蛾的越冬尚未见系统研究报道,但确认春季从南部运输的受小菜蛾感染的甘蓝等种苗是美国北部的重要虫源。小菜蛾在欧洲各地越冬的系统研究未见报道,没有证据表明在英国小菜蛾会发生有显著意义的越冬。目前小菜蛾越冬研究主要采用冬季直接试验观察和基于耐寒性试验的越冬预测两种方法。冬季直接试验观察法包括:(1)利用人工饲养的小菜蛾在田间各种潜在的越冬场所的越冬试验;(2)在秋播、野生或残留的十字花科植物上进行冬季种群的系统抽样调查;(3)越冬前后在前茬为十字花科植物的田块广泛搜寻普查小菜蛾的存活个体。基于耐寒性试验的越冬预测法:在获取小菜蛾越冬场所温度的基础上设计低温处理模式,试验低温处理后小菜蛾的存活率及后续发育和生殖。将试验数据和各地气温或小气候相结合,对小菜蛾在的越冬可能性进行推断。     

浙江临海小菜蛾成虫数量季节消长规律

在浙江临海相隔60km的2个十字花科蔬菜种植区,于2002~2005年利用性诱剂诱捕器每日对小菜蛾Plutella xylostella(L.)雄成虫进行诱集并计数。对两地成虫数量季节消长与种群基数、气温、雨量和日照等因子的相关性做了统计分析,建立了5个预测模型。应用这些模型可预测全年36个旬期的雄成虫数量变化,从而可预测春季高峰期并对全年的发生量和为害程度进行预警,这对提高当地小菜蛾监测预警与综合防治水平具有实践意义。     

萜类化合物对小菜蛾幼虫的拒食活性

采用叶碟浸液法测试从松节油合成的26个不同结构萜类化合物样品对小菜蛾Plutella xylostella(L.)4龄幼虫的拒食活性。结果显示,编号为2,4,8,13,27,31的6个化合物,即诺卜醇、诺卜丙基醚、内型异莰烷基甲醇丙酸酯、4-(1-甲基乙烯基)-1-环己烯-1-乙醇丙酸酯、羟基香茅醛丙酸酯、羟基香茅醛1,2-丙二醇缩醛具有较高的拒食活性。处理有效成分为0.01g/mL时,这6个化合物24h拒食率为70%～100%。其中2,4,8号3个化合物拒食活性最高,处理有效成分为0.01g/mL和0.001g/mL时,24h拒食率分别为98.33%,99.80%,100.00%和85.60%,83.90%,66.97%;并且持效性较好,72h拒食率分别为92.67%,89.97%,98.73%和63.20%,63.30%,45.93%。     

小菜蛾酪氨酸酶生物化学性质研究

采用时间动力学法对小菜蛾Plutella xylostella酪氨酸酶的最适反应条件、对抑制剂的敏感度以及发育期变化规律进行了研究。结果表明：小菜蛾酪氨酸酶反应的最适pH值为6.4,反应线性时间为0～2 min,在22℃和4℃下其活性分别可保持12 h 和72 h,在-20℃下至少可保持7 天。不同发育阶段小菜蛾酪氨酸酶活性的大小依次为预蛹＞4龄幼虫＞蛹＞1龄、2龄、3龄幼虫＞成虫。苯基硫脲（phenyl thiourea,PTU）对小菜蛾阿维菌素抗性品系和敏感品系酪氨酸酶活性的抑制中浓度（I₅₀）值分别为1.1796 μmol/L和1.2795 μmol/L,二者无明显差异。     

颗粒体病毒对不同蔬菜上小菜蛾实验种群的控制作用

通过组建施用颗粒体病毒Plutella xylostellagranulosis virus(PxGV)后4种十字花科蔬菜上的小菜蛾Plutella xylostellaL.实验种群生命表发现:小菜蛾颗粒体病毒除了能引起幼虫感病死亡外,还能显著地降低成虫的产卵量,推迟产卵高蜂1~2 d,但对化蛹及雌蛾寿命无显著影响。在种群水平上,病毒对小菜蛾的控制作用综合表现为压低了小菜蛾种群增长趋势指数,在菜心、芥蓝、芥菜和小白菜上小菜蛾实验种群增长趋势指数在分别为13.7,12.3,10.7和22.7,病毒对小菜蛾种群的干扰作用控制指数(IIPC)分别为0.334,0.177,0.280和0.433,表明病毒对小菜蛾种群的控制能力受到寄主植物的影响,在芥蓝上控制能力最强,其次为芥菜和菜心,而小白菜最次。     

小菜蛾对几种常用药剂的敏感性测定

采用叶片浸溃法,在2001年10月和2002年10月分别测定了福州上街蔬菜基地的小菜蛾,对10种杀虫剂的敏感性。结果表明,上街菜区小菜蛾对灭多威、杀虫双、辛硫磷、毒死蜱、杀灭菊酯和功夫菊酯等农药极不敏感,已不能有效控制小菜蛾的危害。而小菜蛾对氟啶脲、阿维菌素、氟虫腈和虫螨腈比较敏感。通过两次毒力测定比较(2002年10月的LC50;2001年10月的LC50),结果表明,大多数杀虫剂敏感性变化较小。但小菜蛾对阿维菌素敏感性明显下降,毒力倍数达6．65倍,对氟虫腈毒力倍数相差1．48倍。此外,2002年10月小菜蛾对功夫菊酯的LC50为2001年10月的2．02倍。用浸叶法和浸溃法测定多杀菌素、氟虫腈与虫螨腈推荐浓度对小菜蛾幼虫的毒杀作用。结果表明3种药剂浸叶法处理效果均较理想。     

Biological control of the diamondback moth, Plutella xylostella: A review

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive cosmopolitan insect pests of brassicaceous crops. It was the first crop insect reported to be resistant to DDT and now, in many crucifer producing regions, it has shown significant resistance to almost every synthetic insecticide applied in the field. In certain parts of the world, economical production of crucifers has become almost impossible due to insecticidal control failures. Consequently, increased efforts worldwide have been undertaken to develop integrated pest management (IPM) programs, principally based on manipulation of its natural enemies. Although over 130 parasitoid species are known to attack various life stages of DBM, most control worldwide is achieved by relatively few hymenopteran species belonging to the ichneumonid genera Diadegma and Diadromus, the braconid genera Microplitis and Cotesia, and the eulophid genus Oomyzus. DBM populations native to different regions have genetic and biological differences, and specific parasitoid strains may be associated with the specific DBM strains. Therefore, accurate identification based on genetic studies of both host and parasitoid is of crucial importance to attaining successful control of DBM through inoculative or inundative releases. Although parasitoids of DBM larvae and pupae are currently its principal regulators, bacteria-derived products (e.g., crystal toxins from Bacillus thuringiensis) and myco-insecticides principally based on Zoophthora radicans and Beauveria bassiana are increasingly being applied or investigated for biological control. Viruses, nematodes and microsporidia also have potential as biopesticides for DBM. When an insect pest is exposed to more than one mortality factor, there is the possibility of interactions that can enhance, limit, or limit and enhance the various aspects of effectiveness of a particular control tactic. This paper reviews the effectiveness of various parasitoids and entomopathogens against DBM, interactions among them, and their possible integration into modern IPM programs.     

小菜蛾鞣化激素基因克隆及其功能分析

鞣化激素是调节昆虫表皮骨化和翅膀发育的一种神经激素, 尽管已经在许多不同种昆虫上克隆了鞣化激素基因, 但是关于小菜蛾 Plutella xylostella鞣化激素及其基因的研究至今未见报道。本研究克隆了两个小菜蛾鞣化激素基因Pxbursα和Pxbursβ （GenBank 登录号分别为KF498645和KF498646）全长cDNA, 其序列长度分别为537 bp和360 bp, 与已报道的其他昆虫的鞣化激素氨基酸序列一致性分别为51%~68% 和37%~57%。实时定量PCR分析发现Pxbursα和Pxbursβ均在蛹期表达量高, 而在幼虫期和成虫期的表达量低。以Pxbursα部分序列的双链RNA（dsRNA）饲喂小菜蛾4龄末期幼虫, 发现蛹期Pxbursα的表达受到了显著抑制, 小菜蛾的发育停滞在蛹期而无法正常羽化, 并最终死亡。由此推测, 小菜蛾鞣化激素基因在蛹期的大量表达对其生长发育和羽化具有重要的作用。     

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.     

cDNA cloning and induction of tyrosine hydroxylase gene from the diamondback moth,Plutella xylostella

We cloned a full‐length tyrosine hydroxylase cDNA from the integument of the diamondback moth, Plutella xylostella. In the phylogenetic tree, tyrosine hydroxylase (PxTH) clustered with the other lepidopteran THs. Serine residues in the PxTH sequence, namely Ser²⁴, Ser³¹, Ser³⁵, Ser⁵³, and Ser⁶⁵, were predicted to be the target sites for phosphorylation based on PROSITE analysis. In particular, Ser³⁵ of PxTH is highly conserved across a broad phylogenetic range of animal taxa including rat and human. Western blot analysis using both PxTH‐Ab1 and PxTH‐Ab2 polyclonal antibodies verified the expression of PxTH in all life cycle stages of P. xylostella, namely the larval, pupal, and adult stages. To examine the possible immune function of PxTH in P. xylostella, PxTH gene expression was investigated by RT‐PCR and western blotting analysis after challenging P. xylostella with bacteria. PxTH expression was elevated 1 h post‐infection and was continued till 12 h of post‐infection relative to control larvae injected with sterile water. © 2010 Wiley Periodicals, Inc.     

cDNA characterization and expression analysis of two arylphorin-like hexameric protein genes from the diamondback moth, Plutella xylostella (L.)

We cloned and characterized two hexameric storage protein genes, PxAry1 and PxAry2, from Plutella xylostella and investigated the expression pattern in different developmental stages and in response to treatment by a juvenile hormone (JH) analog. The complete coding sequences of PxAry1 and PxAry2 are comprised of 2,097 and 2,094 bp with 699 and 698 amino acid residues, respectively. Signal peptides of 16 amino acids are predicted at the N-termini. According to both the phylogenetic analysis and amino acid composition (>16% aromatic amino acids), PxAry1 and PxAry2 belong to the arylphorin-like protein genes. Analysis using Northern hybridization and RT-PCR showed varying levels of genes expression in the developmental stages with a small difference between sexes. Expression of both genes in fourth instar larvae was suppressed after treatment with a JH-analog. Southern hybridization revealed the presence of multiple arylphorin genes in the genome.     

小菜蛾化学生态学研究现状与展望

本文综述了几种植物吸引或阻抑小菜蛾成虫产卵或幼虫取食的效应。完整的、机械损伤的和菜粉蝶为害的甘蓝类蔬菜释放出数十种醇、醛、酯、酮、硫化物、羧酸类、异硫氰酸酯类和萜烯类挥发性化合物。机械损伤的甘蓝和小菜蛾、菜粉蝶及蜗牛为害的甘蓝释放的挥发物引诱菜粉蝶绒茧蜂。小菜蛾为害甘蓝释放的挥发物引诱菜蛾绒茧蜂。小菜蛾性信息素基本成分是顺 -1 1 -十六碳烯醛和顺 -1 1 -十六碳烯乙酸酯。当顺 -1 1 -十六碳烯醛、顺 -1 1 -十六碳烯乙酸酯和顺 -1 1 -十六碳烯醇以 5∶5∶0 1或顺 -1 1 -十六碳烯醛、顺 -1 1 -十六碳烯乙酸酯、顺 -1 1 -十六碳烯醇和顺 -9-十四碳烯乙酸酯按70∶3 0∶1∶0 0 1比例制成的诱芯诱蛾效果较好。宜深入探究寄主植物—小菜蛾—菜蛾绒茧蜂间的通讯机制 ,开发高效诱芯。     

Molecular cloning and expression profiles of calreticulin gene from the diamondback moth,Plutella xylostella

Calreticulin (CRT) plays pivotal roles in Ca²⁺ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full-length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly-A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.     

小菜蛾对苦皮藤素抗性选育及交互抗性测定

敏感小菜蛾Plutella xylostella (L.)经苦皮藤素20代的抗性选育,其抗性增长21.57倍。选育的小菜蛾抗性品系对杀虫双、杀螟丹和叶蝉散分别有4.63、4.11和3.71倍的交互抗性;对溴氰菊酯、氯菊酯、氯氰菊酯分别有0.22、0.01和0.26倍的负交互抗性。高抗杀螟丹、杀虫双小菜蛾品系对苦皮藤素无明显交互抗性,而高抗溴氰菊酯小菜蛾品系对苦皮藤素有3.61倍的交互抗性。     

小菜蛾对溴氰菊酯抗性相关的DNA片段的克隆与分析

采用代表性差异分析（representational difference analysis, RDA ）方法,以小菜蛾Plutella xylostella (L.)的敏感品系作为对照组,溴氰菊酯抗性近等基因系（near isogenic line）作为测试组,通过三轮消减杂交得到大小为 150～300 bp的差异扩增带,经亚克隆测序并与GenBank等数据库同源比较,发现差异片段与已知抗性相关基因无同源性;以随机选取的差异片段作探针进行Southern blot分析,在测试组扩增子和三轮差异产物中都获得了阳性结果,而在对照组扩增子中的结果是阴性的。这些结果表明测序片段可能是与抗性相关的新基因序列或调控序列。     

2009年我国小菜蛾迁飞路径典型案例分析

小菜蛾Plutella xylostella Linnaeus是一种世界性的十字花科蔬菜重要害虫,具有远距离迁飞的特性,明确小菜蛾在我国的种群发生动态及迁飞路径对其早期预警具有重要意义.本文调查了2009年5月我国南京、故城、安阳、大连、公主岭、沈阳6个地区小菜蛾成虫种群的发生动态,并首次利用HYSPLIT平台对不同地区小菜蛾种群的迁飞峰次进行了轨迹分析.结果表明,2009年5月我国6个地区的小菜蛾成虫种群存在显著地“突增”或“突减”现象,符合迁飞昆虫在迁飞期的种群动态的典型特征;5月14日南京地区起飞的小菜蛾种群可迁飞至大连,5月19日故城地区起飞的小菜蛾种群可迁飞至公主岭,5月20日安阳地区起飞的小菜蛾种群可迁飞至沈阳;首次明确了小菜蛾轨迹分析的设定参数,800-1200 m为小菜蛾适宜迁飞的飞行高度,飞行持续时间一般为2-3d.     

基于质谱和生物信息学分析的小菜蛾蛋白质鉴定

本研究以非模式昆虫小菜蛾Plutella xylostella为材料, 对比2, 3, 4龄幼虫的蛋白质组双向电泳图谱, 得到24个蛋白质差异点, 从中选取了编号为1111的差异表达蛋白质点进行质谱鉴定和生物信息学分析. 采用胶内酶解的多肽进行MALDI-TOF/TOF分析, 获得该点的肽质量指纹图谱（PMF）及串联质谱（MS/MS）图谱。将获得的PMF分别用MASCOT和ProFound等常用软件在NCBInr的Metazoa蛋白质数据库进行搜索, 匹配结果不理想. 进一步用PMF+MS/MS谱图搜索NCBInr的Metazoa蛋白质数据库, 以及小菜蛾EST数据库。 在NCBInr库中匹配结果为拟暗果蝇Drosophila pseudoobscura中的一种假定蛋白GA18218-PA, 而用EST库搜索的结果为家蚕Bombyx mori的ATP合酶的亚基。为验证搜索结果, 将该蛋白质点进行磺基异硫氰酸苯酯（SPITC）化学衍生后de novo测序, 最后确认该点可能为ATP合酶的一个亚基。最后着重讨论了蛋白质的质谱鉴定与生物信息学分析的联合使用, 希望据此选择出最适合于非模式昆虫蛋白质组学鉴定的方法。