Molecular approaches for identification and construction of novel insecticidal genes for crop protection

Summary The insecticidal cry (crystal) genes from Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. Discovery of new insecticidal genes is of importance for delaying the development of resistance in target insects. The diversity of Bt strains facilitates isolation of new types of cry and vip (vegetative insecticidal protein) genes. PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities. PCR together with other methods of analysis such as RFLP, gene sequence determination, electrophoretic, immunological and chromatographic analysis of Cry proteins and insect bioassays for evaluation of toxicity have been employed for identification of new insecticidal proteins. Some other new approaches have also been devised. Many Bt strains with novel insecticidal genes have been found. A desired combination of Cry proteins can be assembled via site-specific recombination vectors into a recipient Bt strain to create a genetically improved biopesticide. For better pest control, the cry genes have been transferred to plants. Stacking of more than one insecticidal gene is required for resistance management in transgenic crops. Modification of Cry proteins through protein engineering for increasing the toxicity and/or the insecticidal spectrum is also a promising approach, but requires detailed understanding of the structure and function of these proteins and analysis of toxin-receptor interactions. More research into this area will provide useful insights for the design of toxins for management of insect resistance. Insecticidal genes from other bacteria and plants are also being examined for their potential for deployment in transgenic crops. Stringent implementation of resistance management is needed for maintaining the efficacy of Bt transgenic crops and deriving maximum economic and environmental benefit.     

Novel bait stations for attract-and-kill of pestiferous fruit flies

A novel, visually-attractive bait station was developed in Hawaii for application of insecticidal baits against oriental fruit fly, Bactrocera dorsalis (Hendel), melon fly, Bactrocera cucurbitae (Coquillett), and Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (all Diptera: Tephritidae). The bait station developed represents a supernormal visual stimulus of papaya foliage and takes advantage of the flies' strong response to the high light-reflecting properties of yellow color and of their need for shelter, while fully protecting the bait against rainfall. Field studies revealed that the behavioral response of female fruit flies, in particular C. capitata and B. cucurbitae , to yellow-painted bait stations sprayed with GF-120 NF Naturalyte Fruit Fly Bait was significantly enhanced compared with similarly sprayed bait stations that mimicked the green color of fully grown papaya leaves. Field studies conducted with B. cucurbitae indicated that the period of bait attractiveness can be extended for at least 1 week after bait application due to the rain-fastness properties of the bait stations and the use of a visually-attractive color. Our studies provide the behavioral basis for the development of improved attract-and-kill bait stations for fruit flies in Hawaii. These devices also provide a standardized way of evaluating bait spray formulations, thus allowing for proper comparisons over time, across species, and among geographical areas.     

欧美杨108号转蜘蛛杀虫肽与Bt毒蛋白嵌合基因的研究

以欧美杨108号为外植体材料,利用农杆菌介导法成功地将抗虫基因（蜘蛛杀虫肽+Bt毒蛋白的嵌合基因）导入受体中。通过PCR检测,凝胶电泳结果显示8个卡那霉素抗性芽中,6个存在目的基因;经PCR-Southern印迹杂交检测,阳性率为50%,进一步表明目的基因成功整合到欧美杨108号基因组内。     

转Bt cry1Ab基因水稻对稻田弹尾虫种群数量的影响

以转Bt cry1Ab基因水稻克螟稻1号(KMD1)和克螟稻2号(KMD2)及其亲本非转基因水稻秀水11(XS11)为材料,于2003年9月和翌春4月,调查了KMD1和KMD2对水稻灌浆期和收割后休田期稻田土表落叶层中弹尾虫种群数量的影响.结果表明,KMD植株表达的cry1Ab杀虫蛋白可在稻田环境中残留160 d以上;在水稻灌浆期采用吸虫器法在稻田落叶层中采集到灰橄榄长角跳虫(Entomobrya griseoolivata)和钩圆跳虫(Bourletiella christianseni)等2种弹尾虫,其中灰橄榄长角跳虫在KMD1和KMD2稻田中的种群密度显著高于XS11稻田;在水稻收割后休田期采用网袋法采集到灰橄榄长角跳虫、钩圆跳虫、球角跳虫(Hypogastrura matura)和等节跳虫(Isotoma monochaeta)等4种弹尾虫,其中转Bt基因水稻稻田中的灰橄榄长角跳虫和球角跳虫的种群密度显著高于XS11稻田,且其植株组织残体生物量损失率显著高于XS11.     

苏云金杆菌杀虫蛋白的多样性

苏云金杆菌是生物防治中应用最为广泛的一种杀虫剂,其杀虫蛋白具有广泛的多样性。本文就苏云金杆菌杀虫蛋白的基因、基因分布、杀虫蛋白结构以及作用机制的多样性进行了概述。     

苏云金芽孢杆菌vip3A基因的鉴定、克隆及活性分析

【目的】鉴定我国自行分离的苏云金芽孢杆菌（Bacillus thuringiensis, Bt）菌株中vip3A基因的分布和基因型,从其中对鳞翅目幼虫表现高毒力的Bt菌株中克隆vip3Aa基因。【方法】利用PCR-RFLP方法确定vip3A基因的分布和鉴定基因型,利用PCR方法克隆vip3A全长基因。【结果】171株野生型Bt菌株中共鉴定出63株含有vip3A基因,均与vip3Aa1类基因相似。从TF9和Bt16菌株中克隆得到2个vip3Aa基因,分别构建了携带vip3Aa基因的表达载体p30vip-26和p30vip-27,SDS-PAGE和Western Blot分析表明,IPTG诱导后均可表达88 kDa左右的Vip3A蛋白,蛋白可溶性分析表明约10%可溶。这两种基因序列已被国际Bt基因命名委员会分别正式命名为vip3Aa26和vip3Aa27。生物测定结果显示,Vip3Aa27蛋白对粉纹夜蛾（Trichoplusia ni）、甜菜夜蛾（Spodoptera exigua）和棉铃虫（Helicoverpa armigera）3种重要鳞翅目害虫初孵幼虫的毒力较高,LC50值分别为0.125 μg/mL,0.238 μg/mL和9.238 μg/mL。而Vip3Aa26蛋白仅对粉纹夜蛾有活性,LC50值为4.423 μg/mL。【结论】本研究中的Vip3Aa27蛋白对粉纹夜蛾、甜菜夜蛾和棉铃虫幼虫均能表现高杀虫活性,而Vip3Aa26蛋白仅对粉纹夜蛾幼虫有活性,实验结果表明Vip3A蛋白个别氨基酸的变化对其杀虫活性影响很大。     

对粉纹夜蛾高毒力cry9Ea基因的克隆及表达

【目的】从本实验室分离的Bt4菌株中克隆cry9Ea基因,并研究其表达和杀虫活性。【方法】以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因。【结果】将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcry9Ea。转化E.coli BL21(DE3),诱导后表达130kDa的蛋白,再将cry9Ea7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定。生物活性测定结果显示Cry9Ea7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC50为0.044μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性。【结论】克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Ea7,并成功构建了工程菌BioHD9Ea7。     

An insect peptide engineered into the tomato prosystemin gene is released in transgenic tobacco plants and exerts biological activity

Tomato systemin is a signalling peptide produced in response to wounding that locally and systemically activates several defence genes. The peptide is released from the C-terminus of prosystemin, the 200 amino acid precursor, following post-translational modifications involving unknown events and enzymes. In tobacco, two systemin molecules have been recently isolated, neither sharing any sequence homologies with the tomato prosystemin gene/protein, but performing similar functions. We modified the tomato prosystemin gene by replacing the systemin-encoding region with a synthetic sequence encoding TMOF (trypsin-modulating oostatic factor), a 10 amino acid insect peptide hormone toxic to Heliothis virescens larvae, and expressed the chimeric gene in tobacco. The results reported here show that transformed leaves contain the TMOF peptide and exert toxic activity against insect larvae reared on them. In addition, subcellular localization studies showed the cytoplasmic location of the released TMOF, suggesting that in tobacco the enzymes responsible for the post-translational modifications of the tomato precursor protein are present and act in the cytoplasm to recognise the modified prohormone. The molecular engineering of the precursor, beside supplying new clues towards the understanding of prosystemin processing, constitutes an useful tool for plant genetic manipulation, by enabling the delivery of short biological active peptides.     

TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post‐translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modern high‐expression transgenic Bt plants possible, with an emphasis on the reasons for the low‐level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.