Identification and functional analysis of two P450 enzymes of Gossypium hirsutum involved in DMNT and TMTT biosynthesis

The homoterpenes (3E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT) and (E,E)‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT) are major herbivore‐induced plant volatiles that can attract predatory or parasitic arthropods to protect injured plants from herbivore attack. In this study, DMNT and TMTT were confirmed to be emitted from cotton (Gossypium hirsutum) plants infested with chewing caterpillars or sucking bugs. Two CYP genes (GhCYP82L1 and GhCYP82L2) involved in homoterpene biosynthesis in G. hirsutum were newly identified and characterized. Yeast recombinant expression and enzyme assays indicated that the two GhCYP82Ls are both responsible for the conversion of (E)‐nerolidol to DMNT and (E,E)‐geranyllinalool to TMTT. The two heterologously expressed proteins without cytochrome P450 reductase fail to convert the substrates to homoterpenes. Quantitative real‐time PCR (qPCR) analysis suggested that the two GhCYP82L genes were significantly up‐regulated in leaves and stems of G. hirsutum after herbivore attack. Subsequently, electroantennogram recordings showed that electroantennal responses of Microplitis mediator and Peristenus spretus to DMNT and TMTT were both dose dependent. Laboratory behavioural bioassays showed that females of both wasp species responded positively to DMNT and males and females of M. mediator could be attracted by TMTT. The results provide a better understanding of homoterpene biosynthesis in G. hirsutum and of the potential influence of homoterpenes on the behaviour of natural enemies, which lay a foundation to study genetically modified homoterpene biosynthesis and its possible application in agricultural pest control.     

基于重组溶葡球菌酶和ATP生物发光技术特异定量检测金黄色葡萄球菌

基于重组溶葡球菌酶和ATP生物发光法建立特异定量检测金黄色葡萄球菌的方法。优化设计合成溶葡球菌酶序列,构建重组表达载体pQE30-Lys,转化至大肠杆菌M15并诱导表达,镍柱纯化得到目的蛋白。利用重组溶葡球菌酶和ATP生物发光法特异定量检测金黄色葡萄球菌并与平板计数对比。成功表达了重组溶葡球菌酶,并建立了特异定量检测金黄色葡萄球菌的方法,与平板计数具有显著线性关系。本研究建立的将重组溶葡球菌酶和ATP生物发光法相结合的检测方法操作快捷简单,具有良好的应用前景。     

棉花植株中的单宁测定方法研究

通过 3种方法测定棉花组织中单宁含量比较表明,Folin酚还原法测定的 4个品种不同组织和不同生育期顶叶的含量显著高于正丁醇盐酸法 (即花色素反应,专门用于缩合单宁的测定)近 2倍,说明这种方法测定出的是相对总酚含量,用于表达棉花缩合单宁的含量是不合适的,而香草醛法测定结果与正丁醇盐酸法差异不显著,可用于棉花组织单宁含量的测定.在棉花各个组织中,花萼、铃皮和叶片缩合单宁含量较高,陆地棉中一般达 5%～10%;花瓣、花柱子房和铃心中含量较低 (2%左右).顶端嫩叶缩合单宁含量从苗期 (1%以下)起不断增加,至吐絮期达最高 (10%左右),表明缩合单宁含量与植物组织成熟衰老和木质化程度密切相关.     

Gene cloning and expression of cadherin in midgut of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and its Cry1A binding region

Cadherins belong to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is related to insect’s resistance to Cry1A. The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm, which is 5581 bp in full-length, encoding 1730 amino acid residues (BtR-harm was deposited in GenBank and the accession number is AF519180). Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23, respectively. The inferred amino acid sequence includes a signal sequence, 11 cadherin repeats, a membrane-proximal region, a transmembrane region and a cytoplasmic region. Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2% identity and highly similar to three other lepidopteran cadherin from Bombyx mori, Manduca sexta and Pectinophora gossypiella, with the sequence identities of 60.3.6%, 57.5% and 51.0%, respectively. The cDNA encoding cadherin gene was expressed successfully in E. coli and the recombinant proteins can bind with Cry1Ac. Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence, which located between amino acid 1217 and 1461. Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H. armigera, very low expressed in foregut and hindgut and was not expressed in other tissues. After H. armigera producing resistance to Cry1Ac, the expression quantity of BtR-harm significantly decreased in midgut of H. armigera. It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H. armigera and the binding region was located on a contiguous 244 amino acid sequence, suggesting that the decrease of expression quantity of BtR-harm is one of the main reasons for H. armigera resistance to Cry1Ac.     

桂南爬树蕨的组织结构研究

本文对桂南爬树蕨的主要器官进行了解剖结构的研究,结果表明：茎的中柱为仅有两个新月型的维管束,中柱内及其周围的机械组织形成“８”字形。与爬树蕨相同,为新的中柱类型,中柱周围的几层皮层组织特化为机械组织的起始位置为对正维管束长轴的中央。而髓部的机械组织分化的起点是在中轴的中间位置,呈线形,同时向两侧分化。木质部分化为沿圆周线方向两端式,为新的分化方式。叶为等面叶,与爬树蕨相比,叶的厚度小,叶肉细胞层数少且细胞排列紧密,机械组织明显少。叶缘近圆形。根为二原型,直径比后者略小些。孢子类型与爬树蕨相同,孢子囊群无囊群盖。     

棉铃虫蛾对寄主植物挥发油的嗅觉电生理及行为反应

本项研究以Tenax TA、Tenax GR为吸附剂,正己烷为洗脱剂,得到了玉米雄穗、玉米花丝、棉花花、棉花顶尖、棉蕾、芹菜、花生叶及花的挥发油。触角电位(EAG)测试结果表明,参试寄主植物及器官的挥发油均有一定的EAG活性,EAG的反应幅度为0.1～1.2mV,雌性棉铃虫Helicoverpa armigera对其产卵寄主或器官的EAG反应在1～10日龄内有随着日龄增加而增大的趋势,交配蛾对棉花花、棉花嫩尖、芹菜、棉蕾的挥发油的EAG反应显著高于处女蛾,雌蛾在经过花生、玉米花丝及棉花顶尖挥发油处理的滤纸上落卵量显著高于对照,表明这几种挥发油中含有可刺激棉铃虫选择其产卵的它感信息化合物。剂量反应曲线表明,交配可显著降低雌蛾对棉花嫩尖和棉花花挥发油的敏感阈值。四臂嗅觉仪测试棉铃虫对不同寄主植物挥发油的行为反应表明,棉蕾、棉花叶、玉米花丝、花生的挥发油均具有-定的诱蛾活性,选择系数分别为0.78、0.61、0.49、0.31。     

中国各棉区棉铃虫滞育解除特点

试验研究了我国华南棉区、长江流域棉区、黄河流域棉区、北部特早熟棉区和西北内陆棉区棉铃虫Helicoverpa armigera(Hubner)13个地理种群的滞育解除特点。随纬度升高地理种群滞育蛹在高温下的发育时间显著延长,羽化格局趋于松散,在30℃下湖南衡阳种群和新疆哈密种群的发育时间分别为(15.6±4.9)d和(41.4±9.0)d。低温处理可明显降低长江流域以北种群的滞育历期,但对华南棉区北部种群则无明显的活化作用。湖南衡阳种群、湖北武穴种群、北京种群和新疆哈密种群活化滞育蛹的发育起点温度和有效积温分别为17.4℃和146.O日度,16.6℃和146.5日度,15.7℃和156.4日度及15.7℃和167.7日度。     

我国新疆和湖南棉铃虫种群的遗传学研究

采自湖南衡阳(HY)和新疆哈密(HM)的棉铃虫Helicoverpa armigera(Hubner)种群被室内杂交和回交,杂交F₁代和回交F₂代雌成虫的产卵量、卵孵化率和产卵时间等繁殖力参数均无明显的变化。在22℃下,HM、HY、HM♀×HY♂、HY♀×HM♂和(HY♀×HM♂)×HY♂后代蛹滞育的临界光周期分别为13h 35 min,11 h,11 h 45 min,11 h 43min和11 h9min。在一15℃下,HM、HY、HM♀×HY♂、HY♀×HM♂、HY♀×(HY♀×HM♂)♂和(HY♀×HM♂)♀×HM♂后代滞育蛹的致死中时间(LT”)分别是159.7h、34.27h、100.65 h、116.75h、90.78h和135.58h,亲本F₁代显性度D(HM♀×HY♂)和D(HY♀XHM♂)分别为0.3998和0.5926。遗传分析表明棉铃虫滞育蛹的抗寒性由多个不完全显性的基因控制。     

Novel indole alpha-methylene-gamma-lactones as potent inhibitors for AKT-mTOR signaling pathway kinases

In an effort to generate novel anticancer agents, a series of hybrids of alpha-methylene-gamma-lactones and 2-phenyl indoles has been synthesized and evaluated for inhibition activities on the phosphorylation of AKT, mTOR, p70S6 kinase, and 4E-BP1. The results indicate that substitutes on the gamma-position of lactones have a rather significant influence on inhibition activities.