小麦淀粉合酶基因I的克隆及反义和RNAi载体的构建

采用RT-PCR方法从小麦品种豫教2号发育的籽粒中克隆出淀粉合酶I基因(starch synthase I,SSI)部分cDNA片段(795 bp)(GenBank No.EF221762),同源性比较结果显示,它与GenBank上已报道的SSI基因有高度同源性。以p WM101质粒为基础,构建了由35S启动子调控的SS I基因的反义表达载体p WM101SSI;另外,还以pFGC5941质粒为基础,构建了SSI基因的RNAi载体pFGC5941SSIsa,这些载体的构建为研究此基因的功能奠定了基础。     

日本血吸虫GSK3蛋白编码cDNA的克隆、表达及其重组蛋白的免疫保护研究

本研究利用生物信息学分析了日本血吸虫(Schistosoma japonicum,sj)Glycogen synthase kinase 3(GSK3)蛋白,并对其中一个GSK3蛋白的编码cDNA进行了克隆和原核表达,制备了特异性的多克隆抗体.同时,还初步评估了重组蛋白的免疫保护效果.生物信息学分析表明在日本血吸虫数据库存在两种GSK3蛋白,且其中一个SjGSK3在日本血吸虫不同发育时期均有转录.Western blot结果表明本研究制备的抗体能特异性识别日本血吸虫SjGSK3重组蛋白,表明该重组蛋白具有良好的免疫原性.动物实验表明免疫SjGSK3重组蛋白的动物与佐剂对照组比较分别获得了平均10.6％减虫率和40.5％肝脏减卵率.     

稻稗HJHL-715种群对五氟磺草胺的抗药性水平及抗性机理分析

为明确东北稻区稻稗Echinochloa oryzoides HJHL-715种群对五氟磺草胺的抗性水平及抗性机制,采用整株生物测定法测定稻稗种群对五氟磺草胺的敏感性,明确抗性种群的交互抗性和多抗性情况,研究3种细胞色素P450抑制剂对其敏感性的影响;并应用分子生物学方法进行稻稗的乙酰乳酸合酶(acetolactate synthase,ALS)离体活性测定、ALS基因序列分析及其表达量测定。结果表明:在东北稻区,五氟磺草胺对稻稗HJHL-715种群鲜重的抑制中剂量GR50为62.53 g/hm^2;稻稗HJHL-715的ALS基因序列中未发现氨基酸突变,其ALS离体活性与敏感种群的ALS离体活性无显著性差异,ALS基因表达量显著低于敏感种群。1-氨基苯并三唑(1-aminobenzotriazole,ABT)、胡椒基丁醚(piperomyl butoxide,PBO)、马拉硫磷3种P450抑制剂显著提高了稻稗HJHL-715种群对五氟磺草胺的敏感性,使其对五氟磺草胺的GR50由原来的62.53 g/hm^2分别下降到5.78、5.02、3.53 g/hm^2。表明东北稻区已经出现了对五氟磺草胺具有高水平抗性的稻稗种群,稻稗HJHL-715种群对五氟磺草胺的抗性很可能是由细胞色素P450介导的代谢增强所致。     

宁夏枸杞八氢番茄红素合成酶基因的克隆与序列分析

类胡萝卜素是植物叶绿体光合作用的辅助色素,并能保护叶绿素免受高温强光的破坏(Bartley et al.,1995).现在越来越多的医学研究表明,类胡萝卜素与人类健康密切相关.约有10%的类胡萝卜素是维生素A的前体(Krinsky,1989).据统计,每年有70多个国家的3 000多万儿童因缺乏维生素A而造成致命性疾病(Cheryl et al.,1997).此外,类胡萝卜素在防癌抗癌、预防心血管疾病、增强人体免疫力等方面起着重要的作用(陶俊等,2002).通过对菲律宾、加拿大和中国的食道癌患者的调查已证明β-胡萝卜素能延缓疾病的进程(韩雅珊,1999).由于类胡萝卜素在许多植物中含量较低,并且难以用化学方法合成.因此,利用基因工程手段调控类胡萝卜素的代谢积累,进而大量生产类胡萝卜素已成为研究者们所共识的研究方向之一.目前通过基因工程手段已获得转类胡萝卜素合成酶基因的"金大米"和"金油菜"(陶俊等,2002).     

Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.     

油茶查尔酮合酶和异构酶基因的cDNA克隆

查尔酮合酶和查尔酮异构酶是类黄酮代谢与色素苷代谢的关键酶.以油茶近成熟种子cDNA文库和EST文库为材料,通过分子克隆方法鉴定了1条查尔酮合酶基因全长cDNA和1条查尔酮异构酶全长cDNA.结果表明:油茶查尔酮合酶基因的cDNA含1479 bp,编码412个氨基酸,为目前最长的查尔酮合酶,与其它物种的查尔酮合酶具有极高的相似性,在进化上高度同源;油茶查尔酮异构酶基因的cDNA含899 bp,编码206个氨基酸,与茶的查尔酮异构酶基因高度同源,但与其它物种的相似性较低.     

Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis

Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs.     

Cross-resistance profile of mesosulfuron-methyl-resistant Italian ryegrass in the southern United States

Diclofop-resistant Lolium species (ryegrass) is a major weed problem in wheat production worldwide. This study was conducted to determine the resistance pattern of diclofop-resistant ryegrass accessions from the southern United States to mesosulfuron-methyl, a recently commercialized herbicide for ryegrass control in wheat; to determine the cross-resistance pattern of a Lolium multiflorum Lam. (Italian ryegrass) accession, 03-1, to acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibitors; and to determine the resistance mechanism of Italian ryegrass to mesosulfuron-methyl. Seventeen ryegrass accessions from Arkansas and Louisiana, including standard resistant and susceptible accessions, were used in this experiment. Fourteen of the 17 accessions were more resistant (four- to > 308-fold) to diclofop than the standard susceptible biotype. One accession, 03-1, was resistant to mesosulfuron-methyl as well as to other ALS inhibitor herbicides such as chlorsulfuron, imazamox and sulfometuron. Accession 03-1, however, did not show multiple resistance to the ACCase inhibitor herbicides diclofop, fluazifop, clethodim, sethoxydim and pinoxaden, nor to glyphosate. The in vivo ALS activity of the 03-1 biotype was less affected by mesosulfuron-methyl than the susceptible biotype. This indicates that the resistance mechanism of Italian ryegrass to mesosulfuron-methyl is partly due to an alteration in the target enzyme, ALS. It is concluded that diclofop-resistant ryegrass in the southern United States can be generally controlled by mesosulfuron-methyl. However, mesosulfuron-methyl must be used with caution because not all ryegrass populations are susceptible to it. There is a need for more thorough profiling of ryegrass resistance to herbicides.     

昆虫几丁质合成关键酶功能及其RNAi技术在害虫防治中的研究进展

几丁质对昆虫的生长发育至关重要,且不存在于植物和哺乳动物中,因此其合成途径的关键酶是较为理想的杀虫剂靶标。近年来,多种害虫的几丁质合成关键酶基因被克隆和鉴定,通过RNAi技术应用于一些重要害虫的防治研究工作中。本文较为系统的总结了近些年害虫几丁质合成关键酶的基因克隆及功能研究进展,重点阐述了几丁质合成酶、海藻糖酶基因基于RNAi技术应用于害虫防治取得的研究进展,分析了RNAi技术通过转基因植物表达dsRNA（RNAi抗虫作物）以及作为核酸农药（dsRNA）直接进行作物喷施的两种应用途径,文章最后还探讨了RNAi在害虫防治中面临的主要瓶颈问题以及主要应对策略。上述较为系统的归纳和总结,目的是为今后基于几丁质合成关键酶的RNAi技术应用于害虫绿色防控研究提供有价值的理论指导。     

中国野生毛葡萄芪合酶基因表达及对葡萄抗白粉病的影响

【目的】克隆中国野生毛葡萄（Vitis quinquangularis）‘丹凤-2’芪合酶（stilbene synthase）基因（STS）并研究其功能,为提高欧洲葡萄（V. vinifera）的白粉病抗性及品质提供依据。【方法】利用同源克隆法获得中国野生毛葡萄‘丹凤-2’芪合酶基因VqSTS9和VqSTS21,构建植物过表达载体;用无核白单芽茎段诱导出分生愈伤组织,作为农杆菌介导法遗传转化的受体材料,获得抗性植株,经过不同水平检测,确定转基因植株;对野生型和转基因植株叶片人工接种葡萄白粉病菌（Uncinula necator）,通过显微技术观察叶片受白粉病菌侵染后的情况,比较两者对白粉病的抗性;利用实时荧光定量PCR（qRT-PCR）分析野生型和转基因植株在自然条件和接种白粉病菌后STS及其相关基因的表达,用高效液相色谱法（HPLC）检测转基因植株中芪类物质的种类与含量。【结果】同源序列克隆得到VqSTS9（JQ868689）与VqSTS21（JQ868677）的cDNA序列,长度为1 179 bp。经PCR和Western blot检测,鉴定出过表达VqSTS9无核白植株4株和过表达VqSTS21无核白植株3株。显微观察发现,与野生型植株相比,转VqSTS9 和VqSTS21植株叶片上的菌丝生长较慢,表现出对白粉病的抗性。qRT-PCR结果表明,自然生长条件下,与野生型植株相比,转VqSTS9和VqSTS21植株STS的表达量提高,STS上游苯丙氨酸裂解酶基因（PAL）、下游白藜芦醇糖基转移酶基因（RSGT）、转录因子基因（MYB14、MYB15）的表达量均不同程度上升,而查尔酮合成酶基因（CHS）表达量降低;人工接种白粉病菌后,与野生型植株相比,转基因植株STS表达量显著上调。高效液相色谱分析表明,自然条件下,芪类物质主要以反式云杉新苷形式存在,转基因植株芪类物质的含量高于野生型植株;在接种白粉病菌诱导表达后,除了反式云杉新苷,还产生了反式白藜芦醇和葡萄素,即转基因植株体内芪类物质的种类和含量均有所增加。【结论】将VqSTS9、VqSTS21转入无核白后,转基因植株STS的表达量增高,芪类物质的含量与种类增加,并抑制白粉病菌的生长。因此,中国野生毛葡萄‘丹凤-2’携带的VqSTS9和VqSTS21能够增强欧洲葡萄对白粉病的抗性,‘丹凤-2’可用作葡萄抗病性育种的种质资源。