拟南芥新型脂转移蛋白AtDHyPRP1的亚细胞定位及其对灰霉菌的抗性

通过遗传转化技术研究了拟南芥脂转移蛋白AtDHyPRP1在细胞中的定位及其对真菌病原体的抗性。采用PCR方法从拟南芥Ws生态型克隆了AtDHyPRP1基因,构建产生pRI101-AN-AtDHyPRP1植物双元表达载体和pCAMBIA1302-AtDHyPRP1-GFP融合表达载体,经农杆菌介导的叶盘和浸花法得到烟草和拟南芥转基因植株。AtDHyPRP1基因能够明显增加烟草对灰霉菌的抗性,转AtDHyPRP1烟草叶片的被侵染部位有大量H2O2积累,激光共聚焦显微观察发现AtDHyPRP1蛋白定位于细胞表面。说明AtDHyPRP1蛋白在合成后被分泌到细胞外执行特殊的功能,与植物抗病防御机制有关。     

拟南芥HyPRP蛋白AZI1在转基因烟草中的亚细胞定位及其对灰葡萄孢的抗性

通过农杆菌转化法得到了整合有拟南芥AZII基因的烟草植株,进一步利用转基因烟草分析了AZI1蛋白的亚细胞定位及其对真菌病原体的抗性特征。在上下游引物5’端分别引入NcoI和SpeI酶切位点,采用高保真耐热DNA聚合酶彤Pfu从拟南芥Co1-0生态型基因组DNA扩增AZII基因的编码序列,用NcoI和Spel对扩增片段和pCAMBIA1302质粒载体进行双酶切,通过T4DNA连接酶构建产生AZII-GFP融合表达载体。用包含融合表达载体的农杆菌细胞转化烟草叶片,经潮霉素选择获得了完整的再生植株,并收取了T。代种子。激光共聚焦显微观察发现,AZI1蛋白主要定位于细胞表面。病原体侵染结果显示,AZI1基因能够明显提高烟草对灰葡萄孢的抗性。说明AZI1蛋白通过分泌途径被定位到细胞表面后,能够抑制真菌病原体对植物组织的侵染过程。     

拟南芥乙酰乳酸合成酶基因(Atals)多位点突变以及转基因过表达植株除草剂抗性分析

乙酰乳酸合成酶(ALS)是支链氨基酸、缬氨酸、亮氨酸和异亮氨酸生物合成途径中的关键酶,也是多种除草剂的靶点。为了研究als基因不同突变位点组合后其抗除草剂抗性的变化,并整合和增强植株对不同类型除草剂的抗性,本研究对已知抗性位点进行组合并进行了拟南芥转基因分析。我们通过重叠延伸PCR技术体外突变扩增四个已知位点突变的P197S/R199A/W574S/S653F拟南芥Atals,克隆到pCAMBIA1 300-GFP载体上,从而构建了四个位点突变的m4Atals-GFP融合蛋白过表达载体。然后用农杆菌介导法转化野生型拟南芥Col-0,获得转基因株系。采用潮霉素抗性筛选鉴定阳性转基因植株,并利用荧光体式显微镜观察过表达植株以及在蛋白水平检测GFP-m4Atals融合蛋白表达情况。对纯合转基因株系进行除草剂抗性分析。分析表明转基因拟南芥具有磺酰脲和咪唑啉酮两种除草剂的整合抗性。此研究有助于系统地分析als基因不同突变位点对抑制剂的抗性,有效避免和应对自然界als单一位点突变的杂草的困扰。     

棉花乙烯合成基因促进拟南芥和烟草不定根发生的研究

从棉花纤维cDNA中克隆获得乙烯合成基因GhACO3,构建了植物过量表达载体p35S::GhACO3.通过花序侵染法和叶盘法分别转化拟南芥和烟草,利用卡那霉素筛选及分子检测获得转基因阳性拟南芥和烟草植株.结果表明,GhACO3基因已整合到拟南芥和烟草基因组中;经过纯合筛选后获得转基因T2代拟南芥植株;与野生型拟南芥相比,GhACO3基因对拟南芥不定根发生具有显著促进作用;与野生型烟草植株相比,转GhACO3基因烟草不定根发生得到了显著的促进.研究表明,GhACO3基因的过量表达能够促进拟南芥和烟草不定根的形成发育,为进一步探讨GhACO3的生物学功能和进行转基因育种奠定了基础.     

盐穗木功能未知蛋白 (HcUKPP)的亚细胞定位

藜科的极端盐生植物盐穗木（Halostachys caspica）的高盐胁迫抑制差减文库中有39%的功能未知蛋白（proteins with obscure features, POFs）,利用亚细胞定位分析可以初步判断其可能的功能.将盐穗木的1个POF-cDNA序列HcUKPP的编码区构建至pCAMBIA1301-GFP植物表达载体上,冻融法将重组质粒pCAMBIA1301-HcUKPP-GFP转化农杆菌EH105A,利用花序浸染法将基因导入拟南芥,经潮霉素筛选获得T1代阳性幼苗.通过激光扫描共聚焦显微镜观察转基因拟南芥植株的根部细胞. 结果显示,表达GFP蛋白的对照转基因植株中,绿色荧光在细胞核、细胞膜以及细胞质中均能检测到,而表达HcUKPP-GFP融合蛋白的转基因植株中,绿色荧光只在细胞质膜上表达,说明HcUKPP蛋白为细胞质膜相关蛋白.本研究为深入探讨盐穗木未知蛋白的功能奠定了基础.     

AtRGS1-GFP融合蛋白对AtRGS1细胞定位的研究

应用Gateway克隆技术构建了以CaMV35S为启动子,含AtRGS1-GFP融合基因的植物表达载体,并分别用根癌农杆菌介导法和PEG介导法转化拟南芥野生型（C01）悬浮细胞系和幼苗叶片原生质体,利用荧光显微镜观察AtRGS1-GFP融合基因在转化受体系统中的表达与定位。结果显示,在含AtRGS1-GFP融合基因的转化细胞系中,GFP绿色荧光在细胞膜（壁）上特异表达;原生质体瞬时表达系统中,GFP绿色荧光在细胞膜上强烈表达,表明AtRGS1蛋白定位于细胞质膜上。     

转甜瓜CmSAMDC基因拟南芥的获得及其耐盐性研究

该研究以哥伦比亚生态型野生拟南芥为材料,将甜瓜CmSAMDC基因构建到植物双元表达载体pCAMBIA1304上,采用农杆菌介导法转入拟南芥,在含有50mg/L潮霉素(Hyg)MS固体培养基上筛选转基因后代,并利用T3代转基因幼苗进行耐盐性分析。结果显示:(1)成功构建了植物超表达载体35S∷CmSAMDC,并经农杆菌介导法转化拟南芥,潮霉素抗性筛选后获得了转CmSAMDC基因拟南芥T3代植株。(2)转CmSAMDC基因拟南芥T3代幼苗在含100、150、200mmol/L NaCl培养基中,侧根长势比野生型植株更为健壮;在200mmol/L NaCl浇灌处理后,转CmSAMDC基因T3代植株仍能维持正常生长,而野生型植株的生长明显受到抑制;在400mmol/L NaCl浇灌处理后16d,野生型植株逐渐死亡,而转基因植株仍能继续存活;对盐胁迫后植株的脂质过氧化程度(MDA)测定显示,野生型植株MDA水平较转基因植株上升更为明显。研究表明,过表达甜瓜CmSAMDC基因增强了转基因拟南芥的耐盐性。     

两种瞬时表达体系研究拟南芥Profilin-1的亚细胞定位

使用两种瞬时表达方法研究Profilin-1(PRF1)的亚细胞定位,并比较了2种瞬时表达体系在亚细胞定位研究中的优缺点。利用拟南芥幼叶作为材料,提取叶片的RNA,采用特异性引物RT-PCR的方法克隆PRF1基因,连接到p CAMBIA1300-GFP的改造载体上,成功的构建p CAMBIA1300-GFP-PRF1的表达载体。然后分别利用PEG转化拟南芥原生质体、农杆菌浸染烟草叶片两种技术进行了瞬时表达,并在激光共聚焦显微镜下观察绿色荧光蛋白(GFP)融合蛋白的表达。研究结果表明,将PRF1基因导入拟南芥的原生质体和烟草表皮细胞后,融合蛋白绿色荧光均能被观察到,PRF1基因与GFP融合蛋白的产物在烟草表皮细胞中主要定位在细胞质和外周细胞器中,在拟南芥的原生质体中的细胞核和细胞质中都有定位。两种不同的瞬时表达体系中PRF1蛋白的定位出现了不同,这可能与同源或异源表达的植物的特性相关。     

拟南芥CYCD2;1基因植物表达载体的构建及在烟草中的遗传转化分析

细胞分裂是生物的基本特征之一,在植物生长发育的过程中,发挥着极其重要的作用,细胞周期蛋白CYCD2;1基因作为一个调控因子,对调节细胞周期具有重要作用。本文以拟南芥细胞周期蛋白(At CYCD2;1基因)作为研究对象,利用PCR技术从拟南芥花序c DNA扩增出At CYCD2;1基因,构建植物表达载体(pROKIIAt CYCD2;1)并利用农杆菌介导的叶盘法转化野生型烟草。转基因植株的PCR检测结果表明,At CYCD2;1基因已经整合到了烟草基因组中。在T2代植株中,通过实时定量荧光PCR检测显示,At CYCD2;1在mRNA水平也均有表达。过量表达CYCD2;1的转基因烟草在花器官中存在明显表型,与野生型相比主要表现为转基因植株花冠宽度变大,花瓣和萼片长度变长,果实变大,上述结果表明At CYCD2;1基因影响花的发育。     

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.     

Constitutive expression of Arabidopsis NPR1 confers enhanced resistance to the early instars of Spodoptera litura in transgenic tobacco

In Arabidopsis , NPR1 ( AtNPR1 ) regulates salicylic acid (SA)-mediated activation of PR genes at the onset of systemic acquired resistance. AtNPR1 also modulates SA-induced suppression of jasmonic acid-responsive gene expression, and npr1 mutants manifest enhanced herbivore resistance. We have raised stable transgenic tobacco lines, expressing AtNPR1 constitutively, which showed elevated expression of PR1 and PR2 genes upon SA treatment. Herbivore bioassays with a generalist polyphagous pest, Spodoptera litura , revealed that the transgenic lines exhibited enhanced resistance compared to the wild-type plants, particularly with respect to younger larval populations. Insect-mediated injury induced several protease inhibitors (PIs), more significantly a 40-kDa serine PI in all the tobacco lines, but the induction was higher in the transgenic plants. We show in this communication that heterologous expression of AtNPR1 provides enhanced resistance to early larval populations of the herbivore, Spodoptera in transgenic tobacco plants.     

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.