油菜抗咪唑啉酮类除草剂基因BnALS1R等位基因特异PCR标记的开发与应用

油菜抗咪唑啉酮类除草剂基因BnALS1R是从抗性突变体M9中克隆获得,抗性基因BnALS1R与野生型基因BnALS1存在1处SNP,即乙酰乳酸合酶第638位丝氨酸残基被天冬酰胺酸替代。为获得油菜抗除草剂基因BnALS1R的分子标记,根据该处点突变,结合获得的BnALS3与BnALS1序列,开发30条等位基因特异PCR (allele-specific PCR,AS-PCR)引物,采用筛选出的3条AS-PCR引物在F₂、BC₁和BC₂群体中进行PCR扩增。结果表明,该标记有效检测出群体中存在的3种基因型,其分离比分别为1∶2∶1、1∶1、1∶1,均遵循单基因遗传规律。应用该标记对获得的抗性恢复系进行PCR扩增,结果发现所有抗性恢复系均能扩增出抗性基因BnALS1R目的条带,表明3条标记引物可应用于抗性基因的检测。AS-PCR标记的获得将促进以抗性基因进行油菜抗除草剂分子标记辅助选择育种。     

酵母表达芥子酶基因TGG4、TGG5的酶学特性差异研究

十字花科植物广泛存在芥子酶,并利用芥子酶以抵御病虫害的侵袭。TGG4和TGG5是在拟南芥中发现的新型芥子酶基因,二者基因序列极为相近。本试验通过对转TGG4和TGG5基因酵母的诱导表达及相应重组蛋白的纯化,探讨两个蛋白的酶学差异性。结果表明:TGG4、TGG5的最大Vc激发浓度都为1mmol/L;但是在最适反应温度和最适反应pH上,两个重组蛋白略有不同,TGG4和TGG5的最适温度分别为67℃和57℃,而最适pH分别为6.5和5.5,TGG4比TGG5要耐高温耐碱性;这两个重组蛋白酶活性都受NaCl的抑制。     

水稻抗咪唑啉酮类除草剂基因ALS功能标记的开发与应用

选育和利用抗除草剂水稻品种具有重要的生产实践意义。通过筛选水稻资源, 发现了抗除草剂材料金粳818, 其ALS基因编码区第1880位碱基存在一个由G到A的碱基变异, 导致丝氨酸突变为天冬酰胺, 从而具有除草剂抗性。本研究基于该位点的碱基变异, 设计了11条等位基因特异PCR (allelic-specific PCR, AS-PCR)引物, 经过优化筛选, 获得两个引物组合F1N (S1/S9)和F1M (S1/S10), 将该标记命名为AS-ALS。利用F₂群体及其亲本和杂交种, 结合AS-ALS标记检测和除草剂抗性分析, 结果表明感除草剂ALS-G等位基因型只能被F1N引物对有效扩增, 抗除草剂ALS-A等位基因型只能被F1M引物对有效扩增, 而杂合基因型能同时被两对引物F1N和F1M扩增, ALS-A纯合或杂合等位基因型都表现抗除草剂, ALS-G纯合基因型表现感除草剂。因此本研究开发的标记能有效区分除草剂抗性基因的3种基因型, 基因型与表型完全对应。该标记用于回交育种, 可以选择ALS-A杂合基因型单株, 剔除ALS-G纯合等位基因型, 在自交的F₂保留ALS-A纯合基因型单株, 连续自交, 能快速获得除草剂抗性稳定的水稻材料。该除草剂抗性基因的功能标记还可用于咪唑啉酮类除草剂抗性资源筛选。     

绿盲蝽膜结合型海藻糖酶ALTre-2基因表达、纯化及酶学特性

以绿盲蝽为材料,在前期获得绿盲蝽膜结合型海藻糖酶(ALTre-2)基因的基础上,构建了ALTre-2原核表达载体(pET28a-ALTre-2),经IPTG诱导表达、变性、复性和蛋白纯化后,获得具海藻糖酶活力的重组蛋白,最后在以海藻糖为底物及重组蛋白浓度为1.1 mg·mL-1的条件下,对纯化得到的重组ALTre-2蛋白进行活性检测,确定了其酶促反应的最佳pH和最适温度.试验结果表明:ALTre-2基因在大肠杆菌BL21(DE3)中能够高效表达一个约60 kD大小的蛋白,SDS-PAGE显示该蛋白以包涵体形式存在,经变性、复性及蛋白纯化获得了具海藻糖酶活力的重组蛋白;该重组ALTre-2蛋白在pH为7.0时活性最高,同时重组ALTre-2蛋白酶活性最适温度是50℃.研究结果为深入探讨绿盲蝽膜结合型海藻糖酶分子调控机制奠定基础.     

耐三唑并嘧啶类除草剂花生种质创制与鉴定

目前耐除草剂花生的种质资源匮乏,这制约了花生栽培模式的多元化发展。为创制耐不同除草剂的花生种质资源,我们利用甲基磺酸乙酯诱变技术创建了一个由55,000多个花生株系组成的诱变群体,随后选择多种不同除草剂对该群体进行筛选,获得多个对不同除草剂具有耐性的花生突变体。其中一个突变体在田间叶面喷施处理和多种实验室条件下的除草剂耐受性评估试验中均表现出对唑嘧磺草胺和双氟磺草胺极强的耐性,但这种耐性性状并未对花生的产量与品质产生不良影响。为明确这种性状是否与除草剂靶标抗性相关联,我们比较分析了该突变体与野生型中这2种除草剂靶标酶(乙酰羟酸合酶,AHAS)的基因序列及其表达量的差异。分子克隆与序列分析显示,花生A10与B10染色体上各含一个与拟南芥AtAHAS序列高度相似的基因,分别命名为AhAHAS1a与AhAHAS1b。花生A08与B08染色体上亦各含一个AHAS基因,分别命名为AhAHAS2a与AhAHAS2b。然而,与野生型相比,突变体中的这些基因并未发生能够致使氨基酸序列发生改变的核苷酸替换。进而发现, AhAHAS基因的表达量在突变体与野生型中没有显著差异。这些结果表明,该花生突变体的除...     

抗除草剂转基因水稻的研究进展及其安全性问题

近年来,抗除草剂转基因作物的研究取得了一些进展。本文简述了抗除草剂转基因作物的发展历史以及除草剂的作用机理,分别介绍了抗EPSPS抑制剂基因、抗ALS抑制剂基因、乙酰CoA转移酶基因、阿特拉津氯水解酶基因、细胞色素P450基因和原卟啉原氧化酶基因这6类抗除草剂基因的来源,抗性机理以及目前它们在转基因水稻上的应用。最后,对转基因水稻的食用安全性,转基因释放、飘移对生态的影响进行了探讨,并对转基因水稻的未来发展进行了展望。     

香蕉内生拮抗细菌KKWB-5的几丁质酶在大肠杆菌中的表达纯化与复性研究

将香蕉内生拮抗细菌KKWB-5的几丁质酶在大肠杆菌中的表达,旨在检测该酶对香蕉枯萎病病原菌4号小种的抗性。PCR扩增该基因连入大肠杆菌表达载体pET22b,导入大肠杆菌BL21(DE3),IPTG诱导重组菌株,SDS-PAGE分析目的蛋白的表达,对目的蛋白进行纯化,并将纯化后的目的蛋白复性,对复性产物进行水解几丁质活性和拮抗香蕉枯萎病活性的分析。SDS-PAGE分析表明目的蛋白在大肠杆菌中以包涵体的形式大量表达,经变性Ni2+柱纯化得到了高纯度的重组蛋白,表达量达293 mg/L;稀释和透析两种复性方法中,透析复性法的目的蛋白得率较高,为84.6 %。复性后的目的蛋白具有水解几丁质的活性,同时对香蕉枯萎病病原菌也具有一定的抗性,但抗性较弱。该几丁质酶在大肠杆菌中得到高效表达,且复性后的几丁质酶对香蕉枯萎病病原菌4号小种有一定的抑制作用,该酶对KKWB-5菌株拮抗香蕉枯萎病菌的能力有一定的贡献。     

乙酰乳酸合成酶及ALS基因研究概述

乙酰乳酸合成酶抑制剂类除草剂以其独有的优点,20世纪90年代以来一直占领世界除草剂市场。抗草甘膦转基因作物的问世,草甘膦的销售量大幅增加,乙酰乳酸合成酶抑制剂类除草剂销量不断减少。然而,大量抗草甘膦杂草的产生,使得人们期望培育出抗不同类型除草剂转基因作物,抗ALS抑制剂类除草剂成为人们的首选。笔者概述了乙酰乳酸合成酶的结构、功能、除草剂作用机理、抗ALS基因的研究现状。期望对抗ALS抑制剂基因的筛选,利用提供参考。     

三七多聚半乳糖醛酸酶抑制蛋白基因的功能分析

多聚半乳糖醛酸酶抑制蛋白(PGIP)是植物抗病防御系统的重要组成部分。在前期研究中,从三七中分离得到一个编码PGIP蛋白的基因PnPGIP,PnPGIP在转录水平响应三七根腐病菌茄腐镰刀菌的侵染。为深入分析PnPGIP的功能,构建PnPGIP的原核表达载体,转入大肠杆菌BL21(DE3)进行大量表达,纯化获得PnPGIP的重组蛋白,并进行体外平板抑菌试验;构建PnPGIP的植物超表达载体,通过根癌农杆菌介导产生了PnPGIP转基因烟草,分析T2转基因烟草离体叶片对茄腐镰刀菌的抗性。PnPGIP原核重组蛋白对茄腐镰刀菌、胶孢炭疽菌、核盘菌和轮枝状镰刀菌的菌丝生长具有很强的抑制作用,并且随着蛋白量的增加,抑制活性也逐渐增强。q RT-PCR分析结果显示,PnPGIP在T2转基因烟草中大量表达。此外,与野生型烟草相比,转基因烟草对茄腐镰刀菌的抗性显著增强。PnPGIP是三七中参与茄腐镰刀菌防御反应的重要抗病基因。     

海马齿甜菜碱醛脱氢酶基因克隆、高效表达及酶学特性分析

在许多渗透调节剂中,甜菜碱是最理想的有机小分子渗透调节物质。甜菜碱在植物体内大量积累不会带来危害,同时能提高植物对环境胁迫的抗性。将海马齿中克隆到的甜菜碱醛脱氢酶基因构建到表达载体pET-28a(+)上,获得重组载体pET-SpBADH并将其成功地转化到BL21(DE3)中得到重组工程菌,经IPTG诱导能高效表达55 kD目的蛋白,表达量可以达到301 μg mL–1。酶学特征分析表明,该蛋白最适pH值为7.2,在偏碱条件下能维持较高的催化活性;SpBADH蛋白对高温敏感,且温度对催化活性影响较大,超过55℃时酶活性只有20%,最适酶催化活性温度为37℃;而有机小分子醇类对酶的催化活性有保护作用,可以通过自身特征维持酶催化活性的微环境。     

油菜抗咪唑啉酮类除草剂基因标记的开发与应用

草害已成为严重制约我国油菜生产的重要因素。种植抗除草剂品种和采用化学除草是防控草害的经济有效途径。为了开展分子标记辅助选择(marker-assisted selection,MAS)育种,加速抗除草剂品种培育,本研究针对已发现的抗咪唑啉酮类除草剂油菜M9中BnALS1R基因编码区第1913位点的SNP变异,开发高通量、低成本的竞争性等位基因特异PCR (kompetitive allele specific PCR, KASP)标记。采用筛选出的KBA1R19681913B标记在2个F_2群体中进行KASP反应。结果表明,该标记能有效检测群体中存在的BnALS1R 3种基因型,其分离比均为1︰2︰1,遵循单基因遗传规律,且基因型与表型完全吻合。将该标记用于BnALS1R抗性纯合基因的回交转育,获得200多个抗咪唑啉酮油菜恢复系。该标记还可在油菜苗期鉴定抗性杂交种的纯度。KASP标记KBA1R19681913B的获得为油菜抗除草剂MAS育种以及抗性新种质的培育提供了技术支撑。     

枯草杆菌YLNF基因编码蛋白产物性质的研究

将枯草杆菌ylnF基因经PCR扩增,酶切后插入表达质粒pET15b中,转化大肠杆菌BL21(DE3),诱导表达,金属螯合亲和层析一步纯化,酶活性测定。结果表明:ylnF基因编码产物是依赖于NAD 的前咕啉-2氧化酶,一个西罗血红素生物合成末端的酶。SDS-PAGE显示YlnF亚基分子量约22kD,全酶分子量约25kD,显示酶为单体酶。将Asp102定点突变Ala102,酶活丧失,表明Asp102在催化中起重要作用。     

Efficacy of Rinskor™ (florpyrauxifen-benzyl ester) on Herbicide Resistant Barnyardgrass (<Emphasis Type="Italic">Echinochloa crus-galli</Emphasis>) in Rice Fields of Mekong Delta,Vietnam

Barnyardgrass (Echinochloa crus-galli) seed samples were collected in rice fields in different locations at Mekong delta in Vietnam for herbicide resistance tests. The ALS-resistant and synthetic auxin-resistant E. crus-galli were confirmed at several locations in the Mekong Delta. The average LD₉₀ value of bispyribac, penoxsulam and quinclorac for assessed weed populations was 33.1, 15.1 and 550.2 g a.i.ha^?1 respectively. There were cross resistant barnyardgrass populations to bispyribac and penoxsulam, the LD₉₀ value of the two ALS inhibitors for E. crus-galli was positively correlated at R²=0.39, the cross resistant population was 33.3% of total sample. The correlation analysis was not useful to evaluate the multiple resistance between quinclorac and the two ALS inhibitors, the R² value was lower than 0.05, however, the percentage of multiple resistance weed was 36.2% of population. There was no cross resistance or multiple resistance among the 3 tested herbicides and the new synthetic auxin herbicide Rinskor?. All tested weed samples, including quinclorac-resistant populations, were effectively controlled by Rinskor?. There was no difference between control from Rinskor? in the different herbicide resistant populations. Average LD₉₀ value of Rinskor? in all tested barnyardgrass was 17.1 g a.i ha^?1.     

Herbicide resistant transgenic flax field test: Agronomic performance in normal and sulfonylurea-containing soils

Summary Two linseed flax (Linum usitatissimum) lines transformed with a mutant Arabidopsis ALS gene conferring resistance to sulfonylurea herbicides were tested in a replicated, randomized field test against its nontransformed commercial cultivar parent (cv. NorLin) in normal soil and in soil containing the commonly used sulfonylurea herbicides chlorsulfuron (Glean^®) or metsulfuron methyl (Ally^®). There were no significant differences between the transgenic lines and the parent for any agronomic trait measured in untreated soil, indicating that there is no detrimental effect of T-DNA or foreign gene expression. Similarly, there were no significant differences for performance of the transgenic lines between the untreated and the herbicide treated soils, indicating that the transferred gene does confer a field level of tolerance to the flax. The control NorLin was devastated by the presence of the herbicides in the soil.Abbreviation ALS acetolactate synthase     

Development of rice (Oryza sativa) lines resistant to aryloxyphenoxypropionate herbicides through induced mutation with gamma rays

The aryloxyphenoxypropionate herbicides (APPs) are graminicides with excellent control of many grass weeds species, including weedy rice (Oryza sativa L.). These herbicides block the fatty acid biosynthesis by inhibiting the enzyme acetyl‐CoA carboxylase (ACCase), resulting in the death of susceptible plants. Inducing mutation with gamma rays to rice seeds, two lines resistant to APPs herbicides were developed. Plant dose–response assays confirmed the resistance to the APPS herbicides quizalofop‐p‐ethyl and haloxyfop‐p‐methyl. The carboxyltransferase domain fragments of ACCase from the resistant biotype and susceptible control were sequenced and compared. A point mutation was detected in the amino acid position 2,027 (Rice Genome Annotation Project: Os05g22940.1). Results indicated that resistance to APPs is a consequence of an altered ACCase enzyme that confers resistance. The use of APPs herbicide‐resistant rice lines represents an innovative and promising alternative for weedy rice control in paddy rice systems.     

春油菜田抗性生物型野燕麦对高效氟吡甲禾灵的抗性机制研究

明确抗性生物型野燕麦对高效氟吡甲禾灵产生抗性的3种酶机制,为治理与延缓杂草抗药性提供科学依据。[方法]采用温室盆栽法,分别测定了抗性和敏感性生物型野燕麦的乙酰辅酶A羧化酶(ACCase)、细胞色素P450还原酶(CYP450)和谷胱甘肽-S-转移酶(GSTs)的活性差异。结果表明,未加药剂时,抗性生物型野燕麦ACCase的活性低于敏感性生物型;随着药剂浓度的增加,2个生物型ACCase的活性逐渐降低,但抗性生物型降低幅度小于敏感生物型,二者ACCase活性IC50分别为78.369 μmol/L和43.469 μmol/L,活性倍数1.8。清水处理时,野燕麦抗性生物型与敏感生物型CYP450还原酶和GSTs活性差异均不明显;施药后,抗性生物型CYP450还原酶和GSTs的活性均高于敏感生物型,药后12天,抗性生物型与敏感生物型CYP450还原酶活性分别113.1 pmol/L和38.9 pmol/L,活性倍数2.91;2个生物型GSTs活性分别为210.6 mIU/L和319.1 mIU/L,活性倍数1.52。[结论]靶标酶活性较高水平的维持及代谢酶活性的增强是野燕麦对高效氟吡甲禾灵产生抗性的重要机制。     

Development,evaluation and genetic analysis of sulfosulfuron herbicide resistance in sorghum

Herbicide tolerant varieties in combination with herbicide seed treatments can be used to manage Striga. However, there are no herbicide resistant sorghum varieties in Kenya. The objectives of this study, therefore, were to develop sulfosulfuron resistance in sorghum, to determine the level of resistance in resultant herbicide tolerant mutants, and to determine the genetic inheritance of herbicide tolerance in sorghum. Five ethyl methane sulphonate (EMS)-derived sulfosulfuron tolerant mutants (designated hb6, hb8, hb12, hb56, and hb462) were identified and selfed to M4 generation. Varying rates of sulfosulfuron, either as a spray or seed coat, were applied to determine the level of tolerance of the mutant lines. Mutant lines were also crossed with the wild-type Seredo and among themselves to determine mode of inheritance. Results showed that the susceptible wild-type Seredo was killed at the lowest herbicide rates of 0.5 g ha^-1 and 1 g ha^-1 sulfosulfuron. Dry matter from the spraying and seed coating experiments showed mutants to be up to 170 times more resistant to sulfosulfuron than the wild-type. The LD₅₀ values indicated a general trend of hb46 > hb12 > hb462 ~ hb56 > hb8 for level of tolerance under both spraying and seed coating experiments. The F₂ progeny of mutant X wild-type crosses segregated in a 1:2:1 fashion for resistant, intermediate, and susceptible, indicating semi-dominant inheritance. Intercrosses between mutant lines did not segregate for resistance in the F₂ generation indicating the same mutation could be responsible for the tolerance in all five mutants.