田旋花对草甘膦的耐药性机制

在温室条件下,测定了草甘膦对田旋花与打碗花生长的抑制作用及其体内莽草酸含量变化的影响,比较了两者5-烯醇丙酮莽草酸-3-磷酸合成酶（5-enolpyruvylshikimate-3-phosphate syn-thase,EPSPS）基因及编码氨基酸的差异,以探讨田旋花对草甘膦的耐药性机制。结果表明,田旋花对草甘膦的耐药性较高,GR50值（有效成分）为2834.04g/hm2;打碗花相对敏感,GR50值为210.63 g/hm2。经有效成分922.50 g/hm2草甘膦处理后,耐药性高的田旋花体内莽草酸积累缓慢,处理后7天达到峰值,为397.69μg/g;而打碗花体内莽草酸含量呈现持续快速升高的趋势,处理后13天达到峰值,为1 299.52μg/g。克隆获得的田旋花和打碗花编码EPSPS cDNA长度相同,均为1 707 bp,编码520个氨基酸,两者在EPSPS保守区内存在不同的氨基酸位点,田旋花的第101位为极性丝氨酸,而打碗花为非极性苯丙氨酸。     

草甘膦对田旋花和打碗花体内莽草酸含量的影响

草甘膦处理后14d,对田旋花、打碗花、大豆体内莽草酸积累量进行分析,可明确田旋花和打碗花对草甘膦的敏感性.草甘膦840ga.i./hm2、420ga.i./hm2处理后,不同植物体内莽草酸的积累量依次为大豆>打碗花>田旋花.田旋花对草甘膦的耐药能力最强,并且随着生长时间的延长对草甘膦的抗药能力增强.打碗花只是在草甘膦剂量比较低时具有一定的耐药能力.     

不同地区田旋花种群对草甘膦耐受水平的差异

在温室条件下,研究了草甘膦对北京、河北等地田旋花种群生长及其莽草酸含量变化的影响。结果表明,不同地区田旋花种群对草甘膦具有普遍的耐受能力,其中河北元氏的田旋花种群对草甘膦的耐受能力最强,GR50值为4 014.92 g a.i./hm2;北京海淀田旋花种群对草甘膦相对敏感,GR50值为957.65 g a.i./hm2。经922.5 ga.i./hm2草甘膦处理后,耐药性最高的河北元氏田旋花种群体内莽草酸含量累积缓慢,处理后14 d所受伤害能自行恢复并保持正常生长;敏感的北京海淀田旋花种群体内莽草酸迅速积累,在处理后7 d莽草酸含量达到最高值,植株生长受到一定程度的抑制。田旋花体内莽草酸含量与其耐药性高低呈负相关,因此可以通过检测田旋花经草甘膦处理后体内莽草酸的含量来明确不同地区田旋花对草甘膦耐受水平的高低。     

田旋花EPSPS基因表达载体的构建及拟南芥转化

根据田旋花(Convolvulus arvensis L.)EPSPS基因(Gen Bank登录号:EU698030)c DNA序列,设计含有酶切位点的特异性引物,以田旋花c DNA为模板,合成EPSPS基因并连接到含有35S启动子和GUS基因的p BI121载体上,成功构建植物超表达载体p BI-EPSPS。采用氯化钙冻融法将其转入根癌农杆菌(Agrobacterium tumefaciens)中,然后用根癌农杆菌介导法转化拟南芥(Arabidopsis thaliana),共获得15株卡那霉素抗性拟南芥苗。对其中的5株进行PCR和RT-PCR检测,结果表明EPSPS基因已整合到拟南芥的基因组中并可以表达。     

转基因抗虫耐除草剂(Cry1Ac+EPSPS)玉米对草甘膦耐受性研究

将具有我国自主知识产权的抗虫、耐除草剂基因转化到玉米中获得兼具抗虫和耐除草剂的复合性状转基因玉米,实现了玉米复杂性状的有效改良,研究了转耐除草剂CC-2基因和可表达Bt毒素的cry1Ac基因的双抗玉米对除草剂的耐受性及目标蛋白表达量。通过调查喷施除草剂后玉米植株存活率及株高和生长发育情况,研究了转抗虫耐除草剂基因玉米‘CC-2×BT799’、抗虫玉米‘BT799’、耐除草剂玉米‘CC-2’,以及常规玉米‘郑58’在田间对除草剂的耐受性,并用ELISA测定了供试品种中CP4EPSPS和Cry1Ac蛋白含量。对草甘膦的耐受性试验表明,转复合基因玉米‘CC-2×BT799’与单抗草甘膦品种‘CC-2’对草甘膦均具有良好的耐受性。心叶期喷施草甘膦推荐施用剂量中量和2倍剂量对其株高和后期生长发育无不良影响。ELISA检测显示,CP4EPSPS蛋白在雄穗中的表达量较叶片稍高。且双抗品种‘CC-2×BT799’较‘CC-2’中的CP4EPSPS蛋白含量稍高。在雄穗中,‘BT799’的Cry1Ac蛋白含量与‘CC-2×BT799’相当。在叶片中,‘BT799’的Cry1Ac蛋白含量约为‘CC-2×BT799’的2倍。抗虫耐除草剂玉米能够有效抵御草甘膦的喷施,Cry1Ac蛋白含量在不同品种不同组织中存在差异。     

利用双重数字PCR检测进口美国大豆中长芒苋的EPSPS基因拷贝数

长芒苋是一种原产北美洲的雌雄异株杂草.在美国,一些长芒苋种群已经对草甘膦产生了抗药性.EPSPS基因拷贝数的增加是长芒苋产生抗药性的主要原因.本文建立了双重数字PCR检测长芒苋EPSPS基因拷贝数的方法,对进口美国大豆中携带的8个长芒苋样品进行检测,发现3个样品的EPSPS相对拷贝数较低,5个样品EPSPS相对拷贝数大...     

田旋花 EPSPS 基因启动子的克隆、序列分析及转化

根据已克隆的田旋花（ Convolvulus arvensis L．） EPSPS基因mRNA序列设计引物,通过染色体步移技术获得了1142 bp的EPSPS-P启动子片段（ GenBank 登录号：KC107822）。对启动子序列分析显示,该片段富含A/T碱基、TATA-box、CAAT-box及其他作用元件如GATA-motif、TC-rich repeats、sp1等。将该启动子与GUS报告基因连接以构建植物表达载体,利用农杆菌介导法获得转基因拟南芥;PCR和GUS组织化学染色分析证明, EPSPS-P已转化到拟南芥中。     

抗草甘膦基因的克隆及在原核表达系统中的功能分析

为了克隆抗草甘膦基因并在原核表达系统中分析其抗性水平,利用200 μmol/L草甘膦平板从草甘膦严重污染的土壤中筛选到1株抗草甘膦菌株G6PP,经电镜和16S rDNA鉴定为恶臭假单胞菌;以该菌株基因组DNA为模板,通过PCR方法扩增出5-烯醇丙酮莽草酸-3-磷酸酯合成酶(EPSPS)基因,该基因编码440个氨基酸,亚克隆到原核表达载体pEET-28a中构建原核表达载体pET-G6;将重组表达载体转化大肠杆菌BL21 (DE3)中,经IPTG诱导,表达出46 ku的融合蛋白;携带pET-G6质粒的大肠杆菌BL21 (DE3)在液体M63培养基中能耐受150 μmol/L草甘膦的抑制.本研究结果表明克隆到的G6基因具有一定的抗草甘膦活性,对抗草甘膦作物的培育具有实践意义.     

苏门白酒草对草甘膦等除草剂的多抗性检测及防治药剂筛选

苏门白酒草Conyza sumatrensis是中国华南地区常见的阔叶杂草,在果园和非耕地常造成严重危害。本研究采用整株剂量反应法,明确了采自广东省广州市的苏门白酒草疑似抗性种群 (GZ-R) 对草甘膦、百草枯和敌草快的抗性水平,比对了GZ-R种群和采自广东省清远市的敏感对照种群 (QY-S) 的草甘膦靶标酶基因EPSPS2片段的差异,并测定了灭草松、氯氟吡氧乙酸等5种茎叶处理剂对不同叶龄苏门白酒草的室内防除效果。结果表明:GZ-R种群对草甘膦和百草枯分别产生了中等水平和高水平抗性,并已对敌草快产生交互抗性,3种药剂对GZ-R种群的LD₅₀值分别是对QY-S种群LD₅₀值的7.2、72.3和6.6倍;与QY-S种群相比,GZ-R种群的EPSPS2基因106位由脯氨酸突变为苏氨酸。在灭草松、氯氟吡氧乙酸或2甲4氯钠推荐剂量下,于4~5叶期施药,苏门白酒草死亡率均为100%,但于6~7叶期和10~12叶期施药,苏门白酒草死亡率显著下降至44.4%~91.7%;而在草铵膦或苯嘧磺草胺推荐剂量下,不同叶龄期施药苏门白酒草的死亡率均为100%,因此在植株生长早期可使用草铵膦和苯嘧磺草胺防除已对草甘膦和百草枯等除草剂产生抗性的苏门白酒草。     

茶树病程相关蛋白基因CsPR5的克隆及表达分析

为探究茶树中病程相关蛋白5(pathogenesis-related protein 5,PR5)在茶树中的功能,采用cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术克隆了PR5基因的全长;通过荧光定量PCR方法检测了CsPR5在龙井43茶树不同组织部位、植物激素(茉莉酸、乙烯利和水杨酸)处理、茶炭疽病菌Colletotrichum camelliae侵染和小贯小绿叶蝉Empoasca onukii为害后的表达特征。结果表明,CsPR5基因开放阅读框为843 bp,编码281个氨基酸。CsPR5基因在茶树根、茎、嫩叶、花和种子5个组织中的相对表达量分别为0.187、3.093、0.928、0.045和0.012,且五者之间差异显著,主要在茎和叶中表达,其分别为根中相对表达量的16.54倍与4.96倍。水杨酸处理6 h及乙烯利处理6、12 h后,茶树CsPR5基因的相对表达量分别为1.622、2.344和1.845,分别比对照显著上调2.20倍、3.18倍和2.35倍;但是外施茉莉酸对茶树CsPR5基因的相对表达量无影响。茶炭疽病菌侵染显著诱导了茶树CsPR5基因的相对表达量,处理后的相对表达量为1.977,是对照的1.90倍;小贯小绿叶蝉取食显著抑制了其为害部位CsPR5的相对表达量,为7.273,仅为对照的38.80%,但该诱导不具系统性。     

EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas

BACKGROUND: Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non‐crop systems. A glyphosate‐resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. RESULTS: The investigation was conducted on resistant and susceptible plants from a population in Desha County, Arkansas (Des03). The amounts of glyphosate that caused 50% overall visual injury were 7 to 13 times greater than those for susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6‐fold higher basal EPSPS enzyme activities than the susceptible plants, but their I₅₀ values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. CONCLUSION: Increased EPSPS gene amplification and EPSPS enzyme activity confer resistance to glyphosate in the Des03 population. This is the first report of EPSPS gene amplification in glyphosate‐resistant Italian ryegrass. Other resistance mechanism(s) may also be involved. Copyright © 2012 Society of Chemical Industry     

Physiological and molecular mechanisms of glyphosate tolerance in an in vitro selected cotton mutant

We have selected an upland cotton (Gossypium hirsutum L.) cell line (R1098) that is highly tolerant to glyphosate. This cell line was developed by in vitro selection with gradually increasing glyphosate concentrations, and its mechanisms conferring glyphosate tolerance were studied. Based on a whole-plant dose–response bioassay, R1098 plants were tolerant to glyphosate at a concentration of 1500 g ae ha⁻¹ glyphosate (1.5× the recommended field rate) whereas the control plants (Coker 312) were unable to survive at 150 g ae ha⁻¹ glyphosate. Coker 312 accumulated 13.1 times more shikimate in leaves at 5 days after glyphosate treatment (1500 g ae ha⁻¹) than that of R1098. Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), EPSPS-1 and EPSPS-2, were isolated from R1098. Both cDNAs were 97.7% identical within the common protein-coding region and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other known higher plants. In comparison to the glyphosate-susceptible cotton Coker 312, sequence analysis of the EPSPS-1 gene indicated that R1098 has an alanine insertion at nucleotide position 1216 resulting in frameshift. It leads to two copy functional EPSPS genes in R1098. There was no difference between R1098 and Coker 312 in EPSPS mRNA levels before glyphosate treatment. However, its treatment caused a 2–4 times increase in the basal EPSPS mRNA level in R1098.     

Characterization of glyphosate resistance in cloned Amaranthus palmeri plants

Glyphosate‐resistant Palmer amaranth from Georgia (GA), USA, possesses multiple copies of the gene that encodes 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), the enzyme target site of this herbicide. The cloned plants of glyphosate‐resistant and glyphosate‐susceptible Palmer amaranth biotypes from Mississippi (MS), USA, and GA were evaluated for glyphosate injury (digital imaging) in leaf disc bioassays. Four groups (three resistant groups: two from MS [G and R] and one from GA [C7]; one susceptible group from GA [C3]) were chosen for cloning to facilitate long‐term studies. After exposure to glyphosate (1.0 mmol L^–1, 144 h), the level of injury (mean value) was low in the resistant groups, while a higher level of injury was found in the susceptible group. However, the individual injury values within all groups varied widely. The mean EPSPS gene copy number of these groups was G ≥ R > C7 >>> C3. However, a higher copy number did not always convey increased resistance in these bioassays. When the copy number was high (>20), 81.5% of the bioassayed plants exhibited little or no injury and only ～20% were significantly injured, while 50% of the plants with a low copy number (<20) remained healthy. Overall, no strong statistical correlation of the copy number versus injury occurred in these cloned plants and no statistical relationship of resistance and copy number with the sex of the MS plants was observed. The results suggest that although an elevated copy number of the EPSPS gene can instill resistance, other mechanisms might contribute to the overall glyphosate resistance of Palmer amaranth in these plants.     

水稻抗纹枯病苗期快速鉴定技术研究

 以抗感反应不同的5个水稻品种为试验材料,在人工气候箱、控温室中进行水稻苗期抗纹枯病接种试验,并与田间相应的成株期抗性试验进行比较,研究水稻苗期快速鉴定技术。结果表明:85%的相对湿度为纹枯病菌侵染危害水稻苗期植株的适宜湿度;苗期5个水稻品种对纹枯病抗性差异极显著,可将其分为相对感病(Lemont、武育粳3号)和相对抗病(YSBR1、Jasmine85、特青)2大类;接种叶龄对发病程度有显著的影响,5个品种在四叶期接种时的平均病级显著高于五叶期接种的平均病级;苗期水稻品种间抗感差异小于田间鉴定试验结果,但两者间品种抗感趋势基本一致。苗期快速鉴定技术可用于大规模水稻品种(组合)的抗性筛选或初步鉴定。