Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha~(–1) of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.     

The EPSPS Pro106Ser substitution solely accounts for glyphosate resistance in a goosegrass(Eleusine indica) population from Tennessee,United States

Previous studies have documented the occurrence of glyphosate-resistant(GR) goosegrass(Eleusine indica(L.) Gaertn.) and, in at least some cases, resistance is due to an altered target site. Research was performed to determine if an altered target site was responsible for GR in a Tennessee, United States goosegrass population(Tenn GR). DNA sequencing revealed a mutation in Tenn GR plants conferring the Pro106 Ser 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) substitution previously identified in other GR populations. F_2 populations were derived from Tenn GR plants crossed with plants from a glyphosate-susceptible population(Tenn GS) and analyzed for their response to glyphosate and genotyped at the EPSPS locus. Plants from the F_2 populations segregated 1:2:1 sensitive:intermediate:resistant in response to a selective dose of glyphosate, and these responses co-segregated with the EPSPS genotypes(PP106, PS106, and SS106). To separately investigate the effect of the Pro106 Ser substitution on GR, glyphosate dose-response curves and 50% effective dose(ED_(50)) values were compared among the three genotypes and the two parental populations. The SS106 genotype was 3.4-fold resistant relative to the PP106 genotype, identical to the resistance level obtained when comparing the resistant and susceptible parental populations. We conclude that the mutation conferring a Pro106Ser EPSPS mutation is solely responsible for GR in the TennGR goosegrass population.     

转基因抗草甘膦棉花及其对草甘膦抗性的时空表达

草甘膦是一种非选择性除草剂,它通过竞争性抑制莽草酸途径中的5-烯醇丙酮莽草酸-3-磷酸合酶(EPSP合酶)干扰芳香族氨基酸的生物合成而发挥毒性作用。虽然获得抗草甘膦棉花的途径有多种,但当前用于生产的抗草甘膦棉花主要是以CP4-EPSPS基因转化棉花获得的。草甘膦对转CP4-EPSPS棉花的营养生长没有不利影响,但4叶期后喷施草甘膦会显著降低花粉粒的活性,抑制散粉、授粉和结实,导致转CP4-EPSPS棉花对草甘膦抗性呈现出一定程度的时空变化。草甘膦对抗草甘膦棉花(雄性)生殖器官的抑制效应一方面源于草甘膦在生殖器官内的大量积累,另一方面在于CP4-EPSPS基因在(雄性)生殖器官的低效表达。促进CP4-EPSPS基因在全株高效表达是提高棉花对草甘膦抗性的重要途径。     

Investigating the mechanisms of glyphosate resistance in goosegrass(Eleusine indica) population from South China

Glyphosate has been used worldwide for nearly 40 years,and 30 types of resistant weeds have been reported.Glyphosate is mass-produced and widely used in China,but few studies and reports on glyphosate-resistant weeds and resistance mechanisms exist.Previous studies found a goosegrass species with high glyphosate resistance from orchards in South China and its glyphosate resistant mechanism was described in this study.The cDNAof 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS,EC 2.5.1.19),the target enzyme of glyphosate,was cloned from the glyphosate-resistant and-susceptible goosegrass,respectively,and referred as EPSPS-R and EPSPS-S.The Pro106 residue was known to be involved in the glyphosate resistance in most goosegrass populations.However,sequence analysis did not find the mutation at the Pro106 residue in the R biotype EPSPS amino acid sequence.The residue 133 and 382 was mutated in the R biotype EPSPS amino acid sequence instead,but it did not affect the EPSPS-S and EPSPS-R genes sensitivities to glyphosate.RT-PCR and Western blot analyses suggested that EPSPS mRNA and protein are mainly present in the shoot tissues both in the R and S goosegrass biotypes.The EPSPS-R rapidly responds to the glyphosate in R-biotype goosegrass and the induced expression was detected at 12 h post glyphosate treatment.The mRNA and protein expression of EPSPS-R increased constantly as the increasing concentration of glyphosate.However,the expression of the EPSPS-S was not induced significantly by glyphosate in the S goosegrass biotype.Quantification of real-time PCR results showed that the copy number of the EPSPS in R-biotype goosegrass was 4.7 times higher than that in the S goosegrass biotype.All the results implied that EPSPS gene amplification might mainly caused the glyphosate resistance of a goosegrass population collected from orchards in South China.     

牛筋草对草甘膦的抗药性

【目的】明确广东省果园或菜田田埂牛筋草（Eleusine indica）对草甘膦（glyphosate）的抗药性水平。【方法】利用整株测定法测定广东省广州市、惠州市、梅州市等地共7个点牛筋草对草甘膦的抗药性水平;采用紫外分光光度计测定抗性水平差异最大的2种牛筋草体内莽草酸含量的差异;采用光纤型双通道PAM-100测定抗性水平差异最大的2种牛筋草叶片各自叶绿素荧光参数的差异。【结果】惠州市杨村番石榴园（以下简称杨村）牛筋草对草甘膦的相对抗性指数达11.0,试验结果与田间实际反映情况相吻合;莽草酸含量测定结果表明,1 845 g a.i./hm2草甘膦处理后0-7 d,杨村牛筋草植株内部莽草酸含量较低,与对照相当,而广州番禺牛筋草植株内部积累了较多莽草酸,草甘膦处理后7 d,其莽草酸含量（623.1 µg•g FW-1）为杨村牛筋草（68.1 µg•g FW-1）的9.1倍;叶片荧光参数结果表明,杨村牛筋草Fv/Fm（PSⅡ最大原初光能转化效率）、α（PSII光响应曲线的初始斜率,代表了植物对光能的利用效率）值均高于广州番禺牛筋草,且值较稳定,而广州番禺牛筋草Fv/Fm和α在草甘膦处理5 d后均降为0。【结论】广东省部分果园或菜田田埂牛筋草对草甘膦已产生不同程度的抗药性,其中杨村牛筋草抗性最高;杨村牛筋草体内积累的莽草酸很低,植株光合系统基本未受到影响,光合作用正常进行。     

基于GR79 EPSPS筛选标记的抗虫抗除草剂表达载体构建及抗性鉴定

虫害和草害是农业生产中两大主要危害。目前,尽管转基因抗虫和抗除草剂作物相继报道和应用,然而我国抗虫抗除草剂复合性状作物培育仍然明显滞后,而且多依靠传统杂交选育的手段,费事费力、周期长。根据苏云金芽孢杆菌（Bacillus thuringiensis, Bt）Cry1Ac蛋白结合结构域和毒性结构域特征,人工合成杀虫基因cry1Ac-2.5。同时,利用抗除草剂基因GR79 EPSPS替换卡那霉素筛选标记,构建基于草甘膦除草剂筛选标记的复合抗虫抗除草剂植物表达载体,并转化烟草。qRT-PCR结果表明转基因烟草cry1Ac-2.5和GR79 EPSPS基因转录水平均成功表达,经Bt和GR79试纸条检测表明上述基因在蛋白水平正确翻译。抗性实验表明,转基因烟草愈伤经草甘膦筛选,阳性率达到80%,且转基因烟草耐受100 mg/L草甘膦处理。抗虫实验表明,饲喂cry1Ac-2.5转基因烟草4 d后,棉铃虫幼虫死亡率为90%左右,表明人工基因cry1Ac-2.5具有显著的的抗虫效果。以上结果表明,构建的抗虫抗除草剂植物表达载体（Cry1Ac-2.5+GR79 EPSPS）将抗虫基因和抗除草剂基因有效合并,对于快速培育抗虫抗除草剂作物具有指导意义。     

牛筋草EPSPS酶联免疫试剂盒的研发及应用

【目的】克隆牛筋草（Eleusine indica）中草甘膦的靶标基因EPSPS，获得牛筋草EPSPS重组蛋白并制备其单克隆及多克隆抗体，组装酶联免疫试剂盒，检测牛筋草植株不同组织EPSPS的表达水平及蛋白含量，为快速鉴定抗性牛筋草提供简便工具。【方法】利用PCR方法克隆EPSPS基因全长；构建pET30a-EPSPS阳性质粒并转化宿主菌BL21，经IPTG诱导表达后获得重组蛋白；利用该重组蛋白免疫小鼠与新西兰大白兔，制备单克隆及多克隆抗体，并利用Western blot检测特异性；利用间接ELISA方法进行免疫配对试验，筛选出最佳配对抗体，优选各试剂工作浓度并组装试剂盒；用研制的试剂盒对不同种群牛筋草进行EPSPS含量检测，并与qPCR结果分析比较。【结果】牛筋草EPSPS的开放阅读框含有1 620 bp的核苷酸，编码540个氨基酸，理论等电点为8.80，该蛋白无跨膜区域。进化树分析结果表明牛筋草EPSPS与水稻EPSPS进化关系最近。诱导蛋白表达的培养条件为1 mmol·L^-1的IPTG，25℃，获得了纯度大于90%，浓度为3 mg·mL^-1的重组蛋白12 mg；编号为R1711和R1712的家兔免疫后产生的多克隆抗体有较高的效价，编号为M171070、M171071、M171072的小鼠产生的单克隆抗体效价较高。进一步筛选出单抗FL-374-08能与多克隆抗体组成最佳配对抗体；酶联免疫试剂盒包被抗体的最优质量工作浓度为2 μg·mL^-1，酶标抗体的最优工作浓度为10 μg·mL^-1；试剂盒线性检测范围为5—80 μg·kg^-1，检出限为5 μg·kg^-1。抗性牛筋草植株叶片中EPSPS的表达量是敏感植株的52.9倍，而茎中EPSPS的表达量是敏感植株的63.0倍。在抗性牛筋草叶片中EPSPS相对拷贝数是敏感植株的53.5倍，而茎中EPSPS的相对拷贝数是敏感植株的78.6倍。Western blot结果表明单抗与牛筋草EPSPS有特异性免疫，并能准确区分不同敏感性植株及不同组织的蛋白含量差异。酶联免疫检测结果发现抗性牛筋草茎中的EPSPS浓度可以达到6.0 μg·L^-1，而敏感植株叶片和茎中EPSPS蛋白的浓度分别为0.22和0.43 μg·L^-1。【结论】获得的牛筋草EPSPS单克隆抗体特异性强、灵敏度高，研发的EPSPS酶联免疫试剂盒能够快速、准确检测牛筋草EPSPS蛋白含量并鉴定牛筋草由EPSPS过量表达导致的草甘膦抗药性。     

巴西橡胶树HbEPSPS基因逆境响应功能解析

草甘膦除草剂的靶标酶为5-烯醇式丙酮莽草酸-3-磷酸合成酶(EPSPS)。为研究HbEPSPS基因在橡胶树中的逆境响应功能,分析其在草甘膦、机械伤害、白粉菌侵染和不同激素处理下的表达模式。结果表明:草甘膦、机械伤害和IAA处理诱导HbEPSPS显著上调;干旱、ABA、SA和ETH处理亦能诱导HbEPSPS显著上调;H2O2处理橡胶树叶片中HbEPSPS表达呈现双峰规律;白粉菌侵染叶片,HbEPSPS表达显著下调。不同部位表达分析结果表明,HbEPSPS基因在花中表达量最高,其次是树皮和叶,在胶乳中表达量最低。此结果表明,HbEPSPS基因在橡胶树草甘膦药害和激素信号响应中具有重要作用,为研究其在橡胶树抗逆机制中的作用奠定良好基础。     

多启动子驱动苘麻epsps优化基因植物表达载体的构建及其转化

转基因抗草甘膦作物是商业化最广泛的转基因作物之一,但是棉花的雄蕊对草甘膦敏感,在四叶期后喷施草甘膦会造成抗草甘膦棉花雄性败育,不能正常授粉,最终导致产量降低。为降低草甘膦对棉花育性的不良影响,有针对性的构建了棉花生殖器官优势表达的arf1启动子、棉花草甘膦高效诱导表达的ag2启动子和组成型启动子CaMV35S驱动苘麻epsps优化基因的串联三表达盒植物表达载体,采用农杆菌喷花法将其转入棉花,对T0代喷施草甘膦筛选获得8株对草甘膦有较强抗性并且生殖发育正常的植株;PCR和RT PCR分析表明外源基因已整合至棉花基因组并正确表达。为探索用不同类型启动子驱动相同基因以扩大外源基因在植物中的表达范围和培育具有自主知识产权的抗草甘膦转基因棉花打下基础。     

肺炎克雷伯氏菌S001草甘膦靶标酶基因的克隆及其抗性验证

【目的】由于草甘膦在农业生产中的广泛使用,抗草甘膦转基因作物的研究一直是转基因作物研究的热点,其中草甘膦抗性功能新基因的挖掘是核心问题。自然界中微生物种类繁多,基因资源丰富,论文拟从广东地区农田土壤中筛选和鉴定出高抗草甘膦菌株,克隆其草甘膦靶标酶基因并进行抗性水平验证,以期获得高抗草甘膦新基因资源用于抗草甘膦转基因作物的研究。【方法】用含有浓度梯度草甘膦的选择培养基从备选土壤中筛选出具有高抗草甘膦特性的菌株;通过显微观察、革兰氏染色以及16S rDNA序列分析结果对菌株种类进行鉴定;利用RT-PCR技术克隆该菌株的草甘膦靶标酶基因aroAS001,并通过序列比对和系统发育树分析aroAS001序列的基本特征;利用重叠 PCR法对aroAS001变异位点进行定点突变获得aroAS001-mut序列后,将aroAS001和aroAS001-mut基因片段转入aroA基因缺陷型菌株DH5α/△aroA中进行基因功能互补和草甘膦抗性水平验证。【结果】分离出一株高抗草甘膦菌株,经形态学和分子生物学鉴定该菌株为肺炎克雷伯氏菌（Klebsiella pneumoniae）,命名为kpS001;克隆该菌株草甘膦靶标酶5-烯醇式丙酮酰莽草酸-3-磷酸合成酶（EPSPS）基因aroAS001,序列分析结果显示由该基因所编码的氨基酸序列具有典型的Class I EPSPS特征,但与已报道肺炎克雷伯氏菌的aroA相比其第227位碱基发生突变,致使对应的氨基酸发生变异。对该基因差异位点进行核酸定点突变获得aroAS001-mut基因片段后,将aroAS001和aroAS001-mut基因片段分别转入缺陷型大肠杆菌菌株DH5α/△aroA进行功能互补验证,与对照菌株相比,转入aroAS001和aroAS001-mut的重组菌株均能在含200 mmol?L-1以下浓度草甘膦的培养基中能够正常生长,表现出良好的抗性,之后随着草甘膦浓度增加其生长状态开始受到抑制,当草甘膦浓度达到350 mmol?L-1时,菌株生长基本完全被抑制。【结论】肺炎克雷伯氏菌kpS001菌株是一株高抗草甘膦菌株,由aroAS001编码的草甘膦靶标酶EPSPS属于Class I EPSP合成酶,aroAS001对草甘膦具有优良抗性,能在含350 mmol?L-1以下浓度草甘膦的培养基中生长,该基因可以作为备选基因资源用于抗草甘膦转基因作物的研究。     

Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase

Glyphosate-resistant crops have been a huge economic success for genetic engineering. The creating of new glyphosate-resistant plants would increase the available choices for planting and lower the price of genetically modified crop seeds. A novel G6 gene from Pseudomonas putida that encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was previously isolated. The G6 gene was transfected into rice via Agrobacterium-mediated transformation. The transgenic rice obtained was confirmed by PCR, Southern, and Western blots. The lab experiment and field trials further confirmed that the transgenic rice can survive glyphosate spraying at a dose of 8 g L⁻¹. In contrast, conventional rice was killed at a weed control glyphosate spray dose of 1 g L⁻¹. Altogether, the present study showed that the G6 gene works well in rice in vivo for glyphosate-resistance.     

核盘菌EPSP合酶基因在大肠杆菌中的表达

不含有内含子的核盘菌arom基因已经被扩增、测序. 该基因编码五功能的AROM蛋白. 为了大量获得核盘菌AROM蛋白的结构域之一5烯醇丙酮酰莽草酸-3磷酸合酶（EPSPS）,将该菌arom基因编码脱氢奎尼酸合酶（DHQS）和EPSPS两结构域的DNA序列和只编码EPSPS结构域的DNA序列分别克隆入载体pGEX-4t-2和载体pET28b中,构建了4个表达载体pGEX-DE、pGEX-E、pET-DE和pET-E, 并将其转入大肠杆菌DH5α、大肠杆菌BL21（DE3）或大肠杆菌JM109中表达. 酶活测定和SDS-PAGE分析结果显示,上述编码序列在大肠杆菌细胞内获得了表达,含有表达载体pGEX-E、pET-DE和pET-E的大肠杆菌BL21（DE3）转化子具有EPSPS的催化活性,说明核盘菌arom基因的这些DNA片段可以被单独表达. 核盘菌EPSPS异源表达系统的 建立为该酶的抑制剂设计奠定了基础.      

转G10aroA棉花株系的获得及分子生物学鉴定

【目的】培育具有抗草甘膦除草剂的棉花材料具有极其重要的意义。利用农杆菌介导法在棉花中转入编码EPSPS酶的抗草甘膦除草剂基因G10aroA,通过体细胞愈伤诱导组织培养技术获得能够稳定遗传的转基因棉花株系材料。【方法】首先,利用不同草甘膦抗性筛选条件比较分析不同棉花受体材料的愈伤诱导效率;其次,以R15材料作为受体,利用含有G10aroA的农杆菌侵染下胚轴切段,在进行体细胞愈伤诱导的组织培养过程中通过草甘膦抗性筛选获得棉花再生植株,对获得的棉花再生植株进行纯合繁育。在此基础上,利用PCR扩增检测证实外源G10aroA在转基因植株中能够稳定遗传;利用RT-PCR分析其外源基因在转基因植株不同组织中的转录水平进行研究、并进一步利用Western-blot对转基因植株中外源蛋白的表达进行分析。【结果】在优化的草甘膦筛选条件下,以草甘膦浓度为2.5 mmol•L-1的抗性条件进行棉花愈伤诱导筛选并获得棉花再生植株;利用特异引物进行PCR检测结果表明,在检测的全部再生植株中,扩增得到1.8 kb预期大小目标条带的阳性株系32株,其中,收获的27个株系的外源目标基因能够在T0、T1转基因植株中稳定遗传;对G10aroA在转基因株系L12、L14的不同组织中的转录表达进行定量RT-PCR分析表明,外源G10aroA在转基因棉花植株的不同组织中表达具有差异,相对表达量高低依次为茎、苞叶、叶和花;另外,蛋白检测结果进一步表明,该外源基因能够在转基因株系L7、L12和L14中植株中正常表达为预期46 kD的EPSPS蛋白。【结论】通过农杆菌介导法转化外源抗草甘膦基因G10aroA,在草甘膦抗性条件下进行棉花体细胞诱导的组织培养,成功获得转外源G10aroA的棉花再生株系,并通过分子生物学方法研究证实外源G10aroA能够在T0、T1转基因株系中稳定遗传、转录以及表达。     

Development and identification of glyphosate-tolerant transgenic soybean via direct selection with glyphosate

Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.     

Evaluation of Chinese rice varieties resistant to the root-knot nematode Meloidogyne graminicola

The root-knot nematode Meloidogyne graminicola, which is distributed worldwide, is considered a major constraint on rice production in Asia. The present study used the root gall index and number of nematodes inside the roots to evaluate resistance/susceptibility to M. graminicola in different subpopulations of Oryza sativa(aus, hybrid aus, indica, hybrid indica, temperate japonica, tropical japonica). Nematode development in highly resistant varieties was also evaluated. Analyses of randomly selected 35 varieties showed the number of M. graminicola nematodes inside the roots correlated very strongly(r=0.87, P≤0.05) with the nematode gall index, and the results from pot and field experiments revealed similar rankings of the varieties for resistance/susceptibility. Among the 136 tested varieties, temperate japonica displayed the highest gall index, followed by tropical japonica, indica, hybrid indica, aus, and hybrid aus. Zhonghua 11(aus), Shenliangyou 1(hybrid aus) and Cliangyou 4418(hybrid indica) were highly resistant to M. graminicola under both pot and field conditions. Further examination of nematode development suggested that compared to susceptible rice, M. graminicola penetrated less often into highly resistant varieties and more frequently failed to develop into females. The promising varieties found in the present research might be useful for the breeding of hybrid rice in China and for the further development of practical nematode management measures.     

鸡胚NANOG、POUV和TWIST2基因不同发育阶段表达谱分析

为了解析鸡胚发育过程中NANOG、POUV和TWIST2基因的调控作用,利用荧光定量RT-PCR方法,研究NANOG、POUV和TWIST2基因在孵化1~6d(E1~E6)的整体胚胎和孵化15和18d(E15、E18)鸡胚的大脑、心脏、肝脏和左侧大腿肌肉组织中的表达谱。结果表明,1)NANOG基因的表达水平在E2~E6整胚和E15、E18胚期的大脑、心脏、肝脏和腿肌中的表达水平均极显著低于E1时期(P0.01)。2)在鸡胚发育的整胚E1~E6和E15、E18时期的大脑、肝脏、心脏和腿肌中,POUV基因的表达水平基本呈现逐渐降低趋势,且从E3时期后均显著低于E1时期(P0.05或P0.01)。3)TWIST2基因在E1~E6整胚中的表达水平呈现逐渐增高趋势,到E6达到最高水平(P0.05或P0.01);在E15和E18时期的上述4种组织中又呈现降低趋势,但均高于E1时期。NANOG、POUV和TWIST2基因在鸡胚发育过程的调控水平存在差异,研究结果可望为鸡的育种和临床研究提供资料。