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.     

Use of chlorophyll fluorescence and P700 absorbance to rapidly detect glyphosate resistance in goosegrass(Eleusine indica)

The rapid detection of glyphosate resistance in goosegrass(Eleusine indica) will enhance our ability to respond to new resistant populations of this major weed. Chlorophyll fluorescence(Fluo) and P700(reaction center chlorophyll of photosystem I) absorbance were analyzed in one biotype of goosegrass that is resistant to glyphosate and in another that remains sensitive to the herbicide. Both biotypes were treated with a foliar spray of glyphosate. Differences in photosystem II maximum quantum yield(Fv/Fm), effective photochemical quantum yield(Y(II)), and non-photochemical quenching(NPQ) between the biotypes increased over time. Values for Fv/Fm and Y(II) differed between the two biotypes 24 h after treatment(HAT). Differentiated activities and energy dissipation processes of photosystem II(PSII) and energy dissipation processes of photosystem I(PSI) were manifested in the two biotypes 24 HAT with 20 mmol L–1 glyphosate. Differentiated energy dissipation processes of PSI were still apparent 24 HAT with 200 mmol L–1 glyphosate. These results indicate that the Fluo parameters related to PSII activity and energy dissipation and the P700 parameters related to energy dissipation are suitable indicators that enable rapid detection of glyphosate resistance in goosegrass.     

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.     

毛竹中NAC转录因子的克隆与序列分析

NAC(NAM,ATAF1/2和CUC2)是植物特有的一类转录因子,在植物的生长发育、器官建成、激素调节和防御抵抗多种生物和非生物胁迫等方面都发挥着重要作用。利用BeNAC1为饵基因,通过NCBI tblastn在毛竹的cDNA文库中筛选到7个与其相似性较高的cDNA全长序列,分别命名为PeNAC1、PeNAC2、PeNAC3、PeNAC4、PeNAC5、PeNAC6和PeNAC7。基因组分析显示,这7个NAC家族转录因子都具有3个外显子,2个内含子;氨基酸序列分析显示其N端都含有典型的NAC保守结构区域,且大多属于ATAF和NAP亚家族。功能分析显示,这些NAC转录因子可能参与毛竹的激素调节、干旱胁迫、盐胁迫、寒冷胁迫等非生物胁迫及昆虫侵害等生物胁迫的防御抵抗,有的还可能参与毛竹的叶片衰老调控。     

山葡萄VaCBF1转录因子基因的克隆与表达分析

【目的】克隆山葡萄(Vitis amurensis)CBF1转录因子基因(VaCBF1),为其功能的深入研究及其在植物抗寒基因工程中的应用奠定基础。【方法】根据植物CBF基因AP2/EREBP保守区设计1对简并引物,利用PCR法从山葡萄cDNA中扩增VaCBF1基因的中间片段。再根据中间片段区域设计2对特异引物,采用反向PCR法扩增VaCBF1基因的5′端和3′端序列。将中间片段与5′端和3′端序列拼接后得到山葡萄VaCBF1基因的cDNA全长序列,据此设计1对特异引物,PCR扩增VaCBF1基因编码区的全长序列,并对其进行生物信息学分析。同时,利用荧光定量PCR分析山葡萄VaCBF1基因在不同逆境胁迫(干旱、低温、盐胁迫)下的表达情况。【结果】成功地从山葡萄中克隆得到VaCBF1基因cDNA全长序列,其长度为762bp,编码253个氨基酸,在GenBank注册号为DQ517296。同源性分析证实,VaCBF1属于CBF转录因子家族。荧光定量PCR分析发现,低温胁迫可以诱导山葡萄VaCBF1基因高表达,而该基因的表达不受盐及干旱处理诱导。【结论】首次从山葡萄中克隆了VaCBF1基因,并证实该基因参与了植物对低温胁迫的应答。     

毛竹PheDof4-1基因克隆及表达分析

Dof(DNA binding with one finger)蛋白是植物特有的一类转录因子,由多基因共同编码,参与植物转录调控、生长发育及胁迫响应等生物学过程。以毛竹为研究对象,克隆了1个Dof基因,命名为Phe Dof4-1,基因长度为1 473 bp,推测其蛋白质产物的分子量是53.2 kDa,等电点为8.05。qRT–PCR结果显示,Phe Dof4-1在低温和250 mmol·L~(-1) Na Cl处理的幼茎中诱导表达,而在20%PEG8000处理的根中表达则受到强烈抑制,在叶片中不同处理条件下呈现不同的表达趋势。研究结果为Phe Dof4-1在非生物胁迫中的作用提供理论依据,为竹子分子育种提供参考。     

海岛棉转录因子基因GbTCP27的克隆及表达特性分析

为探究转录因子GbTCP27在棉花纤维发育中的作用,使用RT-PCR技术从海岛棉‘新海21号’中克隆GbTCP27,并对其进行生物信息学和表达特性分析。结果表明,该基因有1个1 017 bp的开放阅读框,编码338个氨基酸,预测分子量约为35.37 ku,等电点为9.08,分子式为C_(1549)H_(2511)N_(439)O_(483)S_(11),序列中含有一个高度保守的TCP结构域;氨基酸序列对比表明,海岛棉GbTCP27基因在进化过程中是相当保守的,蛋白序列与其他植物中的TCP蛋白序列有较高的一致性;亚细胞定位结果显示,GbTCP27属于核定位基因,推测GbTCP27可能在复制和转录的过程中发挥作用;进化树分析表明,海岛棉GbTCP27基因与亚洲棉GaTCP9基因分布在同一分支上;实时荧光定量PCR表明,GbTCP27基因在开花当天的花瓣和开花当天的胚珠中表达量最高;综上所述,GbTCP27转录因子可能参与棉花纤维起始期胚珠表皮细胞的发育。