【目的】将bZIP类转录因子基因AtTGA4转化小麦创制耐低磷转基因小麦新材料,同时分析AtTGA4提高小麦抗逆性的生理机制,为小麦耐低磷胁迫分子育种奠定基础。【方法】采用最小表达框基因枪转化法将AtTGA4和筛选标记基因Bar共转化受体小麦石...【目的】将bZIP类转录因子基因AtTGA4转化小麦创制耐低磷转基因小麦新材料,同时分析AtTGA4提高小麦抗逆性的生理机制,为小麦耐低磷胁迫分子育种奠定基础。【方法】采用最小表达框基因枪转化法将AtTGA4和筛选标记基因Bar共转化受体小麦石4056,通过PCR检测筛选出无Bar并能稳定遗传AtTGA4的转基因小麦新株系。基于试验地土壤养分含量状况施用不同水平的磷肥,形成一定程度的正常和低磷营养胁迫,对AtTGA4转基因小麦新株系进行低磷胁迫耐受性试验。在开花期进行了光系统Ⅱ原初光能转化效率(light efficiency of the light systemⅡ,Fv/Fm),叶绿素相对含量(soil and plant analyzer development readings,SPAD值)和气冠温差(canopy temperature depression,CTD)等生理指标的测定,在成熟期进行了株高、分蘖数、穗粒数等农艺性状的调查,并在小麦收获后进行了产量及不同组分(根、茎、叶、籽粒)磷浓度和磷吸收、残留总量的测定和统计。【结果】PCR分析结果证明,AtTGA4已在石4056小麦中稳定遗传至T4代,共获得4个稳定转基因株系。根据土壤养分含量测定结果,在正常条件地块施加812.39 kg·hm-2的过磷酸钙,低磷处理地块不施磷肥。产量及农艺性状统计结果显示,AtTGA4转基因株系L1和L2在正常和低磷胁迫条件下的产量相对于受体对照小麦显著增加,正常条件下产量增幅为5.3%—8.6%,低磷胁迫下产量增幅为4.4%—7.7%。在低磷胁迫条件下,过表达AtTGA4的转基因小麦种子千粒重显著比受体显著增加。开花期田间生理指标测定结果显示,转基因株系L1和L2在低磷条件下的Fv/Fm和CTD明显优于受体,而SPAD值没有明显差异。田间调查时发现,低磷条件下受体比转基因材料提早结束灌浆,表现在穗子提早变黄。成熟末期磷含量测定结果显示,转基因株系L1和L2在低磷条件下茎杆磷浓度比受体显著提高,在其他组织中则无显著差异。2个转基因株系在低磷条件下茎、叶和籽粒吸收、残留的总磷含量都要高于受体,地上部总磷含量增幅达6.38%—17.47%。转基因材料AtTGA4表达量分析结果显示,目标基因在株系L2中的表达量较株系L1中的低,是株系L1的0.69倍。【结论】在低磷胁迫条件下AtTGA4可以显著提高转基因小麦对磷元素的吸收及运输,提高转基因小麦的产量,进而提高转基因小麦对低磷胁迫的耐性。展开更多
Under stress conditions such as droughthigh-salinity and low-temperature, the transcription factorof DREB (dehydration responsive element binding proteins)improved efficiently stress resistance by regulating the ex-pr...Under stress conditions such as droughthigh-salinity and low-temperature, the transcription factorof DREB (dehydration responsive element binding proteins)improved efficiently stress resistance by regulating the ex-pression of its downstream genes with various environmentastress resistance in plants. GmDREB gene (GenBank Acces-sion No. AF514908) encoding a stress-inducible transcriptionfactor was cloned by screening a cDNA library of Glycinemax cv. Jinong 27 with yeast one-hybrid method. GmDREBgene was 910 bp in length and encoded 174 amino acids con-taining a conserved AP2/EREBP DNA-binding domain of 58amino acids. Two conserved functional amino acids, valineand glutamic acid, were located on the 14th and the 19thamino acid residues in the conserved structural domain. Analkaline amino acid region (KKR) related to a nuclear local-ization signal was at the N-terminal, while an acidic aminoacid region (DDD) related to trans-activation was at theC-terminal. Plant expression vectors were constructed andtransformed into wheat by bombardment. In total, 13 trans-genic plants with Ubi::GmDREB and 11 transgenic plantswith rd29A::GmDREB were identified from 103 regenerationplants by molecular analysis. The drought and salt tolerancesof T1 transgenic lines with Ubi::GmDREB orrd29A::GmDREB were demonstrated to be improved ascompared to wild type. The result also suggested that bothUbiquitin and rd29A promoters could effectively drive theexpression of the GmDREB gene and enhance drought andsalt tolerance of T1 plants.展开更多
文摘【目的】将bZIP类转录因子基因AtTGA4转化小麦创制耐低磷转基因小麦新材料,同时分析AtTGA4提高小麦抗逆性的生理机制,为小麦耐低磷胁迫分子育种奠定基础。【方法】采用最小表达框基因枪转化法将AtTGA4和筛选标记基因Bar共转化受体小麦石4056,通过PCR检测筛选出无Bar并能稳定遗传AtTGA4的转基因小麦新株系。基于试验地土壤养分含量状况施用不同水平的磷肥,形成一定程度的正常和低磷营养胁迫,对AtTGA4转基因小麦新株系进行低磷胁迫耐受性试验。在开花期进行了光系统Ⅱ原初光能转化效率(light efficiency of the light systemⅡ,Fv/Fm),叶绿素相对含量(soil and plant analyzer development readings,SPAD值)和气冠温差(canopy temperature depression,CTD)等生理指标的测定,在成熟期进行了株高、分蘖数、穗粒数等农艺性状的调查,并在小麦收获后进行了产量及不同组分(根、茎、叶、籽粒)磷浓度和磷吸收、残留总量的测定和统计。【结果】PCR分析结果证明,AtTGA4已在石4056小麦中稳定遗传至T4代,共获得4个稳定转基因株系。根据土壤养分含量测定结果,在正常条件地块施加812.39 kg·hm-2的过磷酸钙,低磷处理地块不施磷肥。产量及农艺性状统计结果显示,AtTGA4转基因株系L1和L2在正常和低磷胁迫条件下的产量相对于受体对照小麦显著增加,正常条件下产量增幅为5.3%—8.6%,低磷胁迫下产量增幅为4.4%—7.7%。在低磷胁迫条件下,过表达AtTGA4的转基因小麦种子千粒重显著比受体显著增加。开花期田间生理指标测定结果显示,转基因株系L1和L2在低磷条件下的Fv/Fm和CTD明显优于受体,而SPAD值没有明显差异。田间调查时发现,低磷条件下受体比转基因材料提早结束灌浆,表现在穗子提早变黄。成熟末期磷含量测定结果显示,转基因株系L1和L2在低磷条件下茎杆磷浓度比受体显著提高,在其他组织中则无显著差异。2个转基因株系在低磷条件下茎、叶和籽粒吸收、残留的总磷含量都要高于受体,地上部总磷含量增幅达6.38%—17.47%。转基因材料AtTGA4表达量分析结果显示,目标基因在株系L2中的表达量较株系L1中的低,是株系L1的0.69倍。【结论】在低磷胁迫条件下AtTGA4可以显著提高转基因小麦对磷元素的吸收及运输,提高转基因小麦的产量,进而提高转基因小麦对低磷胁迫的耐性。
基金This work was supported by the National 863 Project(Grant No.2002AA224081)National Special Project for Plant Transgenic and Industry(Grant No.JY03-A-18).
文摘Under stress conditions such as droughthigh-salinity and low-temperature, the transcription factorof DREB (dehydration responsive element binding proteins)improved efficiently stress resistance by regulating the ex-pression of its downstream genes with various environmentastress resistance in plants. GmDREB gene (GenBank Acces-sion No. AF514908) encoding a stress-inducible transcriptionfactor was cloned by screening a cDNA library of Glycinemax cv. Jinong 27 with yeast one-hybrid method. GmDREBgene was 910 bp in length and encoded 174 amino acids con-taining a conserved AP2/EREBP DNA-binding domain of 58amino acids. Two conserved functional amino acids, valineand glutamic acid, were located on the 14th and the 19thamino acid residues in the conserved structural domain. Analkaline amino acid region (KKR) related to a nuclear local-ization signal was at the N-terminal, while an acidic aminoacid region (DDD) related to trans-activation was at theC-terminal. Plant expression vectors were constructed andtransformed into wheat by bombardment. In total, 13 trans-genic plants with Ubi::GmDREB and 11 transgenic plantswith rd29A::GmDREB were identified from 103 regenerationplants by molecular analysis. The drought and salt tolerancesof T1 transgenic lines with Ubi::GmDREB orrd29A::GmDREB were demonstrated to be improved ascompared to wild type. The result also suggested that bothUbiquitin and rd29A promoters could effectively drive theexpression of the GmDREB gene and enhance drought andsalt tolerance of T1 plants.