转GsERF71基因苜蓿的培育及耐碱性

Cultivation and alkaline tolerance analysis of alfalfa transformed with the GsERF71 gene

基于东北农业大学农业生物功能基因重点实验室前期野生大豆(Glycine soja)碱胁迫转录组数据,从中获得碱胁迫应答基因GsERF71,选用USER载体构建GsERF71植物超量表达载体,利用农杆菌介导的子叶节侵染法转化紫花苜蓿(Medicago savita),最终得到69株抗性植株;其中13株通过PCR检测为阳性植株。Southern blot检测获得2株阳性植株,RT-PCR检测发现,这2个株系均为阳性,说明GsERF71已成功整合至苜蓿基因组并能在转录水平上有表达。对非转基因和转基因株系对野生型和转基因株系进行扦插扩繁,分别用0、100、150mmol·L-1 NaHCO3的营养液处理转基因植株14d,进行耐碱性形态指标及生理指标的分析,包括株高、根长和脯氨酸含量、相对质膜透性、丙二醛叶绿素含量、过氧化物酶活性的分析。结果表明,非转基因植株的相对质膜透性和丙二醛含量显著高于转基因苜蓿(P<0.05),而叶绿素含量、株高、根长、脯氨酸含量以及过氧化物酶活性显著低于转基因苜蓿(P<0.05)。因此,GsERF71基因的超量表达提高了苜蓿的耐碱能力。

In this study,we constructed a USER-based plant expression vector overexpressing the GsERF71 gene,which was identified from our previous RNA-seq data of wild soybean in response to alkaline stress.The GsERF71 gene was transformed into the wild type alfalfa using the Agrobacterium tumefaciens mediated cotyledons method.A total of 69 glufosinate-resistant plants were obtained,and 13 plants were identified by PCR analysis with GsERF71 gene-specific primer.Further southern blot analysis verified the existence of GsERF71 in two transgenic lines,which were further characterized by RT-PCR analysis to confirm the constitutive expression of GsERF71.For alkaline resistance analysis,the wild type and GsERF71 transgenic plants were irrigated with either 0,100,and 150 mmol·L-1 NaHCO 3 solutions every 2 days for a total of 14 days,respectively.The morphological and physiological indices were recorded and measured,including the plant height,root length,proline content,relative plasma membrane permeability,chlorophyll content,malondialdehyde content and peroxidase activity.Quantitative analysis revealed that the relative plasma membrane permeability and malondialdehyde content of the transgenic alfalfa was significantly lower than that of the wild type plants;however the content of chlorophyll,plant height,root length,proline,and peroxidase activity were significantly higher(P<0.05).In summary,this research suggests that overexpression of GsERF71 results in significantly improved alkaline tolerance.