大豆根特异性GmPR1-9启动子的鉴定及其在根腐病抗性中的应用

Identification of the Root-Specific Soybean GmPR1-9 Promoter and Application in Phytophthora Root-Rot Resistance

【目的】鉴定大豆根特异性启动子及其最小调控片段,并利用启动子工程技术构建时空特异人工启动子并评价其在根腐病抗性中的应用价值,为大豆抗疫霉根腐病的遗传改良提供遗传元件。【方法】通过分析大豆根、茎和叶片转录组数据,筛选在根中特异高水平表达的基因,克隆获得其启动子序列。根据顺式元件的分布位置构建截短载体,并驱动GUS报告基因在大豆发状根组织中超表达,筛选控制根特异性表达的核心片段。将获得的核心启动子片段与疫霉菌诱导启动子元件p4XD串联构建人工启动子驱动疫霉抗性相关基因GmNDR1在大豆发状根中超表达,分析转基因组织对疫霉菌抗性水平及目的基因在病原菌侵染过程中的表达水平。利用转基因本氏烟草从整株水平评价转基因材料对疫霉菌的抗性水平。【结果】通过筛选发现6个大豆根特异性表达的PR1同源基因,其中,pGmPR1-9具有最高的启动子表达活性。PLACE在线预测发现其启动子区域含有大量的根特异表达相关顺式元件。对pGmPR1-9启动子进行截短试验,发现5′端截短片段L1、L2、L3、L4和L5均具有启动GUS表达活性,长度为166 bp的L5(-166—-1)片段具有全长启动子80%的活性,并可驱动GUS在转基因烟草根中特异表达;3个3′端截短片段R1、R2、R3和1个双端截短片段M1几乎检测不到GUS酶活性。p4XD-L5融合片段驱动GmNDR1在大豆发状根中超表达后可显著提高大豆发状根对疫霉菌的抗性,超表达发状根接种病原菌后发病程度和病斑长度显著低于对照,疫霉菌丝积累量在接种48 h时减少66.5%。GmNDR1在超表达组织中始终维持在高表达水平,在接种前,表达量是对照组织的39.2倍,接种后,表达量受疫霉菌侵染诱导进一步上调,并在36 h达到最高。GmNDR1在p4XD-L5::NDR1转基因本氏烟草根中的表达量显著高于茎和叶片,表现出明显的根部表达偏好性。超表达株系接种辣椒疫霉菌15 d后的株高、根长和鲜重显著高于对照,同时叶片萎蔫率和病斑长度显著低于对照植株。【结论】鉴定获得一个大豆根特异性表达启动子及其核心序列,融合诱导性和组织特异性启动子核心元件的人工启动子p4XD-L5驱动抗性基因GmNDR1超表达,可显著增强转基因大豆发状根和本氏烟草对疫霉菌的抗病性。

【Objective】The objective of this study is to identify the root-specific promotors and the core regulatory sequence of soybean.Then evaluate the potential application of the synthetic promoter in Phytophthora root-rot resistance.【Method】The genes which specifically expressed in roots with high expression levels were screened based on the transcriptome date of soybean root,stem and leaf tissues in the seedling stage.Based on the distribution of the cis elements,the promoter truncation approach was used to map the minimal promoter controlling root specific expression in soybean hairy roots.The obtained minimal promoter fragment was concatenated with the Phytophthora inducible promoter elements p4XD to construct the synthetic promoter.The synthetic promoter driven over-expression of Phytophthora resistance related gene Gm NDR1 in soybean hairy roots,then the resistance level of transgenic tissue to Phytophthora and the expression profiles of Gm NDR1 during the interaction had been analyzed.Furthermore,the transgenic Nicotiana benthamiana plants were generated to evaluate the resistance at plant level.【Result】Though screening,six soybean PR1 homologues with significant root specific expression manner were identified,and Gm PR1-9 had the highest promoter activity.Numbers of root specific expression related cis elements were identified in promoter sequence using the online tool PLACE.Truncation analysis of the promoter showed that serial 5’end deletions L1,L2,L3,L4 and L5 had different GUS activities.The L5(-166 to-1)fragment had 80%activity of the full-length promoter,and was able to drive GUS expression in roots of transgenic N.benthamiana.GUS enzyme activity was almost undetectable in three 3’end deletions R1,R2 and R3,and the double terminal deletion mutant M1.When the fusion promoter p4XD-L5 driven Gm NDR1 expression in soybean hairy roots,the resistance to P.sojae was significantly enhanced.The disease severity and lesion length were significantly reduced in the over-expression hairy roots when compared with control,and the relative biomass of Phytophthora decreased by 66.5%at 48 h post inoculation.Gm NDR1maintained high expression level in over-expression tissues,with 39.2 times of that in control tissues.The expressions were further up-regulated after inoculation,and reached the highest level at 36 h.In p4XD-L5::NDR1 transgenic N.benthamiana plants,the expression of Gm NDR1 was significantly higher in roots than that in stems and leaves.Fifteen days after P.capsica inoculation,the plant height,root length and fresh weight of Gm NDR1 over-expression plants were significantly higher,and meanwhile the leaf wilting rate and lesion length were significantly lower.【Conclusion】This study obtained a soybean root specific promoter and identified the core regulation sequence.The strategy which driven the expression of Gm NDR1 by the synthetic promoter p4XD-L5combined the inducible and tissue-specific promoter core elements can significantly enhance the resistance of transgenic soybean hairy roots and Nicotiana benthamiana plants to Phytophthora pathogens.