文心兰OnFNR基因的克隆及抗软腐病功能鉴定北大核心CSCD

为了解铁氧还蛋白-NADP+氧化还原酶(FNR)在文心兰抵御软腐病过程中的作用,该研究采用RACE技术从文心兰‘小樱桃’中克隆到一条OnFNR基因(登录号为KX461908),分析其序列特性和编码蛋白亚细胞定位情况,利用实时荧光定量PCR(qRT-PCR)分析其在不同组织部位以及不同软腐病感病阶段的表达模式,构建OnFNR过表达载体并转化文心兰原球茎(PLBs),对转基因原球茎进行软腐病抗性评价。结果显示:(1)文心兰OnFNR基因开放阅读框长为1080 bp,预测可编码含359个氨基酸、分子量为40066.14 Da、等电点为8.72的碱性蛋白;OnFNR具有典型的FAD结合结构域和NADP+结合结构域,定位于叶绿体。(2)多序列比对和进化树分析发现,OnFNR与其他植物LFNR聚为一类,并与小兰屿蝴蝶兰LFNR的相似度最高(89.1%)。(3)qRT-PCR结果显示,OnFNR在文心兰叶中的表达量最高,其次是假鳞茎与花,根中最低;文心兰植株接种软腐病病原菌1 d后叶片和假鳞茎的OnFNR基因表达量呈现极显著下调,并且在5个感病阶段的表达量均低于健康植株。(4)成功构建过表达载体pCAMBIA1301-OnFNR,并经农杆菌EHA105侵染成功转入文心兰PLBs,获得过表达OnFNR转基因原球茎16个;在过表达OnFNR文心兰PLBs中,OnFNR与Fd基因表达均呈现极显著上调,尤其是Fd表达量上调至对照(非转基因PLBs)的3.67倍,且过表达OnFNR的PLBs在软腐病病原菌侵染第4天的存活率仍有48.88%,而对照的存活率只有6.66%。研究表明,文心兰OnFNR是一个定位于叶绿体的光合型铁氧还蛋白-NADP+氧化还原酶,过表达OnFNR能够明显提高文心兰植株的抗病性,推测OnFNR在植物抵抗病毒以及ROS爆发中具有重要作用,且过表达OnFNR可能通过提高LET的电子传递效率,进而提高Fd的表达量达到促进MAPK通路信号传导的效果。

Isolation and Preliminarily Functional Analysis of OnFNR Gene from Oncidium

To classify the role of ferredoxin-NADP+oxidoreductase(FNR)in the process of soft rot resistance in Oncidium,we cloned a OnFNR gene(Genbank accession No.KX461907)from‘Little Cherry’Oncidium by using RACE-PCR.Bioinformatics analysis and subcellular localization analysis of OnFNR were performed,and qRT-PCR was used to analyze the expression pattern of OnFNR in different organs and different soft rot infection stages.Moreover,the overexpression vector of OnFNR was constructed and transformed into protocorm-like bodies(PLBs)to investigate its function in Oncidium soft rot resistance.The results showed that:(1)the OnFNR gene contains an open reading frame of 1080 bp that encodes 359 amino acids.The predicted molecular mass is 40066.14 Da,and the isoelectric point is 8.72.OnFNR contains two typical domains:FAD binding domain and NADP+binding domain,and it is subcellularly located in the chloroplast.(2)Multiple sequence alignment and phylogenetic tree analysis revealed that OnFNR is clustered with other plant LFNRs,and is close to Phalaenopsis equestris LFNR(89.1%).(3)qRT-PCR results showed that the expression level of OnFNR was significantly higher in the leaf than that in flower and pseudobulb,and the lowest expression was found in root.Moreover,after the plants were inoculated with the soft rot pathogen,the expression of OnFNR was significant down-regulated in pseudobulb and leaf at only 1 day post pathogen inoculation(dpi)(P<0.01),and the expression levels of the five susceptible stages were all lower than that of the healthy plants.(4)The overexpression vector pCAMBIA1301-OnFNR was successfully constructed,and it was successfully transformed into Oncidium PLBs by agrobacterium EHA105 infection,and 16 transgenic PLBs overexpressing OnFNR were obtained.In the overexpressed OnFNR PLBs of Oncidium,both OnFNR and Fd gene expression were significantly up-regulated,especially the expression of Fd was increased to 3.67 times of the control(non-transgenic PLBs).Moreover,the survival rate of PLBs overexpressing OnFNR was 48.88%on the 4th day of soft rot pathogen infection,while the control was only 6.66%.This study shown that:OnFNR is a photosynthetic ferridoxin-NADP+oxidoreductase located in chloroplast,and overexpression of OnFNR can significantly improve the disease resistance of plants.It is speculated that OnFNR plays an important role in plant resistance to virus and ROS outbreak,and overexpression of OnFNR may promote signal transduction of MAPK pathway by improving electron transfer efficiency of LET and increasing Fd expression.