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植物呼吸爆发氧化酶(Respiratory burst oxidase homologue, Rboh)即NADPH氧化酶(NADPH oxidase, NOX)是动物NADPH氧化酶核心亚基gp91phox 的同源物[1]。ROS(Reactive Oxygen Species)包括O2.−,H2O2,.OH等,主要由质膜上的Rboh、过氧化物酶和胺氧化酶催化形成。植物在响应逆境胁迫时,Rboh将电子传递给O2生成超氧阴离子,在超氧化物歧化酶的作用下产生H2O2,ROS迅速增加作为信号分子传递到下游基因。Rboh作为产生ROS的关键活性酶,在植物的生长发育起关键作用,调节细胞中的许多重要代谢途径。在拟南芥(Arabidopsis thaliana)中AtRbohD和AtRbohF是AtRbohs基因家族中表达最高的基因,其他的AtRboh基因则保持在低表达水平[2]。高表达的AtRbohD在细胞程序性死亡、细胞壁木质化、对胁迫的信号传导中发挥着重要作用[3-5]。模式识别受体(PRRs)对病原相关分子模式(PAMPs)的感知导致ROS的爆发主要依赖于AtRbohD。RbohF可能在某些反应中与RbohD协同作用[6]。在拟南芥 atrbohd/ atrbohf双重突变体的ROS含量降低,ABA诱导的气孔关闭,ROS积累及Ca2+通道的活跃程度均削弱[3, 7-8]。植物NADPH氧化酶的活性主要受Ca2+、蛋白磷酸化、Rac蛋白及激素调控。Ca2+的拮抗剂EGTA能有效抑制马铃薯(Solanum tuberosum)StRboh蛋白诱导的ROS[9];Ca2+通过与AtRbohD蛋白N端的EF−手性基序结合,改变其蛋白结构,从而调控AtRbohD活性[10]。磷酸化状态的AtCDPK5能进一步磷酸化AtRbohD蛋白,从而激活活性氧的爆发[11]。AtBik1(botrytis-induced kinase1)能够磷酸化AtRbohD蛋白的第343位和第347位丝氨酸,激活其活性,促进ROS的积累,同时正反馈调节Ca2+信号,促进Ca2+的细胞内流,引起气孔关闭[6]。Rac是一种具有GTP酶活性的蛋白,调控ROS的形成,进而影响细胞的各种生理过程和增强植物抗病能力。烟草中NtRac5基因通过负调控Rboh基因活性参与H2O2的合成[12]。研究发现,水稻(Oryza sativa)OsRac1与OsRbohB蛋白的N端相互作用,同时,细胞质中的Ca2+浓度的升高也促进了RbohB的酶活性的提高[13]。水杨酸(SA)、茉莉酸(JA)和脱落酸(ABA)广泛参与调控植物的抗逆响应过程,它们可促进Rboh的表达及酶活性提高。拟南芥外源施加MeJA可以诱导AtRbohD和AtRbohF表达量升高[14]。外源施加ABA可以直接刺激Rboh氧化酶的活性,产生ROS类物质,参与植株抗逆的生理调节过程[7]。
木薯(Manihot esculenta)作是世界三大薯类之一,是重要的粮食作物和生物质能源作物[15]。木薯作为热区作物对低温,干旱等环境因素非常敏感,病害也是影响木薯产量的一个重要因素[16-18]。植物NADPH氧化酶家族中RbohD基因已被证实在植物与病原微生物的互作中主要负责ROS的形成。目前,木薯中还没有关于MeRbohD相关的报道。推测MeRbohD可能作为信号传导中的重要基因参与木薯的抗逆响应途径。笔者在获取MeRbohD的互作蛋白基础上,利用酵母点对点杂交验证方法验证MeRbohD和MeNOSIP(nitric oxide synthase-interacting protein)的互作关系;从木薯cDNA中扩增得到MeNOSIP的CDs序列,利用生物信息学方法初步分析MeNOSIP基因和蛋白的理化性质;同时,利用实时荧光定量PCR分析MeNOSIP在JA,SA,ABA诱导下的表达情况,旨在为木薯的遗传改良提供参考资料。
Verification of the Interaction Between Cassava MeNOSIP and MeRbohD Proteins and Their Response to Exogenous Hormones
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摘要: 为了解木薯(Manihot esculenta)MeRbohD在抗病途径中的功能,笔者在获得MeRbohD候选互作蛋白MeNOSIP(nitric oxide synthase-interacting protein,一氧化氮合酶互作蛋白)的基础上,通过RT-PCR克隆获得MeNOSIP基因编码区序列,并进行生物信息学分析。结果表明,MeNOSIP的CDs序列全长918 bp,编码含305个氨基酸的多肽,属于不稳定的亲水类蛋白质,亚细胞定位预测MeNOSIP在细胞膜上表达。采用酵母杂交验证发现,MeRbohD与MeNOSI真实互作。MeRbohD和MeNOSIP在JA,SA,ABA诱导下基因均上调表达;ABA处理下,MeRbohD和MeNOSIP基因表达变化趋势一致,推测它们可能共同参与了由ABA介导的抗逆通路。Abstract: MeNOSIP (Nitric oxide synthase interaction protein) gene coding region sequence in cassava was obtained by RT-PCR cloning based on the MeRbohD candidate interaction protein MeNOSIP to study the function of cassava MeRbohD in the disease resistance pathway. Bioinformatics analysis revealed that the CDs sequence of MeNOSIP was 918 bp, encoding a polypeptide containing 305 amino acids, and MeNOSIP belongs to an unstable hydrophilic protein. The subcellular localization predicted the expression of MeNOSIP on the cell membrane. Yeast hybridization was used to verify the true interaction between MeRbohD and MeNOSIP. MeRbohD and MeNOSIP genes were up-regulated under the induction of JA, SA, and ABA treatment, and MeRbohD and MeNOSIP gene expression under the ABA treatment tended to be consistent, which indicated that that they might be involved in the ABA-mediated stress resistance pathway.
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Key words:
- MeRbohD /
- interaction protein /
- MeNOSIP /
- expression analysis
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