甘蔗β-胡萝卜素异构酶基因家族的鉴定、定位和表达分析

Identification,localization and expression analysis of beta-carotene isomerase gene family in sugarcane

独脚金内酯(strigolactones, SLs)是一种广泛存在、能够抑制植物分蘖或分枝的植物激素。β-胡萝卜素异构酶(D27)是SLs合成中的关键酶,但是目前关于甘蔗D27基因家族的鉴定与分析鲜有报道。本研究通过挖掘甘蔗原始亲本之一的割手密种基因组数据鉴定了5个割手密种D27基因家族成员。系统进化树分析发现,割手密种D27s分处在3个不同系统发育分支,与高粱D27s高度同源。保守结构域预测揭示,割手密种D27s包含β-胡萝卜素异构酶的典型结构域Pfam:DUF4033。顺式元件分析结果显示,割手密种D27s主要参与调控激素和胁迫响应,以及植物生长发育等。基于甘蔗栽培种转录组数据分析发现,甘蔗割手密种D27基因家族成员(Sspon.06G0012830-1A)的同源基因同时参与甘蔗分蘖调控及黑穗病菌胁迫响应。在此基础上,我们克隆获得了甘蔗栽培种ROC22中的同源基因cDNA序列,命名为ScD27.1 (GenBank登录号为MT499895)。生物信息学分析结果显示, ScD27.1编码266个氨基酸,其蛋白等电点为8.91,分子量为30.00 kD,是不稳定蛋白且定位于叶绿体。二级结构主要包括α-螺旋和无规则卷曲,具有叶绿体转运肽,包含4个泛素化位点和18个磷酸化位点。qRT-PCR表达分析表明,ScD27.1基因受ABA及H2O2显著诱导表达,但对MeJA、SA胁迫响应不明显。亚细胞定位结果显示, ScD27.1蛋白可能定位于细胞膜和叶绿体,参与细胞内膜泡运输或由液泡前体、胞内运输小泡分拣运输。以上研究表明, ScD27.1基因可能参与黑穗病菌侵染诱导的甘蔗分蘖及ABA和H2O2相关信号通路。本研究为了解甘蔗ScD27.1蛋白在胞内运输和分蘖中的作用及其参与甘蔗-黑穗病菌互作提供一定的基础理论。

Strigolactones(SLs),a class of plant hormones,exists widely in plants and involves in the regulation of plant tillering and environmental adaptation.β-carotene isomerase(D27)is a key enzyme for SLs synthesis,but there are few reports about the identification and analysis of D27 gene family in sugarcane.In this study,five members of D27 gene family from one of the original parent of modern sugarcane cultivars,Saccharum spontaneum,were analyzed.Phylogenetic tree analysis showed that these D27 s from S.spontaneum was clustered into 3 different branches and were highly homologous to sorghum D27 s.Conserved domain prediction revealed that D27 s contained a typical domain ofβ-carotene isomerase,Pfam:DUF4033.The results of cis-element analysis showed that D27 s mainly involved in the regulation of hormone response,plant growth and development,and stress response.Based on the transcriptomic data of the modern cultivated varieties,the expression analysis of the homologous transcripts of Saccharum spontaneum D27 s showed that Sspon.06 G0012830-1 A was regulated at tillering stage and under Sporisorium scitamineum infection.Besides,the cDNA sequence of Sspon.06 G0012830-1 A was cloned from sugarcane cultivar ROC22 and named as ScD27.1(Gen Bank accession number:MT499895).Bioinformatics analysis indicated that ScD27.1 encoded an unstable protein of 266 amino acids with an isoelectric point of 8.91 and a molecular weight of 30.00 kD,and might be located in the chloroplast,containing chloroplast transit peptides,4 ubiquitination sites and 18 phosphorylation sites.Its secondary structure mainly included alpha helix and random coils.qRT-PCR analysis demonstrated that Sc D27.1 was significantly induced by ABA and H2O2 and did not respond to MeJA and SA.Subcellular localization revealed that ScD27.1 might be located in cell membrane and chloroplast and involved in the vesicle sorting or transported by sorting vesicle in plant cells.The results suggested that ScD27.1 may involve in the tillering induced by S.scitamineum and signaling pathway of ABA and H2O2.The present study provides a basic understanding of intracellular transport and tillering in sugarcane and the involvement of ScD27.1 during sugarcane-S.scitamineum interaction.