与甘蔗抗黑穗病性相关的14-3-3基因的克隆和表达分析  

Cloning and Expression Analysis of a 14-3-3 Gene Related to Sugarcane Smut Resistance

作　　者：肖新换 阙万才[2] 黄宁 刘峰[1] 苏炜华[1] 吴期滨[1] 苏亚春[1] 

机构地区：[1]福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室国家甘蔗产业技术研发中心,福建福州350002 [2]福建医科大学附属协和医院,福建福州350001

出　　处：《热带作物学报》2016年第1期107-116,共10页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金(No.31340060);福建省杰出青年科学基金(No.2015J06006);福建省高等学校新世纪优秀人才支持计划(No.JA14095);农业部甘蔗生物学与遗传育种重点实验室开放课题(No.MOAFJSBGB-201501)

摘　　要：为了解14-3-3基因在甘蔗黑穗病抗性中的作用,本研究以课题组前期构建的甘蔗抗黑穗病抑制消减杂交文库中筛选出的1个与14-3-3基因同源的EST序列作为探针,利用电子克隆和RT-PCR方法从甘蔗ROC22品种中分离到1个Sc14-3-3基因(Gen Bank Accession Number:KJ577592),并对其序列信息和基因表达情况进行分析。结果显示,该基因ORF长771 bp,编码256个氨基酸残基;Sc14-3-3是定位于细胞质的亲水蛋白,无跨膜螺旋区及信号肽,有10个Ser、3个Thr和7个Tyr潜在的磷酸化位点,并在N-端有4个蛋白结合区和1个多核苷酸结合区,对翻译和能量新陈代谢起着重要作用;该蛋白属非ε类群,其N-端和中间区域在不同物种间相对保守,C-端差异相对较大。基因表达模式分析显示,Sc14-3-3基因可能参与甘蔗对生物逆境的细胞免疫响应,其对Me JA介导的信号途径应答较SA和ABA早;黑穗病菌胁迫下,Sc14-3-3基因在甘蔗抗感品种中的表达模式存在差异,其在抗黑穗病品种YC05-179中上调表达且表达量变化明显,揭示了该基因参与甘蔗对黑穗病的应答反应。以上结果为甘蔗Sc14-3-3基因的功能研究积累了一定的基础资料,并为今后甘蔗的抗病分子育种提供后备基因资源。In order to understand the function of 14-3-3 gene in sugarcane smut resistance, based on an EST highly similar to 14-3-3 gene selected from the suppression subtractive hybridization library corresponding to sugarcane smut resistance, we cloned a Sc14-3-3 gene （GenBank Accession Number： KJ577592） from sugarcane ROC22, by electronic cloning using this EST as the probe to search the dbEST couple with RT-PCR amplification method. Sequence information and expression patterns of Sc14-3-3 gene were analyzed. The results showed that the Sc14-3-3 gene contained a complete ORF （771 bp） encoding 256 amino acid residues. Sc14-3-3 was a hydrophilic protein without signal peptides and transmembrane helix localized in the cytoplasm. The protein contained 10 serine, 3 threonine and 7 tyrosine potential phosphorylation sites and bad four protein binding regions and a polynucleotide binding domain in its N-end, which played an important role in the translation and energy metabolism. Amino acid homology analysis revealed that the protein belonged to non-ε groups, and its N- terminal and middle regions were relatively conservative in different species, C-terminal relatively larger difference. The expression analysis showed that the Sc14-3-3 gene might be involved in the immune response which was caused by biotic stress. The quantitative results suggested an earlier reply to MeJA signaling pathway than in SA and ABA stresses. Under the smut pathogen stress, the expression patterns of Sc14-3-3 were different between sugarcane susceptible cultivar ROC22 and resistant cuhivar YC05-179. Compared with ROC22, Sc14-3-3 gene was up-regulated and had significant expression change in YC05-179. The phenomenon indicated that the Sc14- 3-3 gene involved in the smut resistance of sugarcane cuhivars. This study will accumulate the basic information for its functions to further identified, and provide back-up genetic resources for future molecular disease-resistant breeding in sugarcane.

关 键 词：甘蔗 14-3-3基因 生物信息学 荧光定量PCR 

分 类 号：S566.1[农业科学—作物学]