木薯FRK1类似基因的分离及表达分析

Isolation and Expression Analysis of FRK1-Like Genes from Cassava

FRK1是拟南芥先天免疫下游标记基因,该基因的表达意味着PTI途径的启动。本研究以拟南芥FRK1蛋白序列采用Blastp的方法,从木薯本地基因组数据库中筛选FRK1类似基因共8个,经生物信息学分析发现此8个类似基因均编码类受体蛋白激酶,其氨基酸长度位于804~921 aa之间,蛋白质序列平均长度为863 aa;8个类似基因在木薯染色体上呈不均匀分布,主要分布在11号和4号染色体上。对其基因结构分析发现,8个类似基因可分为两大类,与进化树分析结果一致。根据保守基序的位置、氨基酸残基长度等信息分析发现,8个类似FRK蛋白与AtFRK1具有3个相同的保守基序,且长度位于50~250 aa之间。经与AtFRK1比对,综合蛋白基序、染色体定位以及进化关系等结果,候选MeFRK1基因为木薯基因组中先天免疫下游标记基因。以木薯SC8组培幼苗为材料,分别利用激素SA、JA和病原菌Xam进行处理后,利用荧光定量PCR测定MeFRK1基因在叶片中的表达量。结果表明:3个处理表达量基本均呈先上升后下降的变化趋势。激素SA、JA处理在15 min时均达到最大值,且SA的表达量略高于JA,而在病原菌Xam处理后第4天时达到最大值,此时表达量明显高于SA、JA处理,是SA的2.84倍,JA的3.06倍。由此可知,MeFRK1在响应SA和JA信号途径时,短时间内具有正调控作用,且反应较为迅速。在病原菌Xam胁迫中,能更好地诱导该基因的表达,从而提高木薯对病原菌Xam的抗性。其结果可为进一步建立木薯抗病分子体系奠定基础。

FRK1 is a marker gene downstream of the innate immunity in Arabidopsis thaliana(At),and FRKI’s expres-sion implies the initiation of the PTI pathway.This experiment screened eight FRK1-like genes from the cassava local genomic database via the Blastp method.Bioinformatics analysis reveals that all the eight FRK1-like genes encode re-ceptor-like protein kinases with amino acid lengths between 804-921 aa and an average protein sequence length of 863 aa.The eight genes were found unevenly distributed on cassava chromosomes,mainly on chromosomes 11 and 4.The analysis of the gene structures showed that the eight genes could be classified into two groups,which in agreement with the results of the evolutionary tree analysis.Based on the location of the conserved motifs and the length of amino acid residues,it was found that MeFRK1,MeFRK4 and MeFRK5 shared three conserved motifs with AtFRK1,and the length of the conserved motifs was between 50 and 250 aa.By comparing with AtFRK1 and factoring in the results of protein motifs,chromosome positioning and evolutionary relationships,MeFRK1 was identified as a marker gene downstream of the innate immunity in the cassava genome.The experiment was conducted on cassava SC8 group-cultured seedlings,and the expression of MeFRK1 gene in leaves was measured by fluorescence quantitative PCR after treatment with the hormones SA,JA and the pathogenic bacteria Xam,respectively.The experiment unveiled that the expression of MeFRK1 gene after the treatment of hormones SA,JA and Xam all demonstrated an increasing trend followed by a decreasing one.After the treatment of the hormone SA and JA,the expression of MeFRK1 gene both peaked at 15 min,with the expression under SA treatment slightly higher than that of JA.While after the treatment of Xam,the expression peaked at 4 d at which time the expression was significantly higher than that of SA and JA treat-ments,2.84 times that of the SA treatment and 3.06 times that of the JA treatment.It is evident that MeFRK1 has a posi-tive regulatory effect in response to SA and JA signaling pathways for a short period of time,and the response is rela-tively rapid.Pathogenic Xam could better induce the expression of MeFRKI gene,thus improving the resistance of cas-sava to pathogenic bacteria Xam.The results may lay the foundation for further establishment of a molecular system for cassava disease resistance.