CoCl_(2)胁迫下青稞苯丙氨酸解氨酶基因的克隆与表达分析

Cloning and expression of phenylalanine ammonialyase gene of Hordeum vulgare var nudum under CoCl_(2) stress

为阐明青稞幼苗对钴胁迫抗逆基因的响应模式,以青稞‘昆仑15号’为材料,克隆青稞苯丙氨酸解氨酶(PAL)基因,采用电子同源法克隆青稞苯丙氨酸解氨酶基因cDNA序列,并分析相关抗逆基因的表达变化情况。结果表明,该基因开放阅读框(ORF)序列有2 142bp,编码713个氨基酸,命名为HvPAL,其GenBank登录号为MK695675。经分析HvPAL编码的氨基酸序列有3个高度保守结构域,包含典型的植物苯丙氨酸解氨酶活性中心的保守氨基酸序列,与禾本科植物大麦和小麦的氨基酸序列有较高的相似性(90%)。PAL氨基酸的分子系统进化树显示青稞属于禾本科植物。采用实时荧光定量PCR法分析,与对照相比,70mg/L Co^(2+)处理下青稞幼苗SOD基因表达为最高(P<0.05),150mg/L Co^(2+)处理下PAL、GSH和P5CS基因的表达量达到最大值(P<0.05),200mg/L Co^(2+)处理下POD基因表达量最大(P<0.05)。综上,青稞幼苗中POD、PAL、GSH、P5CS和SOD基因协同表达来抵抗和适应Co^(2+)胁迫。

To study the response pattern of stress resistance gene of Hordeum vulgare var nudum seedlings to cobalt stress,the phenylalanine ammonia-lyase(PAL)gene was cloned and the expression patterns of related stress resistance genes were analyzed.In this study,H.vulgare var nudum ‘Kunlun 15’was used as the material to clone the cDNA sequence of PAL,named HvPAL(GenBank No.MK695675).The length of PAL gene cDNA is 2 142 bp,which contains a open reading frame(ORF)encoding 713 amino acids.The bioinformatics analysis show that there are three highly conserved domains in the amino acid sequence of HvPAL,including the conserved amino acid sequence,which has high homology(90%)with that of barley and wheat.The molecular phylogenetic tree is constructed based on PAL protein sequence,and H.vulgare var nudum and Gramineae were clustered together.The expression of related genes in H.vulgare var nudum seedlings under CoCl_(2) stress was analyzed by real-time fluorescence quantitative PCR.Compared with the control,the expression of SOD gene was the highest under 70 mg/L Co^(2+)treatment(P<0.05),the expression levels of PAL,GSH,and P5 CS genes reached the maximum under 150 mg/L Co^(2+)treatment(P<0.05),and the expression level of POD gene was the highest under 200 mg/L Co^(2+)treatment(P<0.05).In conclusion,the POD,PAL,GSH,P5 CS and SODgenes were coexpressed in seedlings to resist and adapt to Co^(2+)stress.