‘琯溪蜜柚’CmPAL基因的克隆与表达

Cloning and expression analysis of phenylalanine ammonia-lyase(Cm PAL)genes in’Guanximiyou’pummelo

为探究CmPAL基因在蜜柚果实发育过程中的作用与表达规律,以‘琯溪蜜柚’果实为材料,利用反转录PCR技术克隆了3个‘琯溪蜜柚’PAL基因,对它们所编码的蛋白进行了系列生物信息学分析,研究它们在‘琯溪蜜柚’及其两个芽变品种(‘红肉蜜柚’和‘三红蜜柚’)不同发育时期果实汁胞中的表达情况,对3种蜜柚汁胞木质素含量进行了测定.克隆所得到3个CmPAL基因,分别命名为CmPAL1、CmPAL3和CmPAL4,它们的cDNA长度分别为2154 bp、2160 bp和2142 bp,编码717、719和713个氨基酸;生物信息学分析发现3个CmPAL含有相同的保守结构域,均有跨膜螺旋结构与磷酸化位点;进化树分析结果表明,CmPAL1、CmPAL3和CmPAL4分别与甜橙CsPAL1、CsPAL2和CsPAL4的亲缘关系最近,且都归于双子叶植物分组;实时定量PCR结果显示,CmPAL基因蜜柚果实发育过程中表达水平存在显著性差异,不同成员的表达水平和变化趋势各有所不同;相关性分析结果显示,CmPAL3的表达与蜜柚果实木质素的含量存在显著性相关.本研究表明CmPAL基因成员尤其是CmPAL3可能通过调控木质素代谢进而在蜜柚果实成熟过程中发挥较大作用.(图8表4参39)

This study aimed to examine the function and expression patterns of pummelo phenylalanine ammonia-lyase(CmPAL)genes during fruit development.Three CmPAL genes were cloned by reverse transcription PCR;the proteins encoded by these genes were analyzed by a series of bioinformatic methods and the expression patterns at different developmental stages in‘Guanximiyou’pomelo and its bud mutation varieties(‘Hongroumiyou’and‘Sanhongmiyou’)were measured.The lignin contents of the three varieties of pummelo fruit had been previously determined.The complementary DNA(cDNA)lengths of the three CmPAL genes were 2154 bp,2160 bp,and 2142 bp,respectively,encoding 717,719,and 713 amino acids.Bioinformatic analyses showed that the proteins encoded by the three CmPAL genes contained the same conservative domain,and all had a transmembrane spiral structure and phosphorylation sites.Phylogenetic analysis showed that CmPAL1,CmPAL3,and CmPAL4 were closely related to CsPAL1,CsPAL2,and CsPAL4 and belonged to the dicotyledonous plants.Real-time quantitative PCR revealed differences in the expression patterns of the CmPAL genes,which significantly varied across the stages of fruit development.Finally,correlation analysis showed that CmPAL3 expression was significantly correlated with the lignin contents of the pummelo fruit.These results suggest that the CmPAL gene members,especially CmPAL3,may play an important role in the fruit ripening process by regulating lignin metabolism.