杜鹃红山茶CaAPX基因的克隆、表达及功能分析

Cloning,Expression and Functional Analysis of CaAPX Gene from Camellia azalea

[目的]利用分子生物学技术探讨杜鹃红山茶CaAPX基因的表达模式及在模式植物烟草中所起的抗寒、耐热作用,为今后山茶花抗逆育种奠定基础。[方法]根据山茶花同源序列设计特异性引物,利用同源克隆和3’,5’-RACE技术,从杜鹃红山茶嫩叶组织中克隆出抗坏血酸过氧化物酶(APX),命名为CaAPX,基因全长1 097 bp,开放阅读框753 bp,编码250个氨基酸。实时荧光定量PCR对杜鹃红山茶7种组织和温度胁迫下该基因的表达模式进行分析。[结果]表明:CaAPX基因在杜鹃红山茶7种组织中均得到表达,但表达水平不一,表达量由高到低依次为:未成熟果实>嫩叶>花苞>叶芽>种胚>花瓣>花芽,其中,未成熟果实中的表达量是其它组织的2.6811.44倍;温度胁迫处理8 h后该基因呈上调表达,CaAPX基因表达量分别是0 h的3.49和2.67倍。CaAPX基因转化烟草分析表明,过量表达CaAPX基因后,APX活性提高了2.58 4.09倍,抗坏血酸(As A)含量提高了2.673.56倍,并且转基因烟草植株的抗寒、耐热能力也获得提高。[结论]通过过量表达CaAPX基因能够提高烟草植株的抗寒、耐热性,为山茶花抗逆育种提供了科学依据。

[Objective]Using molecular biology techniques to investigate the expression patterns of CaAPX gene in Camellia azalea,and to carry out the functional analysis of CaAPX in tobacco under temperature stress.[Method] On the basis of homologous sequences of C.japonica,an ascorbate peroxidase （APX）gene was isolated from the tender leaf in C.azalea by the 3’,5’-RACE technology named CaAPX.The full-length cDNA of CaAPX is 1 097 bp,containing a 753 bp ORF which encodes 250 amino acids.[Result]Quantitative real-time PCR analysis indi-cated that the CaAPX was expressed differently in all examined camellia tissues,and the expression levels in order were immature green fruit,tender leaf,flower,leaf bud,seed embryo,petal and flower bud.The highest expres-sion level of CaAPX was in immature green fruit,approximately ranging 2.68 to 11.44 times as high as that of the other six groups.It was also found that the expression level was notably up-regulated in leaves of camellia plants subjected to abnormal temperatures.Furthermore,the APX activity in transgenic plants increased by 158% -309%,and the AsA content increased by 167% -256% as higher as wild type plants.In addition,the over ex-pression of CaAPX enhanced cold and heat stress tolerance in transgenic plants under temperature stresses. [Conclusion]The over expression of CaAPX from C.azalea could enhance the cold and heat stress tolerance in transgenic tobacco,and which also provide a basis in molecular breeding of resistance adversity of Camellias in the future.