过量表达杜鹃红山茶CaAPX1的烟草耐热性提高

根据山茶(Camellia japonica)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(C.azalea)嫩叶组织中克隆出抗坏血酸过氧化物酶(ascorbate peroxidase,APX)基因,命名为CaAPX1(Gen Bank登录号:KP635267),基因全长1 097 bp,开放阅读框753 bp,编码250个氨基酸。实时荧光定量分析发现,CaAPX1在4种不同的山茶中均得到表达,其中较耐热的微花连蕊茶(C.minutiflora)表达量最高,其次是中等耐热的杜鹃红山茶和‘耐冬’山茶(C.japonica‘Naidong’),最低的为耐热性较差的‘恨天高’云南山茶(C.reticulata‘Hentiangao’)。结果表明该基因的表达量高低与测试物种的耐热性有关。CaAPX1在杜鹃红山茶4种组织中均得到表达,表达量由高到低依次为:叶片、花蕾、种胚、花芽,其中叶片表达量高达其他组织的1.29~4.26倍。CaAPX1转基因烟草分析表明,过量表达CaAPX1后,APX酶活性提高了2.58~3.81倍,抗坏血酸(Ascorbate,AsA)含量也提高了2.67~3.56倍,并且转基因烟草植株及其种子的耐热能力也提高。

Overexpression of CaAPX1 from Camellia azalea Enhances Hot Stress Tolerance in Transgenic Tobacco

On the basic of homologous sequences of Camellia japonica，an ascorbate peroxidase （APX）gene was isolated from the tender leaf in C. azalea by the 3′，5′-RACE technology named CaAPX1（GenBank Accession ID：KP635267）. The full-length cDNA of CaAPX1 is 1 097 bp，containing a 753 bp ORF which encodes 250 amino acids. Expression of CaAPX1 in four different heat resistant types of camellias were analyzed by fluorescent quantitative real-time PCR. The results showed that APX1 gene was expressed in the four camellias differently，where the expression level of APX1 in C. minutiflora was the highest，medium in C. azalea and C. japonica‘Naidong’，and the lowest in C. reticulata ‘Hentiangao’，which indicated that the expression intensity of the APX1 gene was positively associated with heat resistance in the different camellia. And fluorescent quantitative real-time PCR analysis also indicated that CaAPX1 was expressed differently in all examined camellia tissues，and the expression levels in order were tender leaf，flower，seed embryo and flower bud. The highest expression level of CaAPX1 was tender leaf，approximately ranging 1.29 to 4.26 times as high as other three groups. Furthermore，the APX activity in transgenic plants increased 2.58–3.81 fold，and the AsA content increased 2.67–3.56 fold as higher as wild type. In addition，overexpression of CaAPX1 enhanced heat stress tolerance in transgenic plants and seeds under high temperature stress.