论文部分内容阅读
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。
According to the homologous sequences of Camellia japonica, specific primers were designed and cloned ascorbate peroxidase from the leaves of C. azalea using homologous cloning and 3 ’, 5’-RACE Ascorbate peroxidase (APX) gene was named as CaAPX1 (GenBank accession number: KP635267). The gene was 1 097 bp in length and 753 bp in open reading frame (ORF) encoding 250 amino acids. Real-time fluorescence quantitative analysis showed that CaAPX1 was expressed in four different kinds of camellia, of which the higher expression of C.minutiflora was the highest, followed by moderately heat-resistant azalea C.japonica’Naidong ’, the lowest was C.reticulata’Hentiangao’ with poor heat tolerance. The results show that the level of expression of the gene is related to the heat resistance of the tested species. CaAPX1 was expressed in four tissues of Rhododendron camellia. The expression level of CaAPX1 was leaf, bud, embryo and flower bud from high to low, among which the leaf expression reached 1.29 ~ 4.26 times higher than other tissues. Analysis of CaAPX1 transgenic tobacco showed that the activity of APX increased by 2.58 ~ 3.81 folds, and the content of Ascorbate increased by 2.67 ~ 3.56 folds after overexpression of CaAPX1, and the heat tolerance of transgenic tobacco plants and their seeds also increased.