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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,从云南山茶(C.reticulata)矮生品种‘恨天高’茎尖组织中克隆出GA20–氧化酶(GA20-oxidase,GA20ox)基因,命名为Cr GA20ox1(Gen Bank登录号:KP635268),基因全长1 178 bp,开放阅读框1 146 bp,编码381个氨基酸。实时荧光定量PCR分析发现,GA20ox1在4种不同的山茶中均得到表达,其中乔木类的浙江红山茶(C.chekiangoleosa)GA20ox1表达量最高,其次是岳麓连蕊茶(C.handelii)及金花茶(C.nitidissima),表达量最低的为矮生品种‘恨天高’。结果表明该基因的表达量高低与测试物种的株高有关。GA20ox1在4个物种不同组织中的表达模式基本一致,且在茎尖组织中的表达量均高于叶片。Cr GA20ox1转基因烟草分析结果表明,过表达Cr GA20ox1烟草株高约为野生型的3.6倍,而沉默表达Cr GA20ox1烟草株高约为野生型的一半。
According to the homologous sequences of Camellia japonica, specific primers were designed and cloned from the apical tissues of C. dwarfing var. Sinensis using the homologous cloning and 3 ’, 5’-RACE techniques The gene of GA20-oxidase (GA20ox) was named as Cr GA20ox1 (Gen Bank accession number: KP635268). The gene was 1 178 bp in length and 1 146 bp in open reading frame encoding 381 amino acids. Real-time quantitative PCR analysis showed that GA20ox1 was expressed in four different camellia species, among which the highest expression level of GA20ox1 in C. chekiangoleosa was in arbors, followed by C. handelii and Jin C.nitidissima, the lowest expression of the dwarf variety ’hatred.’ The results showed that the level of expression of the gene is related to the plant height of the tested species. The expression patterns of GA20ox1 in different tissues of four species were basically the same, and the expression level of GA20ox1 in shoot tip tissues was higher than that in leaves. The analysis of Cr GA20ox1 transgenic tobacco showed that the over-expressed Cr GA20ox1 tobacco plant was about 3.6 times higher than that of the wild type, while the silencing expression of Cr GA20ox1 tobacco plant was about half that of the wild type.