矮生山茶‘恨天高’GA20ox1的克隆及其对转基因烟草株形的影响

Overexpression and Silence Expression of CrGA20ox1 from Camellia reticulata ‘Hentiangao’ and Its Effect on Plant Forms in Transgenic Tobacco

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。

On the basic of homologous sequences of Camellia japonica,a GA20-oxdiase（GA20ox）gene was isolated from the stem tip of C. reticulata‘Hentiangao＇by the 3′,5′-RACE technology named Cr GA20ox1（Gen Bank Accession ID：KP635268）. The full-length c DNA of Cr GA20ox1 is 1 178 bp,containing an 1 146 bp ORF which encodes 381 amino acids. Expressions of GA20ox1 in four different plant phenotypes of camellias were analyzed by fluorescent quantitative real-time PCR. The results showed that GA20ox1 gene was expressed in the four camellias differently,where the expression level of GA20ox1 in C. chekiangoleosa was the highest,medium in C. handelii and C. nitidissima,and the lowest in C. reticulata‘Hentiangao＇,which indicated that the expression intensity of the GA20ox1 gene was positively associated with height in the different camellias. The trends of expressions of GA20ox1 gene were almost the same in various organs of the four camellias,and furthermore,the expressions of the gene was higher in the shoot tips than in tender leaves. Phenotypic results of transgenic tobacco showed that the transgenic plant with sense Cr GA20ox1 gene was nearly 3.6 times height than wild type tobacco;Meanwhile,the transgenic plant with anti-sense Cr GA20ox1 gene was about only half height of the wild type tobacco.