猕猴桃ACC氧化酶反义基因转化猕猴桃的研究

Genetic transformation of kiwifruit with its ACC oxidase antisense gene

为建立猕猴桃ACC氧化酶反义基因功能研究技术平台并通过生物技术改良猕猴桃,从海沃德猕猴桃(Actinidia chinensis Planch.var.deliciosa‘Hayward’)叶片中提取总RNA,反转录合成cDNA第一链,并以此为模板,扩增得到ACC氧化酶(ACO)基因片段约1 kb,经限制性内切酶图谱分析,组建亚克隆并进行测序,其开放阅读框长957 bp,编码319个氨基酸,与其他已公布的ACC氧化酶cDNA的核苷酸(AB003514.1)及氨基酸序列(BAA21541.1)同源率分别高达94.0%和95.6%,证明所克隆的cDNA序列的准确性。构建该基因的反义表达载体,并通过农杆菌介导的方法成功转化米良一号猕猴桃(Actinidia deliciosa cv.Miliang-1),得到再生苗。取14株经PCR检测,其中10株检测到ACC氧化酶基因目的条带,阳性植株占71.4%。实时荧光定量PCR分析结果显示,再生苗ACO基因的表达量低于野生型58%～81%。

In order to establish the technology platform for the kiwifruit＇s with Its ACC oxidase antisense gene functions and improve kiwifruit quality through bio-technology, the kiwifruit leaves （ from Actinidia chinensis Planch.var.deliciosa ＇Hayward＇ ） were used to clone a eDNA ofACO gene, and its sequence was analysed.. The sequence was 1003 bp in length, with an open reading frame of 957 bp, encording 319 amino acid residues. The DNA sequence and its deduced amino acid sequence showed 94.0 % and 95.6 % homology and similarity with the sequences of Hayward plants in GenBank, respectively. Ten of the analyzed plants were shown to be positive for the ACO gene insert （71.4 %）. The result of Quantitative Real-Time Fluorescence PCR Technology shows that the ACC oxidase gene expression of re-growth leaves was 58%-81% of the wild type.