苹果果实愈伤转化体系的建立及其在基因功能研究中的应用  

Establishment of gene transformation system in fruit callus and Its application in gene functional analysis for apple plant

作　　者：李兴亮[1] 丁宁[1] 贾美茹 魏灵芝[1] 姜金铸[1] 李冰冰[1] 贾文锁[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193

出　　处：《中国农业大学学报》2015年第2期108-113,共6页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(31171921)

摘　　要：以苹果‘金冠’为试材,对苹果果实愈伤转化体系的建立及其在苹果基因功能分析中的应用进行研究。首先优化了苹果果实表面消毒的方法、外植体大小和形状、愈伤和继代培养基的组成,以及愈伤和继代培养的适宜条件,最后成功建立了苹果果实愈伤培养体系。在此基础上,将CaM 35S驱动的GUS报告基因转化苹果果实愈伤,发现GUS基因大幅度表达,同时GUS活性强烈提高。证明所获得的苹果果实愈伤适用于基因转化。为进一步证明该体系适用于苹果基因功能研究,分别构建了CaM 35S驱动的乙烯合成关键酶MdACO1过表达载体和RNAi载体,MdACO1的过量表达促进了乙烯合成并调控了一系列乙烯信号转导相关的基因表达,相反MdACO1的RNAi抑制了乙烯的合成,同时也改变了一系列与乙烯信号转导有关的基因表达。With apple ‘Golden Delicious＇ as research material,this work was aimed at developing a transient gene transformation system in callus and confirming whether it might be applicable for the functional analysis of the genes in apple plants.The research firstly optimized the sterilization method,the size and shape of explants,the culture medium for callus growth and regeneration,and finally developed the system for agrobacterium mediated gene transient transformation in apple plants.Application of gene transformation was tested with GUS reporter gene driven by CaM 35S.The results showed that there were both high expression of GUS gene and strong GUS activity,suggesting that the developed system could be used for gene manipulation.In order to further demonstrate the reliability of the system,an over-expression construct as well as an RNAi construction of MdACO1,a key gene in ethylene biosynthesis pathway,was respectively transformed into the callus.The expression analysis demonstrated that over-expression of MdACO1 caused dramatic increase in MdACO1 transcript level.In contrast,RNAi caused a significant decrease in the MdACO1 transcript level.Moreover,both over-expression and RNAi of MdACO1 resulted in changes of ethylene production and expression patterns of a series of genes involved in ethylene signaling.

关 键 词：苹果 愈伤 基因转化 乙烯 

分 类 号：S661.1[农业科学—果树学]