苹果MdcyMDH过量表达对光合、激素和生长的影响  

Impacts of Mdcy MDH Overexpression on Photosynthesis, Hormone and Growth in Apple

作　　者：王庆杰[1] 金仲鑫 周李杰[1] 郝玉金[1] 姚玉新[1] 

机构地区：[1]山东农业大学园艺科学与工程学院/作物生物学国家重点实验室,山东泰安271018

出　　处：《中国农业科学》2015年第14期2868-2875,共8页Scientia Agricultura Sinica

基　　金：国家"863"计划(2011AA100204);中国博士后科学基金特别资助项目(2012T50588);山东省现代农业产业技术体系水果创新团队(SDAIT-03-022-03)

摘　　要：【目的】获得过量表达苹果细胞质苹果酸脱氢酶基因(Mdcy MDH)的苹果植株,评价其过量表达对转基因株系生长的影响,并通过光合特性和和激素水平来探讨其调控生长的机理,为进一步揭示该基因调控生长发育的机制奠定基础。【方法】从苹果叶片中克隆Mdcy MDH编码区,连接植物表达载体p BI121,转化农杆菌LBA4404;利用农杆菌侵染‘嘎啦’苹果叶片,通过叶片再生的方法获得Mdcy MDH过表达的转基因苹果株系。以分化的苹果组培苗叶片为材料,提取基因组DNA,利用来自35S启动子和转化基因序列的引物,通过PCR初步筛选转基因株系;然后,提取初筛的转基因苹果组培苗叶片RNA,分别采用半定量和荧光定量PCR的方法检测Mdcy MDH表达量,来确定Mdcy MDH过表达株系。以继代培养40和60 d的野生型和转基因苹果组培苗为材料,进行生根处理,30 d后测定Mdcy MDH过表达对组培苗表型以及株高、叶片数量、根重等生长参数的影响。以驯化的、在大田生长一年的野生型和转基因苹果苗为材料,测定叶片光合参数和叶绿素含量的变化;以组培苗为材料,利用液质联用仪测定叶片内源激素含量的变化。【结果】以苹果叶片基因组DNA为材料,PCR初步筛选获得了3个转基因株系,即Line2、Line 3和Line 4;半定量和荧光定量PCR检测发现Line 2和Line 4中Mdcy MDH表达量分别比野生型提高了12倍和5倍,因此确定以上两个株系为Mdcy MDH过表达株系。此外,Mdcy MDH过量表达也提高了叶绿体苹果酸脱氢酶基因(Mdch MDH)表达量。组培苗生根培养30 d后,Mdcy MDH过表达对转基因株系的株高、叶数目、茎粗度、地上部重量未造成显著性影响,但小幅提高了转基因株系的株高和地上部总重量;与野生型相比,Mdcy MDH过量表达显著提高了根系的重量和总鲜重。Mdcy MDH过表达显著提高了转基因株系的光合速率,Line 2和4光合速率分别提高了13.2%和15.1%【Objective】 The objective of this study is to obtain Mdcy MDH-overexpressed apple in vitro shoot cultures, to evaluate the impacts of Mdcy MDH overexpression on growth, and to disclose the corresponding mechanism by determining photosynthesis and hormone level. The potential results are expected to lay a basis for further unraveling the mechanism related to growth regulation by Mdcy MDH.【Method】The open reading frame of Mdcy MDH was amplified by PCR from apple leaves. The PCR products were cloned into the expression vector p BI121. The resultant construct was introduced into the Agrobacterium LBA4404 and transformed into apple leaves by Agrobacterium mediated transformation method. The transgenic plants would be produced by leaf regeneration. The transgenic lines were preliminarily screened by PCR using the specific primer pairs from the sequence of 35 S promoter and Mdcy MDH. The transgenic lines were further confirmed by semi-quantitative and quantitative RT-PCR. The apple in vitro shoot cultures at 40 and 60 days after subculture were selected to subject to rooting culture. The growth parameters were determined at 30 days after rooting treatment, such as stem height, leaf quantity and fresh root weight. The photosynthesis and chlorophyll content were determined using the leaves from the wild type and transgenic apple trees grown in the field; the chlorophyll content was determined by spectrophotometer. The hormones in the leaves of apple in vitro shoot cultures were detected by LC-MS.【Result】The PCR using DNA template preliminarily identified three transgenic lines, i.e., line 2, line 3 and line 4, which were confirmed by expression analysis with semi-quantitative and quantitative RT-PCR. It was found that Mdcy MDH expression was increased by 12 folds and 5 folds in line 2 and line 4, respectively, compared to the wild types. The expression levels of chloroplast malate dehydrogenase gene（Mdch MDH） were also elevated in the transgenic lines. As to the apple in vitro shoot cultures at 30 days aft

关 键 词：苹果 Mdcy MDH 转基因 生长势 光合特性 脱落酸 

分 类 号：S661.1[农业科学—果树学]