苹果MdcyMDH过量表达对光合、激素和生长的影响

【目的】获得过量表达苹果细胞质苹果酸脱氢酶基因（MdcyMDH）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MdcyMDH编码区，连接植物表达载体pBI121，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MdcyMDH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MdcyMDH表达量，来确定MdcyMDH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MdcyMDH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Line 2、Line 3和Line 4；半定量和荧光定量PCR检测发现Line 2和Line 4中MdcyMDH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MdcyMDH过表达株系。此外，MdcyMDH过量表达也提高了叶绿体苹果酸脱氢酶基因（MdchMDH）表达量。组培苗生根培养30 d后，MdcyMDH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，MdcyMDH过量表达显著提高了根系的重量和总鲜重。MdcyMDH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%；转基因株系的蒸腾速率和气孔导度总体上都有显著性提高，但其胞间CO₂浓度显著降低；MdcyMDH过量表达显著提高了叶绿素a、叶绿素b和叶绿素总含量，与对照相比，Line 2和4株系叶绿素总含量分别提高了20.4%和15.9%。叶片激素含量测定表明，MdcyMDH过量表达显著抑制了ABA水平，其含量约为对照的1/2。【结论】MdcyMDH过量表达通过提高光合能力和降低ABA水平促进了转基因苹果组培苗的生长，尤其是促进了生根。

Impacts of MdcyMDH Overexpression on Photosynthesis,Hormone and Growth in Apple

【Objective】 The objective of this study is to obtain MdcyMDH-overexpressed apple in vitro shoot cultures, to evaluate the impacts of MdcyMDH 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 MdcyMDH.【Method】The open reading frame of MdcyMDH was amplified by PCR from apple leaves. The PCR products were cloned into the expression vector pBI121. 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 35S promoter and MdcyMDH. 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 byexpression analysis with semi-quantitative and quantitative RT-PCR. It was found that MdcyMDH 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 (MdchMDH) were also elevated in the transgenic lines. As to the apple in vitro shoot cultures at 30 days after rooting culture, MdcyMDH overexpression did not generate significant impacts on plant height, leaf number, stem diameter and above-ground fresh weight; by contrast, plant height and above-ground weight were increased to a small extent for the transgenic lines. In addition, it was found that the photosynthesis was significantly increased compared with the control, and the increments of 13.2% and 15.1% were found in line 2 and line 4, respectively. By contrast, stomatal conductance and transpiration rate were also significantly increased, while CO₂ concentration was significantly reduced. Besides, MdcyMDH overexpression led to 20.4% and 15.9% increments of total chlorophyll contents in line 2 and line 4, respectively. On the other hand, there was no significant difference in IAA and GA₃ between the control and transgenic lines. By contrast, MdcyMDH overexpression significantly inhibited ABA levels and produced nearly 50% decline of ABA content.【Conclusion】MdcyMDH overexpression promoted growth of the transgenic plants and roots in especial by increasing photosynthesis and decreasing ABA level.