关键词: 苹果, MdcyMDH, 转基因, 生长势, 光合特性, 脱落酸

Abstract: 【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.

Key words: apple, MdcyMDH, transgene, growth vigor, photosynthesis, ABA