Gao M, Tao R, Miura K, Dandekar A M., Sugiura A
Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, 606-8502, Kyoto, Japan
Plant Sci. 2001 Apr;160(5):837-845. doi: 10.1016/s0168-9452(00)00458-1.
Japanese persimmon (Diospyros kaki Thunb. cv Jiro) was transformed with apple (Malus x domestica Borkh.) cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) by an Agrobacterium-mediated leaf-disc transformation system. Integration and expression of the transgene were confirmed by genomic DNA blot and immunoblot analyses. Sorbitol accumulation in five of six transgenic plants obtained was confirmed by GC-MS. The amount of sorbitol in the leaves of transgenic plants varied from 14.5 to 61.5 µmol g(-1) fr wt(-1). Sorbitol was not found in leaves of non-transformed 'Jiro' or the line PS7 that produced S6PDH protein with no S6PDH activity. Eventually, two transformed lines producing high (PS1) and medium (PS6) amounts of sorbitol, one control transformed line (PS7), and non-transformed 'Jiro' were selected and evaluated for salt-stress tolerance. Under NaCl stress, the activity of photosystem II in leaves was determined in terms of the ratio of the variable (Fv) to the maximum (Fm) fluorescence of chlorophyll. The rate of decline in Fv/Fm under NaCl stress was lower in PS1 than the other three lines, suggesting that PS1 is more tolerant to NaCl stress than the other three lines. The factors that caused enhanced salt stress tolerance in PS1 are discussed in relation to sorbitol biosynthesis and its growth.
通过农杆菌介导的叶盘转化系统，将编码依赖于NADP的山梨醇-6-磷酸脱氢酶（S6PDH）的苹果（Malus x domestica Borkh.）cDNA导入日本柿（Diospyros kaki Thunb. cv Jiro）。通过基因组DNA印迹和免疫印迹分析证实了转基因的整合和表达。通过气相色谱-质谱联用（GC-MS）证实了获得的六株转基因植物中有五株积累了山梨醇。转基因植物叶片中山梨醇的含量在14.5至61.5 μmol g(-1)鲜重(-1)之间变化。在未转化的‘次郎’或产生无S6PDH活性的S6PDH蛋白的品系PS7的叶片中未发现山梨醇。最终，选择了两个分别产生高（PS1）和中等（PS6）量山梨醇的转化株系、一个对照转化株系（PS7）和未转化的‘次郎’，并对其耐盐胁迫能力进行了评估。在NaCl胁迫下，根据叶绿素可变荧光（Fv）与最大荧光（Fm）的比值测定叶片中光系统II的活性。在NaCl胁迫下，PS1中Fv/Fm的下降速率低于其他三个株系，这表明PS1比其他三个株系更耐NaCl胁迫。结合山梨醇生物合成及其生长情况，讨论了导致PS1耐盐胁迫能力增强的因素。
