Chen Ji Bao, Yang Jian Wei, Zhang Zhao Yuan, Feng Xiao Fan, Wang Shu Min
Nanyang Normal University, Wolong Road 1638, Wolong District, Nanyang City 473061, People's Republic of China.
J Genet. 2013 Dec;92(3):461-9. doi: 10.1007/s12041-013-0292-5.
Many plants accumulate proline in response to salt stress. Δ-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme in proline biosynthesis in plants. Plasmid DNA (pCHF3-PvP5CS1 and pCHF3-PvP5CS2) containing the selectable neomycin phosphotransferase gene for kanamycin resistance and Phaseolus vulgaris P5CS (PvP5CS1 and PvP5CS2) cDNA was introduced into Arabidopsis plants using Agrobacterium-mediated gene transfer. Southern blot, northern blot and RT-PCR analyses demonstrated that the foreign genes were integrated into Arabidopsis chromosomal DNA and expressed. Single-gene transformants were analysed in this study. Transgenic plants expressed higher levels of PvP5CS1 and PvP5CS2 transcripts under salt stress conditions than under normal conditions. When treated with 0, 100 and 200 mM NaCl, the average proline content in leaves of transgenic plants was significantly higher (P < 0.01) than control plants. The average relative electrical conductivity (REC) of transgenic lines was significantly lower (P < 0.01) than control plants under salt stress condition. Biomass production of transgenic lines was significantly higher (P < 0.05) than control plants under 200 mM NaCl stress treatment. These results indicated that introducing PvP5CS1 and PvP5CS2 cDNA into transgenic Arabidopsis caused proline overproduction, increasing salt tolerance. Although the expression of PvP5CS1 in L4 lines and PvP5CS2 in S4 lines was the same under salt stress condition, the S4 lines accumulated 1.6 and 1.9 times more proline than the L4 lines under 100 and 200 mM NaCl treatments, respectively. The REC of S4 plants was 0.5 (100 mM NaCl) and 0.6 times (200 mM NaCl) that of L4 plants. The biomass production of S4 plants was 1.6 times (200 mM NaCl) more than in L4 plants. Total P5CS enzyme activity of S4 was significantly higher than that of L4. These results implied that the PvP5CS2 protein had stronger capacity to catalyze proline synthesis than PvP5CS1 under salt stress condition.
许多植物会在盐胁迫下积累脯氨酸。Δ-吡咯啉-5-羧酸合成酶（P5CS）是植物脯氨酸生物合成中的限速酶。利用农杆菌介导的基因转移方法，将含有用于卡那霉素抗性的可选择新霉素磷酸转移酶基因以及菜豆P5CS（PvP5CS1和PvP5CS2）cDNA的质粒DNA（pCHF3-PvP5CS1和pCHF3-PvP5CS2）导入拟南芥植株。Southern杂交、Northern杂交和RT-PCR分析表明，外源基因已整合到拟南芥染色体DNA中并得以表达。本研究对单基因转化体进行了分析。转基因植株在盐胁迫条件下比正常条件下表达更高水平的PvP5CS1和PvP5CS2转录本。当用0、100和200 mM NaCl处理时，转基因植株叶片中的平均脯氨酸含量显著高于对照植株（P < 0.01）。在盐胁迫条件下，转基因株系的平均相对电导率（REC）显著低于对照植株（P < 0.01）。在200 mM NaCl胁迫处理下，转基因株系的生物量产量显著高于对照植株（P < 0.05）。这些结果表明，将PvP5CS1和PvP5CS2 cDNA导入转基因拟南芥中会导致脯氨酸过量产生，从而提高耐盐性。尽管在盐胁迫条件下L4株系中PvP5CS1和S4株系中PvP5CS2的表达相同，但在100和200 mM NaCl处理下，S4株系积累的脯氨酸分别比L4株系多1.6倍和1.9倍。S4植株的REC分别是L4植株的0.5倍（100 mM NaCl）和0.6倍（200 mM NaCl）。S4植株的生物量产量比L4植株高1.6倍（200 mM NaCl）。S4的总P5CS酶活性显著高于L4。这些结果表明，在盐胁迫条件下，PvP5CS2蛋白催化脯氨酸合成的能力比PvP5CS1更强。
