普通菜豆PvP5CS2基因对逆境胁迫的应答

A Response of PvP5CS2 Gene to Abiotic Stresses in Common Bean

为了探索逆境条件下脯氨酸积累的分子遗传机理,改善作物的抗逆能力,应用实时荧光定量PCR(RT-qPCR)技术和水杨酸法分别检测干旱、高盐(200mmol L-1NaCl)和冷(4℃)胁迫条件下普通菜豆幼苗叶和根中脯氨酸合成酶基因PvP5CS2表达量和脯氨酸含量的变化。结果显示,3种胁迫条件下普通菜豆幼苗叶和根中PvP5CS2的转录水平快速上升,干旱处理4d,叶中PvP5CS2表达量达到最大值;高盐处理下,叶和根中PvP5CS2的表达高峰分别出现在2h和6h;冷胁迫下,叶和根中的表达高峰都出现在2h;随着胁迫时间的延长,PvP5CS2基因的转录水平逐渐下降。普通菜豆在逆境胁迫下,幼苗叶和根中脯氨酸大量积累,积累高峰出现在PvP5CS2基因表达高峰之后。这些结果说明,PvP5CS2基因的表达受干旱、高盐和冷胁迫诱导,脯氨酸积累受PvP5CS2基因转录水平的调控。PvP5CS2基因在洋葱表皮细胞中的瞬时表达结果显示PvP5CS2蛋白定位在细胞核和膜上。

Dramatic accumulation of proline under a variety of stress conditions such as drought, salt and cold has been documented in many plants. Understanding the molecular mechanism of the accumulation of proline in plants may be helpful for improving abiotic stress tolerance of crop plants.△^1-pyrroline-5-carboxylate synthetase （P5CS） catalyzed the first two steps of proline biosynthesis in plants, is encoded by P5CS gene in higher plants. In this paper, the responses of PvP5CS2 gene in common bean （Phaseolus vulgaris） seedlings to drought, salt and cold stresses were investigated by Real-time quantitative PCR （RT-qPCR） approach. The accumulation of proline in common bean seedling was measured using aqueous sulfosalicylic acid method. In addition, subcellular localization of PvP5CS2 in onion epidermal cells was studied. The results showed that abiotic stresses significantly triggered the expression of PvP5CS2 gene in leaves and roots of common bean seedling, and caused the accumulation of proine. PvP5CS2 mRNA transcript level in common bean seedling leaves steadily increased to the maximum that was 1.51 times of control under the 4 d drought stress and then rapidly decreased to below control level in later period of drought stress. The proline content in leaves increased from the second day and up to the maximum （300.14 μg g^-1FW） at 8 d of drought stress. Treatment with 200 mmol ^L-1 NaCl caused a dramatic up-regulation of PvP5CS2 expression to the peak value, 7.67 fold of con-trol at 2 h in leaves and 6.14 fold of control at 6 h in roots. The peak of proline accumulation appeared at 6–9 h （2.5 fold） in leaves and 9 h （14.2 fold） in roots when salt stress. A significant expression of PvP5CS2 was detected at 2 h under cold （4℃ ） stress, which was 2.38 fold of control in leaves and 7.55 fold of control in roots. The accumulation of proline steadily increased to the maximum at 24 h of treatment in leaves （43.29 μg g^-1FW） and at 12 h of treatment （28.90 μg g^-1FW） in roots. In addition, the accumulation of PvP5CS2 mRNA preceded the accumulation of proline in three stress treatments. These results indicated that PvP5CS2 gene is an abiotic stress-inducible gene with different transcript levels in response to abiotic stresses, which regulates the accumulation of proline in common bean seedlings under drought, salt and cold stresses. Transient expression of PvP5CS2-GFP fusion gene in onion epidermal cells showed that PvP5CS2 protein was distributed in nucleus and plasmalemma.