转PvP5CS1基因拟南芥植株对干旱和盐胁迫的反应

Response of PvP5CS1 Transgenic Arabidopsis Plants to Drought-and Salt-Stress

为探索普通菜豆脯氨酸合成酶基因P5CS1在植物渗透胁迫中的作用,本研究应用农杆菌介导法,将PvP5CS1基因转入拟南芥,获得6株阳性转基因株系;通过检测转基因植株与野生型植株在干旱和盐胁迫下种子发芽率,幼苗脯氨酸含量、株系电导率、相对根长和成株死亡率,分析了PvP5CS1基因的表达对改善拟南芥抗渗透胁迫的效应。结果表明,在150mmolL-1NaCl和150mmolL-1甘露醇渗透胁迫下,转基因植株平均相对发芽率分别是野生型的1.60倍和1.62倍;150、250mmolL-1甘露醇和150mmolL-1NaCl处理下,转基因拟南芥植株平均脯氨酸含量分别是野生型的2.68、1.30和1.30倍;平均相对电导率分别是野生型植株的85%、77%和85%;平均相对根长分别是野生型植株的1.2、1.3和1.2倍;300mmolL-1NaCl处理下,转基因植株的平均死亡率为42%,显著低于野生型(90%)(P<0.05);干旱胁迫下,转基因植株的平均死亡率为56%,显著低于野生型(70%)(P<0.05),说明PvP5CS1基因在拟南芥中的表达明显改善了转基因植株的抗旱性和耐盐性。

Under adverse environment, many plants increase their cell osmotic potential through accumulation of intracellular organic osmolytes-proline. The proline accumulation in plants can not only increase cell osmotic potential but also stabilize proteins, membranes, and subcellular structures as well as protect cells against oxidative damage by reactive oxygen species. Pyrroline-5-carboxylate synthetase （P5CS）, a rate-limiting enzyme involved in the biosynthesis of proline in higher plants, is encoded by P5CS gene. The full length cDNA sequence of PvP5CS1 gene was subcloned into pCHF3 vector and transformed into wild Arabidopsis via Agrobacterium tumefaciens. A total of six positive transgenic plants were obtained. The result of RT-PCR showed that the PvP5CS1 gene was normally expressed in transgenic plants. The T4 generation purified lines of transgenic plants and wild Arabidopsis were used for osmotic stress experiment. Seed relative germination rates of transgenic plants under treatment or non-treatment conditions were all significantly higher than those of wild plants under stress conditions （P〈0.001）. The average seed relative germination rate of transgenic seedlings was 1.62 and 1.60 times higher than those of wild plants treated with 150 mmol L^-1 mannitol and 150 mmol L^-1 NaCl, respectively. Proline content, conductivity rate and root length of eight-day transgenic seedlings under osmatic stress were determined. The results showed that introduction of PvP5CS1 gene into Arabidopsis resulted in significant accumulation of proline. The relative proline content in transgenic plants under stress non-treated conditions were all significantly higher than those in wild type plants （P〈0.05）. Under four treatments （CK, 150, 250 mmol L^-1 mannitol and 150 mmol L^-1 NaCl）, the average relative proline contents in transgenic plants were 1.38, 2.68, 1.30, and 1.30 times of those in wild type plants. The transgenic plants had longer root and less cell damage than wild plants under osmotic stress conditions. Under 150, 250 mmol L^-1 mannitol- and 150 mmol L^-1 NaCl-stress conditions, average relative conductivity rate in transgenic plants were 85%, 77%, and 85% of that in wild type plants, respectively. The average relative root length in transgenic plants was 1.2, 1.3, and 1.2 times of that in wild type plants, respectively. After 300 mmol L^-1 NaCl treatment for 15 days, the seedling death rate was 42% and 90% for transgenic and wild type plants, respectively. In the condition of drought stress for 25 days and following by rewatering for 5 days, the seedling death rate of transgenic plants was also significantly lower than that of wild types （P〈0.05）, which was 56% for transgenic plants and 70% for wild plants. These results indicated that over-expression of PvP5CS1 in transgenic Arabidopsis plants increased plants tolerance to salt and drought stresses.