转录因子OsbZIP5负调控水稻的耐旱性

Transcription Factor OsbZIP5 Negatively Regulates Drought-tolerance in Rice

干旱是影响水稻(Oryza sativa)等农作物生长和产量的主要非生物胁迫因素。克隆和鉴定耐旱相关基因,研究水稻耐旱机制对提高粮食产量有重要的意义。本研究以水稻为材料,克隆1个水稻bZIP家族转录因子的编码基因OsbZIP5,并对其耐旱功能进行初步研究。本文构建p35SOsbZIP5-GFP4、pGBKT7-OsbZIP5、pCAMBIA1300-OsbZIP5-OE和pTCK303-OsbZIP5-RNAi等不同的表达载体,分别转化了烟草、酵母、拟南芥和水稻。对OsbZIP5-GFP4转基因烟草进行激光共聚焦(confocal)观察,发现OsbZIP5定位在细胞核。对pGBKT7-OsbZIP5转化的酵母进行报告基因表达分析,表明该蛋白质能够激活报告基因的表达,具有转录激活活性。对OsbZIP5过表达转基因拟南芥在幼苗期和生殖生长时期的耐旱性进行检测。结果表明,OsbZIP5过表达降低了转基因拟南芥在聚乙二醇(polyethyleneglycol,PEG)和脱落酸(abscisic acid,ABA)处理条件下的根长及成株的耐旱性。利用20%PEG模拟干旱胁迫,对OsbZIP5 RNAi转基因水稻幼苗期的耐旱性和耐逆相关生理指标进行统计分析发现,不同OsbZIP5 RNAi转基因水稻株系的平均存活率分别是65%、58.30%和73.30%,而野生型的为21.67%;在正常生长条件下,RNAi转基因水稻的脯氨酸、叶绿素和丙二醛含量与野生型的各项指标基本一致,而在聚乙二醇处理后,RNAi转基因水稻的上述指标的平均值分别是159.54μg/g FW、0.24 mg/g FW和7.44 nmol/g FW,而野生型的平均值分别是102.56μg/g FW、0.13 mg/g FW和13.61 nmol/g FW。进一步分析还发现,干旱条件下,不同OsbZIP5 RNAi株系的水稻叶片的全闭气孔的比例均在66%以上,而野生型的约为50%,RNAi株系的失水率也低于野生型。由此表明,OsbZIP5下调表达改善转基因水稻的生理指标,降低气孔开度和失水率,提高RNAi转基因水稻的耐旱性。上述结果表明,OsbZIP5是水稻干旱胁迫耐受的负调节因子。

Drought is a major abiotic stress factor which affects the growth and yield of rice(Oryza sativa)and other crops seriously.Cloning and identifying drought-tolerant genes and exploring the drought-tolerant mechanism are of great significance for improving grain yield.In this paper,a bZIP family transcription factor encoding gene,OsbZIP5,was cloned from rice and we conducted a preliminary study on its drought tolerance function.We constructed different expression plasmids,including p35SOsbZIP5-GFP4,pGBKT7-OsbZIP5,pCAMBIA1300-OsbZIP5-OE and pTCK303-OsbZIP5-RNAi,which were transformed into tobacco,yeast,Arabidopsis or rice,respectively.Confocal observation of OsbZIP5-GFP4 transgenic tobacco showed that OsbZIP5 was located in the nucleus.The reporter gene expression analysis of pGBKT7-OsbZIP5 transformed yeast showed that the protein can activate the expression of reporter genes and OsbZIP5 has transcription activational activity.The drought tolerance of OsbZIP5 overexpression transgenic Arabidopsis during the seedling and reproductive growth stages was analyzed,and the results showed that OsbZIP5 overexpression reduced the root length of seedlings under polyethyleneglycol(PEG)or abscisic acid(ABA)treatment and the drought tolerance of adult plants in transgenic Arabidopsis.The drought tolerance and stress tolerance related physiological parameters were tested using 20%PEG to simulate drought stress.It was found that the survival rates of different OsbZIP5 RNAi transgenic rice lines were 65%,58.30%,and 73.30%,respectively,while that of the wild type was 21.67%.Under normal growth conditions,the content of proline,chlorophyll and malondialdehyde in RNAi transgenic rice are basically the same as those of wild type.However,after PEG treatment,the average values of the parameters above of RNAi transgenic rice were 159.54μg/g FW,0.24 mg/g FW and 7.44 nmol/g FW,respectively,while the average value of the wild type were 102.56μg/g FW,0.13 mg/g FW and 13.61 nmol/g FW,respectively.Statistical analysis of stomata and water loss rate under drought treatment of rice leaves revealed that the proportions of fully closed stomata of all OsbZIP5 RNAi lines were above 66%while that of the wild type is about 50%.And the water loss rate of rice leaves in RNAi rice seedlings were lower than that of wild type.We showed that down-regulation of OsbZIP5 ameliorated the physiological parameters,reduced stomata opening and water loss rate,and improved the drought tolerance of RNAi transgenic rice.The results above suggested that OsbZIP5 was a negative regulator of drought stress tolerance in rice.