pib基因启动子及其诱导启动性初探

将pib基因上游5.7 kb区段取代pCAMBIA1301中gus基因上游的35S启动子构建了pib拟启动区-GUS+ 35S-hpt 基因表达载体pNAR604。经农杆菌介导转化水稻成熟胚愈伤，获得了转基因抗潮霉素愈伤和36株转基因水稻植株。 转基因抗性愈伤和转基因植株根的组织化学GUS活性检测表明，光照培养下的抗性愈伤和转基因植株根不能使X-gluc显色，而暗处理24 h后的抗性愈伤和定植后转基因植株的根能使X-gluc显色。转基因植株GUS荧光定量分析结果表明，GUS表达具有器官特异性，黑暗处理前根的GUS活性最高、茎次之，分别是是叶片的7倍和3倍，叶片中仅有痕量本底。24 h黑暗处理后根、茎、叶中GUS活性都有增加，且叶片中的增加比例最大，其活性仅次于根。5 mmol/L水杨酸和0.3 mol/L NaCl叶面喷施转基因植株24 h后叶片中GUS活性分别为处理前的2.7和3.6倍。初步确定pib拟启动区是一个诱导型启动子。黑暗、水杨酸和NaCl能诱导该启动子启动活性。

An Initial Exploring for Promoter of pib Gene and its Inductive Activation

A 5.7 kb putative promoter region of pib gene was isolated from the pib genomic clone and substituted for the 35S promoter upstream of gus gene in plasmid pCAMBIA1301 to construct a new plant expression vector pNAR604(putative pib promoter-GUS+35S-hpt).From Agrobacterium-mediated transformation and hygromycin selective culture in vitro,hygromycin resistant calli and 36 transgenic rice(Oryza sativa L.) plants were obtained.Histochemical assays of GUS activity showed that no expression was observed in the resistant calli and roots from transgenic rice if cultured under light,but after 24 h dark treatment there was strong GUS staining.Fluorimetric quantitative analysis indicated that GUS expression was organ-specific in transgenic rice.Without the dark treatment,GUS activity in roots and stems were about 7 and 3 times higher than in leaves in which GUS activity was only trace detected.After 24 h dark treatment,GUS activity in roots,stems and leaves of transgenic plants were all promoted and the largest increase was observed in leaves.Twenty-four huors after spraying with 5 mmol/L SA(Salicylic Acid) or 0.3 mol/L NaCl,GUS activity in leaves of the transgenic plants was 2.7 or 3.6 times respectively higher than untreated control.It was confirmed that an inductive promoter was involved in this 5.7 kb upstream region of pib gene,and dark,SA and NaCl treatments were inductive factors for pib promoter.