Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene ( pl ) and Parasponia hemoglobin gene ( phb ) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pl and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pl gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pl gene was expressed in 3 out of 18 plants with 17% and 7.5%in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum bv. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.
豌豆凝集素和血红蛋白基因对水稻的转化和表达
张静娴1王忆平2沈世华1 王逸群1 高越峰1 单雪琴1 荆玉祥1*
（1. 中国科学院植物研究所，北京100093；2. 北京大学生命科学学院，北京100871）
摘要：为了扩大根瘤菌的宿主范围和试探根瘤菌在非豆科植物上的固氮作用 ,将豌豆凝集素基因 (pl)和Parasponiaandersonii 血红蛋白基因 (phb)构建在同一个植物表达载体上 ,用基因枪法将其导入水稻 (OryzasativaL .ssp .japonica)。经PCR扩增和Southern杂交分析 ,证明外源目的基因已整合到水稻基因组中。GUS组织化学染色及豌豆凝集素基因的Western印迹实验和表达产物的原位杂交 ,证实外源基因在转基因水稻中表达。在 40个转化植株中 18株有pl和phb基因的PCR产物 ,得率为 45 %。再用 18株植物做pl基因的Westernblot检测 ,有 3株有翻译表达 ,占 40株的 7.5 % ,18株的 17%。为水稻与根瘤菌的相互作用和固氮作用的可能性研究奠定了一定的基础
关键词： Parasponiaandersonii 血红蛋白基因；豌豆凝集素基因；基因枪；转化作用；基因表达；转基因水稻
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