番红花组织基因枪转化瞬时表达检测

Transient expression of gus gene via particle bombardment in Crocus sativus L.

用高压氦气式基因枪将CaMV35S启动子驱动下的gus基因导入经济植物番红花的叶鞘和叶、芽来源的愈伤组织块中 ,4 8h后染色检测到了gus基因的瞬间表达 ,2 0d后仍检测到极少量表达 .研究发现 ,轰击前在加有 0 5mol L甘露醇的液体愈伤组织继代培养基上处理 6h ,明显增加了gus基因表达的数量 ;选择 110 0psi× 9cm的轰击条件 ,转化效果最好 ;高浓度卡那霉素 (5 0 0～ 80 0mg L)可以作为筛选剂 .结果表明 ,基因枪法是有效的番红花遗传转化方法 ,gus基因可以作为番红花基因工程研究的报告基因 。

The gus gene(uid A) followed by CaM35S promoter is introduced into the callus and sheath of the economic plant Crocus sativus L. using Biolistic PDS-1000/He microjectile bombardment system. Transient expression of gus gene is observed in the sheath and callus by hischemical staining for 48 h and 20 d after transformation. The results show that the amount of transient gus expression increases when calli are subjected to a 0.5 mol/L mannitol subculture liquid medium,pre-treatment for 6 h prior to bombardment and the highest transient gus expression is obtained when selecting helium of 1 100 psi×9 cm target distance as physical bombardment parameters. The concentration of kanamycin used in resistant calli selection is 500--800 mg/L.The results suggest that gus gene can be used as a useful reporter gene in genetic engineering of Crocus sativus L. and the CaM35S promoter is an effective promoter without tissue specificity.