紫云英细胞转化条件的研究

STUDY ON THE CONDITIONS OF CELL TRANSFORMATION IN ASTRAGALUS SINICUS

研究了紫云英（ＡｓｔｒａｇａｌｕｓｓｉｎｉｃｕｓＬ．）细胞遗传转化的条件。根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍ ｅｆａｃｉｅｎｓ）经含乙酰丁香酮的低ｐＨ／ＰＯ３－４ 诱导培养后，用来感染紫云英下胚轴原生质体，随后的细胞ＧＵＳ瞬间表达活性显著提高，间接证明了上述预培养诱导活化了细菌ｖｉｒ基因，促进了Ｔ－ＤＮＡ 向植物细胞转移。在ＰＥＧ介导的ＤＮＡ 转移中，较高的ｐＨ 和Ｃａ２＋ 浓度能够提高细胞ＧＵＳ活性。质粒ＤＮＡ 浓度及启动子类型对外源基因在植物细胞内表达也有一定影响。采用外植体－农杆菌共培养法，获得ＧＵＳ和ＮＰＴ

The conditions of genetic transformation of cells in Astragalus sinicus were studied.The experimental results showed that Agrobacterium tumefaciens strain C 58 (pKIW 105), when incubated in medium of low pH and low phosphate concentration in presence of acetosyringone could be induced and activated.When the activated bacteria were used to infect A.sinicus, the GUS gene transient expression in the hypocotyl protoplasts of A.sinicus was immediately and remarkably enhanced.This indicated that the vir gene of A.tumefaciens was activated under the above mentioned incubation conditions which facilitated T DNA transfer.In PEG mediated DNA direct transfer, transient expression of GUS gene was promoted by higher pH and higher Ca 2+ concentration of fusion medium.In the same experimental condition, expression of GUS gene under the control of MAS CaMV 35S chimeric promoter was more effective than that under the control of CaMV 35S promoter, and intensity of GUS gene expression was positively correlated with the amount of foreign plasmid DNA in the range of 10—100 μg.Adventitious shoots were induced from cotyledon and hypocotyl explants treated with Agrobacterium tumefaciens strain PGV 2260 (pBI 121) and were subcultured on MS medium containing 50 mg/L kanamycin to select transformants, and then the transformed shoots were rooted.Stable expression of the foreign genes in the transformed plants was confirmed by assay of neomycin phosphotransferase Ⅱ (NPT Ⅱ) and β glucuronidase (GUS) activity.