石防风原生质体遗传转化及抗除草剂植株的再生  

Genetic Transformation of Protoplasts from Peucedanum terebinthaceum and Regeneration of Herbicide_resistant Plantlets

作　　者：王秀凤[1] 杨爱芳[1] 张举仁[1] 

机构地区：[1]山东大学生命科学学院

出　　处：《Acta Botanica Sinica》1999年第7期706-710,共5页Acta Botanica Sinica（植物学报：英文版）

摘　　要：以石防风（Ｐｅｕｃｅｄａｎｕｍｔｅｒｅｂｉｎｔｈａｃｅｕｍ（Ｆｉｓｃｈ．）Ｆｉｓｃｈ．ｅｘＴｕｒｃｚ．）叶柄愈伤组织为材料建立以致密细胞团为主的悬浮培养物，用酶解法获得原生质体。用ＰＥＧ法将拟南芥菜（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）抗除草剂基因导入原生质体后进行液体浅层培养。转化细胞经除草剂ｃｈｌｏｒｓｕｌｆｕｒｏｎ筛选，通过胚状体途径产生抗性小植株。转化处理１０６个原生质体，以加入４０～５０μｇ／ｍＬ质粒ＤＮＡ，ＰＥＧ终浓度为１０％，于１ｍＬ转化介质中，２６℃黑暗下处理２０ｍｉｎ的效果最好。ｃｔＤＮＡ的加入对转化结果无明显影响。分子杂交试验表明，突变的ａｌｓ基因已整合进转化植株的基因组中。转基因植株的细胞在脱分化过程中仍具有抗ｃｈｌｏｒｓｕｌｆｕｒｏｎ的能力；移栽成活的转基因小苗仍表现出抗除草剂特性，而未转化的植株在低浓度除草剂下即逐渐死亡。表明转入的ａｌｓ基因在石防风细胞内能够稳定表达。Calli produced from the segments of young petioles of Peucedanum terebinthaceum Fisch. ex Turcz. were transfered into liquid medium for suspension culture.The protoplasts were isolated from cells of clusters by enzymes digestion.The mutant als (acetolactate synthase) gene of Arabidopsis thaliana (L.) Heynh. was transferred into protoplasts of P.terebinthaceum with PEG method. A transformation medium containing 40～50 μg/mL DNA and 10% PEG and an incubation condition of 26 ℃ for 25 minutes in dark revealed the best result. Supplementation with 0～60 μg/mL ctDNA into the medium gave no remarkable effect on transformation frequency.After cell clumps formed from the treated protoplasts in liquid medium, they were transferred onto proliferation medium containing 0.01 mg/L chlorsulfuron. The resistant clones grew 20 days later, and were transferred onto differentiation medium containing 0.03 mg/L chlorsulfuron. Plantlets regenerated via embryoid pathway. Southern blot hybridization with the 32 P labeled 2.5 kb fragments of p35s_als demonstrated that the mutant als gene had inserted into P.terebinthaceum genome. Chlorsulfuron_resistant calli which grew normally were produced when segments of transgenic plantlets were cultured on the proliferation medium containing 0.01 mg/L chlorsulfuron, whereas the growth of untransgenic explants were inhibited. Meanwhile, the transgenic plants could stand high density of chlorsulfuron up to 0.06 mg/L, but not the wild plants. These results indicated that the exogenous als gene had integrated into P. terebinthaceum genome and could transcript and translate stably.

关 键 词：原生质体 遗传转化 CHLORSULFURON 石防风 

分 类 号：S567.239[农业科学—中草药栽培]