多年生黑麦草高频再生体系的建立及农杆菌介导PEPC基因的转化

摘要

In order to establish a highly efficient regeneration and transformation system of perennial ryegrass (Lolium perenne L. ) , its mature seeds were used as explants. Result showed that the best induction rate was 61. 33%. Callus differentiated after 2~3 generation. The optimum medium for callus differentiation was MS+6-BA at 1. 0 mg · L-1 with 61. 33% regeneration rate. The best medium for rooting was 1/2 MS+NAA at 0. 5 mg · L-1 and the ratio of rooting was 100%. Based on this regeneration system, the embryonic callus cells were used for Agrobacterium-mediated transformation. Transformed tissues were regenerated with media containing hyg as a selective solvent. PCR and Real-Time PCR analysis of transformants showed that PEPC gene was successfully integrated into the genome of perennial ryegrass and transformation rate was 8. 67%.%以多年生黑麦草(Lolium perenne L.)成熟种子作为外植体,建立其高频再生体系,通过农杆菌介导法将磷酸烯醇式丙酮酸羧化酶(PEPC)基因转入,为多年生黑麦草的抗逆转基因工作奠定基础.结果表明:种子充分灭菌后在附加6.0 mg· L-1 2,4-D的培养基中诱导愈伤组织,诱导率可达61.33％.愈伤组织在2,4-D浓度减半的培养基上继代培养长势良好,分化培养基为MS培养基附加1.0mg· L-16-BA,分化率最高达到61.33％,在1/2 MS+0.5 mg· L-1 NAA培养基中进行生根培养,生根率达100％.以该再生体系为基础,将获得的愈伤组织作为外植体进行遗传转化,经筛选培养后得到再生植株,通过PCR及实时荧光定量(Real-Time)PCR验证表明PEPC基因已成功转入,转化率为8.67％.试验结果将为多年生黑麦草抗性品种的研究奠定基础.