erium-Mediated Transformation of 4CL Gene from Neosinocalamus affinis into Dendrocalamus farinosus | 农杆菌介导慈竹4CL基因遗传转化梁山慈竹

Chinese. 以梁山慈竹2种类型成熟胚的愈伤组织为材料,采用农杆菌遗传介导的方法,将已构建好的具有降低木质素含量的PBI121-4CL-RNAi表达载体导入愈伤组织,探讨愈伤组织预培养时间、菌液浓度、侵染时间、共培养时间和温度对遗传转化的影响。研究结果表明,淡黄色、颗粒状、疏松易碎的胚性愈伤组织是较好的遗传转化材料。以在愈伤组织培养基上预培养8天的淡黄色、颗粒状、疏松易碎的胚性愈伤组织为转化受体,在菌液浓度为OD600=0.05的EHA105中侵染20min后,在25℃、黑暗条件下共培养2天（共培养基表面加一层无菌滤纸）,在含有卡那霉素为55mg.L-1的抗性筛选培养基上筛选30天,抗性愈伤组织率为90%,经PCR检测,慈竹4CL基因已导入梁山慈竹愈伤组织中。抗性愈伤组织在芽诱导培养基上诱导30天,可获得丛生芽,待丛生芽长至3～5cm后,在生根培养基上经过20～30天的诱导,可产生1～8条根,获得再生植株。经PCR检测,慈竹4CL基因已导入梁山慈竹再生植株中,获得了转基因植株,转化效率为9%。RT-PCR检测结果表明,转4CL基因的梁山慈竹愈伤组织和植株的内源4CL基因的表达受到抑制,且表达量比对照明显降低。

English. The effects of precuhure time, bacteria concentration, infection time, time and temperature of co-culture on Agrobacterium-mediated transformation were studied through introducing expression vector PBI121-4CL-RNAi, that decreases lignin content, into callus from a mature embryo of Dendrocalamus farinosus. Our purpose is to establish an Agrobacterium-mediated transformation method of Dendrocalamus farinosus and to obtain transgenic plants. This study could lay a foundation for transformation of Dendrocalamus farinosus. The results showed that the loose and fragile callus of Dendrocalamus farinosus with pale yellow color and granular shape was a good donor to be used for Agrobacterium-mediated transformation. The callus was precultured in the inducing medium for 8 days, before it was immersed for 20 min in Agrobacterium suspension （EHA105, Agrobacterium concentration is OD600 = 0.05） , and then it were co-cultured in the co-cultivation medium with single filter paper at 25 for 2 days in the dark. The co-cultured callus were transferred into Kana-resistant medium containing Kana （55 mg.L-1） for 30 days. The Kana-resistant callus was accounted for 90% of the total callus. PCR identification of the Kana-resistant callus showed that the 4CL gene from Neosinocalamus affinis was introduced into Dendrocalamus farinosus callus. The Kana-resistant calli were transferred to the shooting medium and cultured for 30 days to obtain clustered shoots. After growing to 3-5 cm, the shoots were transferred into the rooting medium for 20-30 days. The regeneration plants with 1-8 roots were obtained. PCR identification of resistant plants showed that the 4CL gene from Neosinocalamus affinis was introduced into the resistant plants. The transgenic plant was obtained. The transformation efficiency with the system was 9%. The results showed that the endogenous 4CL gene expression in the transgenic callus and plants was effectively suppressed. The expression in the transcription level is significantly lower than the control by RT-PCR detection.