摘要
本研究以甘薯品种栗子香的胚性悬浮细胞为材料，用根癌农杆菌菌株EHA101对根癌农杆菌介导的甘薯遗传转化进行了系统的研究。该菌株所携带的pROA93质粒上含有新霉素磷酸转移酶基因(NPTⅡ)和β-葡萄糖苷酸酶基因(GUS)。结果表明，将达到对数生长期的根癌农杆菌菌液稀释至OD_(600nm)=0.5，与继代培养3d后的胚性悬浮细胞共培养，转化效率最高，适宜的共培养时间为4d。将共培养后的材料在含有300mg/L Carb和2.0mg/L 2，4-D的MS液体培养基中培养5～8d，再进行选择培养，有利于转化细胞的增殖。将转化材料在含有300mg/L Carb、25～75mg/L Kan和2.0mg/L 2,4-D的MS液体培养基中进行选择培养，结果表明，Carb和Kan的适宜浓度分别为100mg／L和50mg/L。选择培养2～4周后，共获得2453个直径约1mm的小细胞团，将这些小细胞团转移到添加100mg／L Carb、50mg／L Kan和2.0mg/L 2,4-D的MS固体培养基上，经过8～20周的选择培养，获得46个Kan抗性愈伤组织。将得到的Kan抗性愈伤组织转移到添加100mg/L Carb、25mg／L Kan和1.0mg／L ABA的MS固体培养基上，Kan抗性愈伤组织分化形成体细胞胚并发芽，最后转移到添加50mg/L Kan的MS基本培养基上，获得了138株再生植株。PCR和PCR-Southern检测结果表明，其中的104株(75.4％)再生植株是转基因植株。
利用该转化体系，对水稻巯基蛋白酶抑制剂(OCI)基因的遗传转化进行了研究。根癌农杆菌LBA4404携带质粒pBinh，该质粒携带OCI和NPTⅡ基因。将达到对数生长期的根癌农杆菌菌液稀释至OD_(600nm)=0.5，与继代培养3d后的胚性悬浮细胞共培养4d。将共培养后的材料在含有300mg/L Carb和2.0mg/L 2,4-D的MS液体培养基中培养5～8d，然后将转化材料在含有300mg/L Carb、50mg/L Kan和2.0mg/L 2,4-D的MS液体培养基中进行选择培养，总共获得1847个直径约1mm的小细胞团。将这些小细胞团转移到添加100mg/L Carb、50mg／L Kan和2.0mg/L 2,4-D的MS固体培养基上，经过8～20周的选择培养，获得19个Kan抗性愈伤组织。将得到的Kan抗性愈伤组织转移到添加100mg/L Carb、25mg/L Kan和1.0mg/L ABA的MS固体培养基上，Kan抗性愈伤组织分化形成体细胞胚并发芽，最后转移到添加50mg/L Kan的MS基本培养基上，获得了31株再生植株。PCR和PCR-Southern检测结果表明，其中的16株(51.6％)再生植株是转基因植株。通过对OCI转基因植株和阴性对照植株中水稻巯基蛋白酶抑制剂的SDS聚丙烯酰胺凝胶电泳，结果表明OCI蛋白在转基因植株中有一定量的累积。
将GUS转基因植株和OCI转基因植株转移到温室，结果发现大部分转基因植株没有出现形态特征的变异。然而，在部分OCI转基因植株和GUS转基因植株上都发现叶片脉基色从淡褐色变为淡绿色、茎颜色从淡绿色出现局部淡褐色的变异。其中，GUS转基因植株中还出现1株丛生型的株型变异。这些变异究竟是由于基因转化造成的、或是由于体细胞无性系变异等其他原因造成的，还需要进一步的研究。
An Agrobacterium tumefaciens-mediated transformation system of sweetpotato, Ipomoea batatas (L.) Lam., was established by using embryogenic suspension cultures of the cultivar Lizixiang. A.tumefaciens strain EHA101 harboring a binary vector pROA93 with p-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes was used in the present study. Embryogenic suspension cultures of 3 d after subculture were cocultivated with EHA101 (OD600nm=0.5) for 4 days. After cocultivation, the infected suspension cultures were first cultured for 1 week in MS medium with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/L carbencillin but without kanamycin and then transferred into MS medium with 2 mg/L 2,4-D, 50 mg/L kanamycin and 300 mg/L carbencillin for the selection culture. 2~4 weeks after selection, 2453 kanamycin-resistant cell aggregates about 1 mm in size from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/L 2,4-D, 50 mg/L kanamycin and 100 mg/L carbencillin. and after 8 ~ 20 weeks of selection formed 46 kanamycin-resistant embryogenic calluses. After transfer to MS medium supplemented with 1 mg/L abscisic acid (ABA), 50 mg/L kanamycin and 100 mg/L carbencillin, these embryogenic calluses formed 138 plantlets via somatic embryogenesis. PCR and PCR-Southern blot analysis indicated that 104 regenerated plants (75.4%) were transgenic plants.
Using such gene transfer system, the transfer of OCI gene to sweetpotato cv. Lizixiang was also conducted. A.tumefaciens strain LBA4404 harboring a binary vector pBinh with OCI and neomycin phosphotransferase (NPT II) genes was used in the study. Embryogenic suspension cultures of 3 d after subculture were cocultivated with LBA4404 (OD600nm=0.5) for 4 days. After cocultivation, the infected suspension cultures were first cultured for 1 week in MS medium with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/L carbencillin but without kanamycin and then transferred into MS medium with 2 mg/L 2,4-D, 50 mg/L kanamycin and 300 mg/L carbencillin for the selection culture. 2-4 weeks after selection, 1847 kanamycin-resistant cell aggregates about 1 mm in size from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/L 2,4-D, 50 mg/L kanamycin and 100 mg/L carbencillin. and after 8-20 weeks of selection formed 19 kanamycin-resistant embryogenic calluses. After transfer to MS medium supplemented with 1 mg/L abscisic acid (ABA), 50 mg/L kanamycin and 100 mg/L carbencillin, these embryogenic calluses formed 31 plantlets via somatic embryogenesis. PCR and PCR-Southern blot analysis showed that 16 regenerated plants (51.6%) were transgenic plants. SDS-PEG analysis of OCI from transgenic plants with OCI negative control indicated that accumulation was found in transgenic plants with OCI gene.
These transgenic plants with gusA or OCI gene were transferred to greenhouse. No obvious morphological variations were observed in most of transgenic plants. However, some variations in vein base color and stem color were observed in part of transgenic plants with gusA or OCI gene. Also, one transgenic plaint with gusA gene showed variation in plant type. It needs further study if these morhphological variations are due to the transformation or the somatic mutations.
引文
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