农杆菌介导冷调节基因(Cbcor15a)遗传转化甘蔗体系的建立  

Establishment of sugarcane genetic transformation system by Agrobacterium tumefaciens-mediated cold regulated gene(Cbcor15a)

作　　者：滕峥[1,2] 李鸣[3] 崔永祯[1,2] 方位宽[3] 梁朝旭[3] 何姗珊[3] 梁俊[3] 吴凯朝[3] 叶燕萍[1,2] 李容柏[1,2] 

机构地区：[1]广西亚热带生物资源保护利用重点实验室,南宁530005 [2]广西大学农学院,南宁530005 [3]广西农业科学院甘蔗研究所,南宁530007

出　　处：《南方农业学报》2014年第8期1333-1339,共7页Journal of Southern Agriculture

基　　金：广西科学研究与技术开发计划项目(桂科能1347012-1;桂科重14121005-1-2);南宁市科学研究与技术开发计划项目(20131038);广西农业科学院甘蔗研究所基本科研业务专项项目(G2008005)

摘　　要：【目的】利用农杆菌介导法,将外源冷调节基因Cbcor15a导入甘蔗愈伤组织,建立快速、高效的甘蔗遗传转化体系,为培育具抗寒性甘蔗品种奠定基础。【方法】以新台糖22号(ROC22)为材料,通过对愈伤组织诱导、分化、生根等培养基进行优化,筛选出外源基因转化甘蔗的适合激素种类与用量、PPT用量、抗生素种类与用量;利用甘蔗转基因二元植物表达载体pCambia1300-Cbcor15a-bar,通过农杆菌介导法将目的基因Cbcor15a导入甘蔗愈伤组织。【结果】当农杆菌菌液OD600为0.4、侵染20 min时有利于愈伤组织分化;甘蔗愈伤组织诱导和分化培养阶段的最佳PPT为0.50mg/L。侵染后在共培养中添加500.00 mg/L抗生素Cef能有效抑制农杆菌,添加300.00 mg/L抗生素Cef能促进愈伤组织分化成苗,添加200.00 mg/L抗生素Cef能促进幼苗生根。MS培养基中添加低量NAA更有利于甘蔗愈伤组织的分化;在促进细胞分裂时KT的效果明显优于6-BA。MS培养基中添加5.00 mg/LNAA和70.00g/L蔗糖能有效促进分化苗生根。利用建立的遗传转化体系可获得286株转冷调节基因Cbcor15a的甘蔗转化植株;选取83株通过PPT抗性筛选后长势良好的转化植株进行阳性检测,其中有4株呈阳性。【结论】利用农杆菌介导的Cbcor15a基因转化甘蔗的遗传转化体系能成功将Cbcor15a基因整合到甘蔗基因组。【Objective】A cold-regulated gene Cbcor15 a isolated from Chorispora bungeana was transferred into sugarcane embryogenic callus by Agrobacterium tumefaciens-mediated method to establish high-efficient genetic transformation system in order to provide basis on breeding sugarcane varieties with cold resistance. 【Method】Sugarcane variety ROC22 was used as tested material to optimize culture media for callus induction, bud differentiation and plantlet rooting, and screen suitable type and dosage of hormone, PPT and antibiotics dosage, which adapted for exogenous gene transferring to sugarcane. By using a bivalent plant expression vector named pCambia1300- Cbcor15a-bar,Cbcor15 a gene was transferred into callus of sugarcane using Agrobacterium tumefaciens-mediated method. 【Result】The results showed that OD6000.4 of Agrobacterium tumefaciens and 20 min of infecting time were beneficial to callus differentiation. The optimal dosage of PPT for callus induction and differentiation was 0.50 mg/L. After infected,adding 500.00 mg/L antibiotic Cef in co-culture could effectively inhabit growth of Agrobacterium tumefaciens, while adding 300.00 mg/L Cef could promote callus differentiation, and 200.00 mg/L Cef could promote plantlet rooting.Low dosage of NAA in MS medium was more beneficial to callus differentiation, and KT was superior to 6-BA in promoting cell division. By adding 5.00 mg/L NAA and 70.00 g/L sugar in MS medium could effectively promote plantlets rooting. After the preliminary selection using PPT, 286 anti- PPT regenerated plantlets transferred with Cbcor15 a gene were obtained. Eighty-three plantlets with well growing were selected to detect by PCR, out of which four anti-PPT regenerated plantlets were positive. 【Conclusion】The Agrobacterium tumefaciens-mediated genetic transformation system was established successfully for exogenous gene transferring to sugarcane, and Cbcor15 a gene had been integrated into genome of sugarcane.

关 键 词：农杆菌介导 冷调节基因 甘蔗遗传转化体系 建立 

分 类 号：S566.1[农业科学—作物学]