发根农杆菌介导的尾巨桉遗传转化体系的建立  

Establishment of Agrobacterium rhizogenes-mediated Genetic Transformation System of Eucalyptus urophylla×E.grandis

作　　者：罗萍 张昊楠 徐建民[1] 胡冰 王晓萍 李光友[1] 范春节[1] LUO Ping;ZHANG Haonan;XU Jianmin;HU Bing;WANG Xiaoping;LI Guangyou;FAN Chunjie(State Key Laboratory of Tree Genetics and Breeding,Key Laboratory of State Forestry and Grassland Administration on Tropical Forestry,Re‐search Institute of Tropical Forestry,Chinese Academy of Forestry,Guangzhou 510520;Nanjing Forestry University,Nanjing 210037;State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University,Harbin 150040)

机构地区：[1]林木遗传育种国家重点实验室,国家林业和草原局热带林木培育重点实验室中国林业科学研究院热带林业研究所,广州510520 [2]南京林业大学,南京210037 [3]林木遗传育种国家重点实验室,东北林业大学,哈尔滨150040

出　　处：《植物研究》2022年第3期512-520,共9页Bulletin of Botanical Research

基　　金：中国林业科学研究院基本科研业务费专项资金项目(CAFYBB2020ZB004);广东省基础与应用基础研究基金项目(2020A1515011059)。

摘　　要：发根农杆菌(Agrobacterium rhizogenes)的建立对植物功能基因的验证具有重要意义,为了在桉树(Eucalyptus)中建立发根农杆菌介导的遗传转化体系,本研究以不同的发根农杆菌菌株侵染尾巨桉(Eucalyptus urophylla×E.grandis)的叶片和茎段,确定合适的农杆菌菌株和外植体类型,在此基础上开展农杆菌浓度、侵染时间对毛状根诱导的影响。结果表明:采用发根农杆菌菌株MSU440,以叶片为外植体进行发根诱导,最高获得了81.0%的毛状根诱导率,毛状根平均根长达到3.23 cm。在发根农杆菌浓度为OD_(600)=0.3、侵染时间为30 min时,共培养48 h后经过20 mg·L^(-1)卡那霉素筛选培养,通过PCR分子鉴定和GUS染色证实外源基因稳定地整合在桉树毛状根基因组中,转化率达20.2%。初步建立了发根农杆菌介导的桉树遗传转化体系,为桉树基因功能鉴定和进一步的转基因育种奠定基础。Agrobacterium rhizogenes-mediated genetic transformation system plays an important role in the identification of functional genes.To establish Agrobacterium rhizogenes-mediated genetic transformation system in Eucalyptus,the different strains infected the leaves and stems of Eucalyptus urophylla×E.grandis were used to select the appropriate A.rhizogenes strains and explant types,and the effects of Agrobacterium concentration and infection time on hairy root induction were explored.The results showed that MSU440 was the optimal A.rhizogenes strain and leaves was the preferred explants for hairy root induction.The highest hairy root induction rate reached 81.0%with the average root length of hairy roots was 3.23 cm.The leaves were infected by MSU440 which grown to a bacterial density of 0.3(OD_(600))for 30 min,then they were co-cultured for 48h and transferred the selection medium supplemented with 20 mg·L^(-1)Kanamycin.The exogenous genes were stably integrated in the genome of Eucalyptus hair roots which confirmed by PCR analyses and GUS staining,and the transformation efficiency reached 20.2%.In our study,an A.rhizogenes-mediated genetic transformation system of Eucalyptus was preliminarily established,which laid a foundation for gene function identification and further transgenic breeding of Eucalyptus.

关 键 词：桉树 发根农杆菌 毛状根 转基因 转化 

分 类 号：S184[农业科学—农业基础科学]