摘要
闽楠[Phoebe bournei(Hemsl.)Yang]为我国重要的珍贵树种。为建立闽楠遗传转化体系,以闽楠幼苗为材料,利用pCAMBIA1300-GFP质粒转化的发根农杆菌介导植株转化。在比较注射和菌液浸泡两种侵染方式侵染效率的基础上,进一步利用正交[L9(3^(3))]试验从菌株种类、菌液侵染浓度和侵染植株苗龄三个方面优化了遗传转化体系。另外,对不同光强影响闽楠毛状根诱导及转化率情况进行了分析。结果表明,注射法的毛状根诱导率和遗传转化率分别为80.0%、66.7%;而菌液浸泡法的诱导率和遗传转化率分别为41.2%、35.3%。正交试验中,影响闽楠毛状根诱导率和遗传转化率的主要因素为菌株种类。最大诱导率和转化效率分别达到了87.5%、70.6%。该体系的最优组合为:K599菌株在菌液浓度OD_(600)=1.2条件下注射侵染三叶期的闽楠幼苗。此外,较弱光强(50μmol·m^(-2)·s^(-1))的培养条件比较强光照(100μmol·m^(-2)·s^(-1))更有利于闽楠幼苗的生长和遗传转化。本研究创建并优化了一套高效、稳定的闽楠遗传转化体系,成功获得了具有转基因毛状根的闽楠植株,为闽楠重要基因挖掘、新种质创建和遗传改良奠定了基础。
Phoebe bournei(Hemsl.)Yang is an important and precious tree species in China.This study aimed to establish a genetic transformation system for P.bournei,pCAMBIA1300-GFP plasmid was constructed and transformed into seedlings with Agrobacterium rhizogenes.To optimize the transformation process,injection and soaking in bacterial solution were compared.Subsequently,an orthogonal[L9(3^(3))]experiment was employed to determine the optimal combination A.rhizogenes strain type,bacterial solution concentration,and seedling age.In addition,the effects of light intensity on induction and transformational rate of P.bournei hairy roots were also examined.The results showed that hairy roots induction and transformation rate with injection method were 80.0%and 66.7%respectively,whereas only 41.2%and 35.3%with soaking method.The main factors affecting the hairy root induction rate and genetic transformation of P.bournei were strain species.Notably,strain type exhibited a substantial influence on both induction and transformation efficiency of hairy roots.The maximum induction and transformation rates were 87.5%and 70.6%respectively.The optimal strain was K599 with concentration of OD_(600)=1.2,and the seedlings at the three-leaf stage was the best The seedlings growth,hairy roots induction and transformation efficiency of P.bournei seedlings under lower light intensity(50μmol·m^(-2)·s^(-1))were more favorable for seedling growth and transformation than high light intensity(100μmol·m^(-2)·s^(-1)).This study established an efficient and steady transgenic system for P.bournei mediated by A.rhizogenes,thereby obtained transgenic hairy roots plants.That provided a useful platform for molecular genetic research of P.bournei and laying the foundation for important gene exploration,new germplasm development and genetic improvement of P.bournei.
作者
吴梦洁
洪家都
李芳燕
周生财
林二培
程龙军
WU Mengjie;HONG Jiadu;LI Fangyan;ZHOU Shengcai;LIN Erpei;CHENG Longjun(College of Forestry and Biotechnology,Zhejiang Agriculture and Forestry University/State Key Laboratory of Subtropical Silviculture,Hangzhou,Zhejiang 311300;Lishui Vocational&Technical College,Lishui,Zhejiang 323000)
出处
《核农学报》
CAS
CSCD
北大核心
2023年第8期1516-1522,共7页
Journal of Nuclear Agricultural Sciences
基金
浙江省重点研发计划项目(2021C02054)。
关键词
闽楠
遗传转化
发根农杆菌
Phoebe bournei
genetic transformation
Agrobacterium rhizogenes
