褐角苔遗传转化体系的建立

Establishment of a Genetic Transformation System for Folioceros fuciformis

关键词:  褐角苔  外植体  农杆菌介导法  遗传转化体系

Key words:  Folioceros fuciformis  explants  Agrobacterium-mediated method  genetic transformation system

摘要:

本研究旨在筛选适用于农杆菌(Agrobacterium)介导的角苔植物遗传转化体系，为今后研究早期陆生植物的进化提供理论基础。实验使用携带pCAMBIA1305.2质粒的农杆菌AGL1菌株对褐角苔(Folioceros fuciformis)外植体进行侵染，将潮霉素作为筛选标记，通过比较外植体预培养时间、菌液OD值、菌液添加量、乙酰丁香酮(AS)浓度和共培养时间来构建该物种的遗传转化体系。结果表明，褐角苔抗性植株筛选所用潮霉素的最适浓度为15 mg/L，预培养4 d的外植体侵染后状态最好。最佳侵染条件为共培养基中添加80 μL OD₆₀₀为0.8的农杆菌菌液，AS浓度为100 μmol/L，共培养时间为3 d。通过潮霉素筛选、PCR、实时荧光定量PCR(RT-qPCR)和β-葡萄糖苷酸酶(GUS)染色鉴定获得转基因植株褐角苔。研究结果可为后续角苔植物基因功能的研究提供技术支持。

Abstract:

The selection of Agrobacterium-mediated genetic transformation system for hornworts provides a theoretical basis for the future study of early terrestrial plant evolution.In this study,Agrobacterium AGL1 carrying the plasmid pCAMBIA1305.2 was used to infect the explants of Folioceros fuciformis.With hygromycin as a screening marker,the pre-culture time of explants,the OD value and addition amount of the bacterial suspension,the concentration of acetosyringone (AS),and the co-culture time were optimized for the establishment of the genetic transformation system.The results showed that the optimum concentration of hygromycin was 15 mg/L for the screening of resistant seedlings,and the state of the explants was the best after 4 d of pre-culture.The optimal infection conditions were addition of 80 μL Agrobacterium suspension with OD₆₀₀=0.8 in the co-culture medium,AS concentration of 100 μmol/L,and co-culture time of 3 d.The transgenic plants were identified by hygromycin screening,PCR,RT-qPCR and beta-glucuronidase (GUS) staining.The findings provide technical support for the subsequent research on the gene functions of hornworts.