谷子茎尖体外遗传转化体系的建立与优化  

Establishment and Optimization of a Shoot Tip-based Genetic Transformation System for Foxtail Millet

作　　者：杨澜 刘雅 项阳 孙秀娟 颜景畏 张阿英[1] Lan Yang;Ya Liu;Yang Xiang;Xiujuan Sun;Jingwei Yan;Aying Zhang(College of Life Science,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学生命科学学院,南京210095

出　　处：《植物学报》2021年第1期71-79,共9页Chinese Bulletin of Botany

基　　金：国家自然科学基金(No.31871534)。

摘　　要：以谷子(Seteriaitalica)豫谷一号为实验材料,建立了一套简便、稳定的体外茎尖遗传转化体系。通过根癌农杆菌(Agrobacteriumtumefaciens)介导的茎尖转化法,对转化受体采取不同的处理方式,待拟转化株长到三叶期后进行PCR鉴定。探明了草丁膦(Basta)喷施处理用于谷子转基因幼苗筛选的最适浓度,以及2种不同检测方式(直接PCR和喷施Basta+PCR)鉴定转基因植株的效果。在上述基础上,对影响谷子遗传转化体系的多种因素进行优化。结果表明,菌液浓度(OD600)=1.4、侵染液中乙酰丁香酮浓度为800μmol·L–1、侵染压强为0.05 MPa、侵染40分钟有利于谷子茎尖的遗传转化。同时,采用上述优化系统获得谷子转SiCBL4基因植株,通过喷施草丁膦和实时荧光定量PCR对T2代转基因植株进行遗传稳定性分析,可节约检测时间。综上,该研究初步建立了稳定的谷子体外茎尖遗传转化体系,并开发了一种便捷的检测后代转基因植株的组合方法。In this study,a simple and stable genetic transformation system of foxtail millet(Seteria italica)was established and optimized,in which shoot tips were used as the explant.We transformed Yugu 1,an elite millet cultivar,by Agrobacterium-mediated transformation,and tested different treatments to boost transformation efficiency.We used a PCR-based assay to screen transformants in third-leaf stage seedlings.We determined an optimal lethal concentration of glufosinate(Basta)when sprayed to millet seedlings,and tested the different PCR-based genotyping methods with or without Basta spary.Using the newly established pipeline,we further optimized various crucial factors that affect genetic transformation efficiency.We found that an optimal concentration of bacterial culture was OD600=1.4,an optimal concentration of acetolsyringone was 800μmol·L–1.We also obtained high transformation efficiency with an infecting pressure at 0.05 MPa,and an infecting time of 40 min.We used the above-mentioned transformation method to transform a Seteria italica calcineurin B-like protein 4(Si CBL4)overexpression construct.Genetic stability analysis on T2 generation transformed plants was performed by the combination assay of Basta resistance and real-time quantitative fluorescence RT-PCR,which can save the time of genotyping.Altogether,this study establishes a shoot tip-based stable genetic transformation system for foxtail millets,and also develops a robust pipeline to detect transgenic offsprings.

关 键 词：谷子 遗传转化 草丁膦 体外 茎尖 

分 类 号：S515[农业科学—作物学]