文献类型：期刊文献

中文题名：黑木相思毛状根遗传转化体系的建立

英文题名：Establishment of the genetic transformation system of hairy roots of Acacia melanoxylon

作者：卫昊钰[1,2,3] 高佳惠[1,2,3] 王学君[3] 曾炳山[3] 马旭俊[1,2] 范春节[1,2,3]

第一作者：卫昊钰

机构：[1]东北林业大学林木遗传育种国家重点实验室,黑龙江哈尔滨150040;[2]中国林业科学研究院林木遗传育种国家重点实验室,北京100081;[3]中国林业科学研究院热带林业研究所国家林业和草原局热带林木培育重点实验室,广东广州510520

年份：2025

卷号：45

期号：12

起止页码：176-183

中文期刊名：中南林业科技大学学报

外文期刊名：Journal of Central South University of Forestry & Technology

收录：;北大核心:【北大核心2023】；

基金：国家重点研发计划项目(2022YFD2200205-1)。

语种：中文

中文关键词：发根农杆菌;黑木相思;遗传转化;毛状根

外文关键词：Agrobacterium rhizogenes;Acacia melanoxylon;genetic transformation;hairy roots

分类号：S792.15

摘要：【目的】黑木相思是我国华南地区广泛种植的珍贵树种,杂交育种周期长且资源较少导致其新种质创建困难。建立黑木相思遗传转化体系对缩短育种周期和开展基因功能研究具有重要意义。【方法】分别以黑木相思优良无性系SR14和SR17增殖苗为材料,通过采用携带RUBY的过表达质粒,进行发根农杆菌菌株(ArA4、Ar1193、MSU440)、侵染浓度(OD_(600)=0.1、0.3、0.5、0.7、0.9、1.2)、侵染时间(15、30、45、60、90 min)以及乙酰丁香酮浓度(0、100、200μmol·L^(-1))等条件的筛选及优化,进一步使用分子检测外源基因的导入,确定最佳诱导率和转化率,最终建立发根农杆菌介导的黑木相思毛状根的遗传转化体系。【结果】黑木相思无性系SR14在采用发根农杆菌菌株ArA4,侵染浓度为OD_(600)=0.9,侵染时间为45 min,乙酰丁香酮浓度为100μmol·L^(-1)条件下,毛状根诱导率及转化率最高,分别达到50.00%和39.39%。而黑木相思SR17则在选择菌株MSU440,侵染浓度为OD_(600)=0.5,侵染时间为60 min条件下达到毛状根最高诱导率(42.42%)和最高转化率(38.38%)。【结论】经过筛选及优化,将获得的毛状根经过PCR技术验证RUBY外源基因的整合情况,观察到根部颜色变红,证明RUBY蛋白已在黑木相思毛状根中表达,初步建立了发根农杆菌介导的黑木相思优良无性系的毛状根高效遗传转化体系,为黑木相思进一步的转基因育种和基因功能解析奠定基础。
【Objective】Acacia melanoxylon is widely planted in south China,but it is difficult to create new germplasm due to the long hybrid breeding cycle and few resources.The establishment of the genetic transformation system is of great significance for shortening the breeding cycle and identifying gene function in A.melanoxylon.【Method】In this study,the superior clones SR14 and SR17 of Acacia melanoxylon were used as materials,and the overexpression plasmids carrying RUBY were used to perform Agrobacterium rhizogenes strains(ArA4,Ar1193,MSU440),infection concentration(OD_(600)=0.1,0.3,0.5,0.7,0.9,1.2),infection time(15,30,45,60,90 min)and acetosyringone addition(0,100,200μmol·L^(-1))and other conditions,and further molecular detection was used to determine the optimal induction rate and conversion rate,and finally the genetic transformation system of Agrobacterium rhizogenes-mediated hairy roots of Acacia nigra was established.【Result】A.rhizogenes strain ArA4 was used for A.melanoxylon clone SR14,with an OD_(600)=0.9,45 min infection with addition of 100μmol·L^(-1) AS,the hairy root induction rate and transformation efficiency reached 50.00%and 39.39%,respectively.Meanwhile,the highest induction rate and transformation efficiency of hairy roots were 42.42%and 38.38%with Agrobacterial strain MSU440,OD_(600)=0.5 and 60 min infection.【Conclusion】After screening and optimization,the obtained hairy roots were identified by PCR amplification and RUBY genes were detected.Furthermore,the transgenic root showed reddish,indicating that the RUBY protein had been expressed in the hairy roots of A.melanoxylon.Finally,an efficient genetic transformation system of hairy roots of A.rhizogenes-mediated excellent clones of A.melanoxylon was preliminarily established,which laid a foundation for further transgenic breeding and gene function analysis of A.melanoxylon.