根癌农杆菌介导山葡萄VaCBF3基因转化欧美杨111的研究
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摘要
以欧美杨111为受体材料,利用农杆菌介导的叶盘法进行了山葡萄VaCBF3基因的遗传转化,分析了预培养时间、菌液浓度、侵染时间、共培养时间对转化效率的影响,并对获得抗性植株进行PCR检测。结果表明:欧美杨111遗传转化过程中适宜的预培养时间为3 d,适宜菌液浓度为OD600为0.3～0.4,适宜侵染时间为2～4 min,适宜共培养时间为3 d。经过对转化子的PCR鉴定,获得4株阳性转基因植株,初步说明外源山葡萄CBF3基因已导入至欧美杨111号中,转化体系已成功建立。
The Populus euramericana 'Bellotto' was used as the acceptor material, the genetic transformation of Vitis amurensis CBF3 gene was carried out by using Agrobacterium tumefaciens mediated blisk method. The effects of preculture time, bacterial concentration, infection time and co-culture time on transformation efficiency were studied, and the obtained resistant plants were detected by PCR. Results show that the suitable preculture time is 3 d, the OD600 of suitable bacterial concentration is 0.3-0.4, the suitable infection time is 2-4 min and the suitable co-cultivation time is 3 d for genetic transformation process of the P. euramericana 'Bellotto'. After PCR identification of transformants, 4 positive transgenic plants are obtained, which preliminarily indicated that V.amurensis VaCBF3 gene is introduced into the P. euramericana 'Bellotto'. The conversion system has been successfully established.
The Populus euramericana 'Bellotto' was used as the acceptor material, the genetic transformation of Vitis amurensis CBF3 gene was carried out by using Agrobacterium tumefaciens mediated blisk method. The effects of preculture time, bacterial concentration, infection time and co-culture time on transformation efficiency were studied, and the obtained resistant plants were detected by PCR. Results show that the suitable preculture time is 3 d, the OD600 of suitable bacterial concentration is 0.3-0.4, the suitable infection time is 2-4 min and the suitable co-cultivation time is 3 d for genetic transformation process of the P. euramericana 'Bellotto'. After PCR identification of transformants, 4 positive transgenic plants are obtained, which preliminarily indicated that V.amurensis VaCBF3 gene is introduced into the P. euramericana 'Bellotto'. The conversion system has been successfully established.
引文
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[19]贾会霞,李建波,孙佩,等.胡杨CBF基因家族的鉴定及表达特性分析[J].分子植物育种,2017,15(2):492-500.
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[22]冯连荣,王占斌,宋立志.2种启动子驱动下的VaCBF3基因植物表达载体构建[J].沈阳农业大学学报,2011,42(5):559-564.
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[26]甄成.毛果杨组培再生及遗传转化体系研究[D].哈尔滨:东北林业大学,2016.
[27]朱伟康.转Bt基因欧洲黑杨组织培养再生体系优化及其TA29-Barnase基因遗传转化[D].杨凌:西北农林科技大学,2016
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[2]刘巍,蔄胜军,纪纯阳,等.辽宁省杨树灾害发生的原因及对策[J].防护林科技,2014(6):70-73.
[3]丁莉萍,王宏芝,魏建华.杨树转基因研究进展及展望[J].林业科学研究,2016,29(1):124-132.
[4]Arisi A C M,Cornic G,Jouanin L,et al.Overexpression of iron superoxide dismutase in transformed poplar modifies the regulation of photosynthesis at low CO2 partial pressures or following exposure to the prooxidant herbicide methyl viologen[J].Plant Physiology,1998,117(2):565-574.
[5]李春霞.抗冻蛋白基因对山杨等植物遗传转化的研究[D].哈尔滨:东北林业大学,2003.
[6]Benedict C,Skinner J S,Meng R G,et al.The CBF1-dependent low temperature signalling pathway,regulon and increase in freeze tolerance are conserved in Populus spp.[J].Plant,Cell and Environment,2006,29(7):1259-1272.
[7]周洲.转脂肪酸去饱和酶基因PtFAD2和PtFAD3银腺杨84K的抗寒性研究[D].北京:中国林业科学研究院,2007.
[8]许东.杨树抗寒转录因子PlMBF1b的克隆及功能分析[D].泰安:山东农业大学,2016.
[9]Stockinger E J,Gilmour S J,Thomashow M F.Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proceedings of the National Academy of Sciences,1997,94(3):1035-1040.
[10]李健,王雅晴,刘洋,等.CBF转录因子在植物抗逆和生长发育中的重要功能[J].植物生理学报,2017,53(12):2045-2056.
[11]Gilmour S J,Sebolt A M,Salazar M P,et al.Overexpression of the Arabidopsis CBF3Transcriptional activator mimics multiple biochemical changes associated with cold acclimation[J].Plant Physiology,2000,124(4):1854-1865.
[12]马刘峰,陈芸,任羽,等.棉花CBF2基因克隆和超表达CBF2棉花增强抗冷性[J].植物生理学报,2018,54(2):255-264.
[13]高启明,王斌,赛买提·吐尔逊,等.农杆菌介导的CBF基因转化哈密大枣[J].北方园艺,2016(18):94-98.
[14]王沛文,朱文哲,刘阳,等.多毛番茄冷诱导转录因子CBF1转化番茄的研究[J].江苏农业科学,2015,43(4):30-35.
[15]谭克,赵福顺,吴慧杰,等.冷诱导基因转录因子CBF1转入黄瓜的研究[J].北方园艺,2015(9):79-82.
[16]孟平红,万发香,王永清,等.冷诱导转录因子CBF3转化茄子的初步研究[J].中国蔬菜,2013(10):36-43.
[17]徐春波,王勇,赵海霞,等.冷诱导转录因子AtCBF1转化紫花苜蓿的研究[J].草业学报,2012,21(4):168-174.
[18]丁咚,陈亚娟,崔进荣,等.毛果杨CBF/DREB1基因家族生物信息学分析[J].西南农业学报,2018,31(3):457-461.
[19]贾会霞,李建波,孙佩,等.胡杨CBF基因家族的鉴定及表达特性分析[J].分子植物育种,2017,15(2):492-500.
[20]姜洋.大青杨PuCBF遗传转化基础及功能验证[D].哈尔滨:东北林业大学,2017.
[21]Wang Z B,Feng L R,Wang J J,et al.Vitis amuerensis CBF3 gene isolation,sequence analysis and expression[J].Agricultural Sciences in China,2010,9(8):1127-1132.
[22]冯连荣,王占斌,宋立志.2种启动子驱动下的VaCBF3基因植物表达载体构建[J].沈阳农业大学学报,2011,42(5):559-564.
[23]冯连荣,张兴芬,尹杰,等.杨树农杆菌介导遗传转化中抗生素浓度的筛选[J].西南林业大学学报,2014,34(4):31-35.
[24]王关林,方宏筠.植物基因工程[M].2版.北京:科学出版社,2002
[25]冯连荣,宋立志,张妍,等.根癌农杆菌介导杨树遗传转化的影响因素[J].西北林学院学报,2015,30(3):120-126.
[26]甄成.毛果杨组培再生及遗传转化体系研究[D].哈尔滨:东北林业大学,2016.
[27]朱伟康.转Bt基因欧洲黑杨组织培养再生体系优化及其TA29-Barnase基因遗传转化[D].杨凌:西北农林科技大学,2016
[28]赵芳方.毛白杨再生体系的建立及Barnase基因转化的初步研究[D].郑州:郑州大学,2014.
[29]孙伟博,于娟,潘惠新,等.‘南林895杨’遗传转化体系的优化[J].林业科技开发,2013,27(6):85-88.