东南景天转运Cd蛋白HMA3基因载体的构建及栾树遗传转化体系的建立  

Construction of the transformation vector of Sedum alfredii HMA3 and establishment of the genetic transformation system of Koelreuteria paniculata

作　　者：周韬[1] 白磊 何其浩 王平[1] ZHOU Tao;BAI Lei;HE Qihao;WANG Ping(College of Life Sciences&Techology,Central South University of Forestry&Technology,Changsha 410004,Hunan,China)

机构地区：[1]中南林业科技大学生命科学与技术学院,湖南长沙410004

出　　处：《中南林业科技大学学报》2022年第10期148-157,共10页Journal of Central South University of Forestry & Technology

基　　金：国家重点研发计划“水-旱轮作系统镉、砷中轻度污染农田‘边萃取边生产’新技术模式与效益评估”(2016YFD0800805-4);湖南省重点研发计划项目“微生物-植物协同修复土壤重金属污染及配套支撑技术”(2016SK2030);湖南省科技重点研发计划项目“农村生活污水生态治理技术创新与示范”(2020SK2039)。

摘　　要：【目的】构建栾树遗传转化体系并通过转入东南景天HMA3基因对复羽叶栾树的基因进行定向改良,以提升其对镉(Cd)的富集能力与转运能力,为之后复羽叶栾树逆境抗性分子机制的研究提供基础。【方法】制备复羽叶栾树单细胞悬浮液,检测其基因组的大小;构建表达载体并转入农杆菌中,调节激素配比诱导复羽叶栾树形成愈伤组织,并通过农杆菌介导进行遗传转化;将筛选得到的阳性愈伤组织制备成原生质体,并用激光共聚焦观察;最后通过Cd胁迫试验检测栾树对Cd的富集能力与转运能力。【结果】通过与内参番茄的比较发现栾树的基因组大小约为335 Mb;在诱导复羽叶栾树愈伤组织时,加入聚乙烯吡咯烷酮(PVP)可以有效地防止愈伤组织发生褐化,加入10%椰汁后,复羽叶栾树愈伤组织的诱导成功率及生长速度均达到最佳;激光共聚焦观察复羽叶栾树阳性愈伤组织发现HMA3定位在细胞膜上;Cd胁迫试验显示,转入HMA3基因的复羽叶栾树,其Cd的富集系数提升了40.3%,转运系数提升了29.3%。【结论】复羽叶栾树较小的基因组决定了其在遗传过程中,有更大的可能保留外源基因,有效避免了外源基因的丢失;PVP及椰汁的加入大幅提升了复羽叶栾树愈伤组织诱导的成功率,并有效较少了愈伤组织的褐化;转入的HMA3成功在细胞膜上表达,对转基因复羽叶栾树的富集能力和转运能力都有显著提升。【Objective】The genetic transformation system of Koelreuteria paniculata was constructed,and the targeted genetic improvement was carried out by the transformation of Sedum alfredii HMA3 to improve the Cd enrichment and transport capacity of Koelreuteria paniculata.【Method】The single cell suspension of Koelreuteria paniculata was prepared and its genome size was detected.The expression vector was constructed and transferred into Agrobacterium tumefaciens to induce callus formation of Koelreuteria paniculata by regulating the ratio of hormones,and the genetic transformation was carried out through Agrobacteriummediated transformation.The positive callus was prepared into protoplasts and observed by the laser confocal microscopy.Finally,the Cd enrichment and transport capacity of Koelreuteria paniculata were detected by the Cd stress test.【Result】Compared with the reference tomato,we found that the genome size of Koelreuteria paniculata was about 335 Mb.In the callus induction of Koelreuteria paniculata,PVP can effectively prevent the browning of callus,and 10%coconut water can contribute to the best success rate of induction and the highest growth rate of callus.Laser confocal observation of the positive callus of Koelreuteria paniculata showed that HMA3 was located on the cell membrane.The Cd stress test showed that the enrichment coefficient and transport coefficient of Koelreuteria paniculata transformed with the HMA3 gene increased by 40.3%and 29.3%,respectively.【Conclusion】The relatively small genome of Koelreuteria paniculata determines that it is more likely to retain and avoid the loss of exogenous genes.PVP and coconut water significantly increase the success rate of callus induction,and effectively reduced the browning of callus.The transferred HMA3 gene was successfully expressed on the cell membrane,which can significantly improve the Cd enrichment and transport capacity of the transgenic Koelreuteria paniculata.

关 键 词：复羽叶栾树 遗传转化体系 HMA3基因 

分 类 号：S792.99[农业科学—林木遗传育种]