纳米磁性颗粒介导的辣椒遗传转化体系建立  

Establishment of Magnetic Nanoparticles-Mediated Genetic Transformation System in Capsicum annuum L.

作　　者：孙国胜[1] 杨宇 许可翠 张昌伟[2] 戴忠良[1] 孙春青[1] 马志虎[1] SUN Guosheng;YANG Yu;XU Kecui;ZHANG Changwei;DAI Zhongliang;SUN Chunqing;MA Zhihu(Zhenjiang Institute of Agricultural Sciences in Hilly Area of Jiangsu Province,Jurong,Jiangsu 212400;College of Horticulture,Nanjing Agricultural University,Nanjing,Jiangsu 210095)

机构地区：[1]江苏丘陵地区镇江农业科学研究所,江苏句容212400 [2]南京农业大学园艺学院,江苏南京210095

出　　处：《核农学报》2021年第6期1307-1312,共6页Journal of Nuclear Agricultural Sciences

基　　金：镇江市重点研发计划—现代农业(NY2017003)。

摘　　要：为建立一套可操作性强、广谱性强、高效的基因编辑辣椒遗传转化体系,以辣椒B2为试验材料,磁性纳米颗粒(magnetic nanoparticles, MNPs)和载体质粒pCas9-TPC按质量比1∶1复合30 min后,加入混有辣椒花粉的培养基中,于MagnetoFACTOR plate24磁板下室温放置30 min,在磁场作用下导入花粉。将携带CRISPR/Cas9基因载体的花粉进行人工授粉,果实成熟后收获种子。通过载体质粒DNA上携带的抗除草剂(草铵膦)抗性标记,检测转化情况。提取T0代植株的DNA和RNA及T2代DNA,以pCas9-TPC为模板设计引物进行PCR扩增,检测T0和T2代植株中是否存在pCas9-TPC载体,对T0代植株进行Southern杂交检测基因编辑载体与辣椒基因组的整合情况。结果显示,平均转化效率约为63.70%,PCR检测显示T0代植株DNA和RNA及T2代DNA均扩增出CRISPR/Cas9载体,随机选取8株阳性植株进行Southern杂交,随机出现1~3条条带。本研究表明纳米磁性颗粒介导的花粉转化法可以在辣椒上建立高效的基因编辑转化体系,为辣椒的遗传育种提供更好的工具。In order to provide a controlled, broad-spectrum and high efficiency method of genetic transformation for gene editing of pepper. Using B2 as the experimental material, magnetic nanoparticles(magnic nanoparticles, MNPs) and vector plasmid pCas9-TPC were added to the pollen medium, the pollen was introduced into the magnetic field at room temperature for 30 min under the action of the MagnetoFACTOR plate 24. The pollen carrying the CRISPR/Cas9 vector was artificially pollinated and the seeds were harvested after the fruit matured. The transformation was detected by the resistance marker of herbicide resistant(ammonium phosphine) carried on the vector plasmid DNA;the DNA and RNA of T0 generation and DNA of T2 generation plants were extracted, and the primer of CRISPR/Cas9 was designed for PCR amplification. The presence or absence of CRISPR/Cas9 vector in T0 and T2 generation plants was detected. The integration of T0 generation plants with southern hybridization detection gene editing vector and cayenne genome was observed. The results showed that the average conversion efficiency was about 63.70%, T0 generation plants DNA and RNA and DNA of T2 generation, CRISPR/Cas9 vectors were amplified by PCR. 8 positive plants were randomly selected for southern hybridization, and 1-3 bands were randomly present. The results show that the nano-magnetic granule-mediated pollen transformation method can establish a high-efficiency gene editing and transformation system on pepper, and provide a better tool for the genetic breeding of pepper.

关 键 词：辣椒 磁性纳米颗粒 花粉 CRISPR/Cas9 

分 类 号：S641.3[农业科学—蔬菜学]