巴西橡胶树小孢子单细胞体外扩增体系的优化  

Optimization of Microspore Single Cell Amplification System in vitro in Hevea brasiliensis

作　　者：王宇翔 杨星星 刁艳茹 刘红东 王英[1] 庄南生[1] 高和琼[1] Wang Yuxiang;Yang Xingxing;Diao Yanru;Liu Hongdong;Wang Ying;Zhuang Nansheng;Gao Heqiong(College of Tropical Crop,Hainan University,Haikou,570228;Rubber Research Institute,Chinese Academy of Topical Agricultural Sciences,Danzhou,571737)

机构地区：[1]海南大学热带作物学院,海口570228 [2]中国热带农业科学院橡胶研究所,儋州571737

出　　处：《分子植物育种》2021年第17期5869-5877,共9页Molecular Plant Breeding

基　　金：2019年海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC132);国家天然橡胶产业技术体系(CARS-33)共同资助。

摘　　要：高质量单细胞的获得且进行高效率的扩增是单细胞测序的前提,而植物细胞因为存在细胞壁使得单细胞扩增变得困难。本研究以巴西橡胶树‘热研7-33-97’幼嫩雄花为试验材料,使用5种不同的酶液在不同的时间和渗透压稳定剂下探索获得高质量且易扩增的巴西橡胶树小孢子单细胞,同时对单细胞扩增过程进行相应的探索优化。结果表明,使用8%纤维素酶+6%果胶酶+3%蜗牛酶混合酶,0.6 mol/L甘露醇作为渗透压稳定剂,37℃酶解45 min后可以得到品质优良的巴西橡胶树小孢子单细胞;单细胞扩增过程确定细胞裂解酶用量为0.5μL,细胞裂解时间为80 min,预扩增循环数为10个循环,可以得到准确且覆盖度高的单细胞扩增产物,经Dot-blot杂交验证其确实来自于巴西橡胶树基因组。本研究为后续巴西橡胶树单细胞全基因组测序,构建巴西橡胶树高分辨SNP遗传图谱提供基础性技术支持。Obtaining high-quality single cells and performing high-efficiency amplification is the prerequisite for single-cell sequencing,but plant cells have cell walls that make single-cell amplification difficult.In this study,young male flowers of the Hevea brasiliensis'Reyan 7-33-97'were used as test materials.Five different enzyme solutions were used under different time and osmotic pressure stabilizer to explore and obtain single cells of Hevea brasiliensis with high quality and easy amplification.At the same time,the single-cell expansion process was optimized accordingly.The results showed that:under the condition of 45 minutes and 37℃,8%cellulase+6%pectinase+3%snailase mixed enzyme,0.6 mol/L mannitol as an osmotic pressure stabilizer,high-quality Hevea brasiliensis single cells can be obtained;during the single-cell amplification process,the dosage of cell lysis enzyme was 0.5μL,the cell lysis time was 80 min,and the number of pre-amplification cycles was 10 cycles.Accurate and high-coverage single-cell amplification products can be obtained,verified by Dot-blot hybridization.It does come from the Hevea brasiliensis genome.This study will provi de basic technical support for the subsequent single-cell whole genome sequencing of Hevea brasiliensis and construction of a high-resolution SNP genetic map of Hevea brasiliensis.

关 键 词：巴西橡胶树(Hevea brasiliensis) 四分体 单细胞扩增 Dot-blot杂交 

分 类 号：S794.1[农业科学—林木遗传育种]