摘要
巴西橡胶树是一种重要的热带作物。以巴西橡胶树‘热研7-33-97’处于减数分裂四分体时期的幼嫩雄花为试验材料来获取单细胞,利用5%纤维素酶、5%果胶酶、5%β-葡聚糖酶、5%纤维素酶与4%果胶酶混合酶4种酶液,探索酶解四分体胼胝质的最佳方案。结果表明:在37℃条件下酶解1 h,5%纤维素酶与4%果胶酶混合酶的酶解效果最佳,可得到品质良好的小孢子单细胞。通过对拉制针尖细长度、磨针斜口直径以及显微操作分离体系的不断探索与试验,成功建立一套适合巴西橡胶树‘热研7-33-97’小孢子单细胞的显微分离技术体系。小孢子单细胞是减数分裂的直接产物,对小孢子单细胞进行分离并分析可精确解析橡胶树减数分裂过程中基因的重组与交换,确定橡胶树基因组的变异情况。该研究结果将为橡胶树单细胞全基因组测序提供理论与技术支持。
Hevea brasiliensis is an important tropical crop.In this study,the young male flowers of‘Reyan 7-33-97’in the meiotic tetrad period were used as the experimental materials to obtain single cells.Four enzymatic solutions,5%cellulose,5%pectinase,5%β-glucanase,5%cellulase and 4%pectinase mixed enzyme solution,were used in the experiment to explore the best scheme for the degradation of tetrad callose.As a result,under the condition of 1 h and 37℃,5%cellulase and 4%pectinase mixed enzyme solution had the best enzymatic hydrolysis effect,and the single microspore with good quality could be obtained.Through the continuous exploration and experiment on the fine length of the needle tip,the diameter of the sharpening of the needle and the micro-operation separation system,a set of microdissection technology system suitable for the single microspore of‘Reyan 7-33-97’was successfully established.Single-microspores were the direct products of meiosis.The separation and analysis of single cells could accurately resolve gene recombination and exchange during the meiosis of rubber trees,and determine the genomic variation.The study would provide theoretical and technical support for single-cell whole genome sequencing of H.brasiliensis.
作者
杨星星
王英
庄南生
高和琼
王宇翔
YANG Xingxing;WANG Ying;ZHUANG Nansheng;GAO Heqiong;WANG Yuxiang(College of Forestry,Hainan University,Haikou,Hainan 570228,China;College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China;Rubber Research Institute,Chinese Academy of Topical Agricultural Sciences,Danzhou,Hainan 571737,China)
出处
《热带作物学报》
CSCD
北大核心
2020年第8期1522-1528,共7页
Chinese Journal of Tropical Crops
基金
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(No.2019RC132)
海南省研究生创新科研课题(No.Hys2019-19)
国家天然橡胶产业技术体系(No.CARS-33)。
关键词
巴西橡胶树
显微分离
单细胞
四分体
Hevea brasiliensis
microdissection
single cell
tetrad
