巴西橡胶树单个花粉细胞显微分离  

Microscopic Isolation of Single Pollen Cells from Hevea brasiliensis

作　　者：毕时时 谭江华 王春 王英[1] 庄南生[1] 高和琼[1] Bi Shishi;Tan Jianghua;Wang Chun;Wang Ying(Zhuang Nansheng 1 Gao Heqiong 11 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》2023年第21期7178-7185,共8页Molecular Plant Breeding

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(2019RC132);国家天然橡胶产业技术体系项目(CARS-33)共同资助。

摘　　要：巴西橡胶树是一种重要的热带产胶作物。因巴西橡胶树基因高度杂合,容易受到遗传或环境影响,为了不受群体效应的干扰,以巴西橡胶树‘热研7-33-97’的幼嫩雄花为试验材料,利用显微分离技术获取单个花粉细胞,使用5%纤维素酶+4%果胶酶混合酶和8%纤维素酶+6%果胶酶+3%蜗牛酶混合酶在不同条件(温度,时间)下,探索花粉细胞壁的最佳酶解体系。结果表明:8%纤维素酶、6%果胶酶和3%蜗牛酶混合酶在42℃条件下水浴15 h酶解效果最佳;同时利用随机引物(S2147和S1402)对酶解后的单个细胞进行酶解去壁效果的RAPD-PCR扩增鉴定,结果揭示该酶解体系能对花粉细胞进行去壁,使核酸物质得以释放并扩增。本试验以单个花粉细胞为研究对象,进行花粉细胞的显微分离,酶解去壁及RAPD-PCR扩增鉴定,为后续进一步开展‘热研7-33-97’单细胞全基因组测序、构建遗传图谱及橡胶树分子辅助育种等提供了科学依据。Hevea brasiliensis is an important tropical rubber crop.In this study,the young male flowers of Hevea brasiliensis'Reyan7-33-97'were used as experimental materials to obtain single pollen cells by microscopic separation technology.The optimal enzymatic hydrolysis system of pollen cell wall was explored by using 5%cellulase+4%pectinase mixed enzyme and 8%cellulase+6%pectinase+3%snail enzyme mixed enzyme under different conditions(temperature and time).The results showed that 8%cellulase,6%pectinase and 3%snailase had the best enzymatic hydrolysis at 42℃for 15 h.At the same time,random primers(S2147 and S1402)were used to carry out RAPD-PCR amplification identification of the enzymolysis effect of enzymolysis on the single cells after enzymolysis.The results showed that the enzymatic hydrolysis system could remove the walls of pollen cells successfully,and the nucleic acid substances could be released and amplified.In this experiment,single pollen cells were taken as the research object,and pollen cells were microseparated,dewalled by enzymatic hydrolysis and identified by RAPD-PCR amplification.which providing scientific basis for the further development of'Reyan7-33-97'single-cell whole genome sequencing,the construction of genetic map and molecular assisted breeding of Hevea brasiliensis.

关 键 词：巴西橡胶树 花粉 显微分离 酶解 RAPD-PCR 

分 类 号：S794.1[农业科学—林木遗传育种]