橡胶树叶片原生质体分离条件的优化  

Optimized Conditions for Protoplast Isolation of Leaves of Rubber Tree

作　　者：戴雪梅[1] 杨先锋[1] 辛士超[1] 成镜 彭素娜 华玉伟[1] DAI Xuemei;YANG Xianfeng;XIN Shichao;CHENG Jing;PENG Suna;HUA Yuwei(Rubber Research Institute, CATAS/Ministry of Agriculture Key Laboratory of Biology of Rubber Tree, Danzhou, Hainan 571737)

机构地区：[1]中国热带农业科学院橡胶研究所/农业部橡胶树生物学重点开放实验室,海南儋州571737

出　　处：《热带农业科学》2018年第3期46-52,共7页Chinese Journal of Tropical Agriculture

基　　金：海南省自然科学基金面上项目-PEG介导GFP基因转化橡胶树原生质体的研究(No.20163136);国家自然科学基金青年科学基金项目-橡胶树原生质体遗传转化体系的建立及Hb CS4基因的导入(No.31600528)

摘　　要：分别以橡胶树GT1种子实生苗古铜期、变色期、淡绿期和稳定期4个不同发育阶段的叶片为材料进行原生质体的酶解分离研究,同时分析比较不同酶组合、酶解时间和甘露醇浓度等主要因素对橡胶树叶片原生质体产量和活力的影响,建立高效稳定的橡胶树叶片原生质体分离体系,为进行橡胶树外源基因瞬时表达及CRISPR基因编辑等研究快速提供高产优质的原生质体。结果表明:在同等条件下,变色期叶片的原生质体产量和活力最高,分别为14.8×10~7个/g FW和97.3%;其次为古铜期叶片,可达到8.6×10~7个/g FW和95.2%;淡绿期和稳定期叶片原生质体的产量非常低,活力也相对较低,分别仅有3.4×10~5个/g FW和89.5%、7.2×104个/g FW和80.6%。单因素实验结果表明,最适合变色期叶片原生质体分离的条件为:酶组合为2%纤维素酶+0.6%离析酶、酶解时间为6 h、甘露醇浓度为0.6 mol/L,此时橡胶树叶肉原生质体的产量可高达19.7×10~7个/g FW,活力约为97.6%。The leaves of rubber tree（Hevea brasiliensis Mull. Arg） at the bronze phase, color-changing phase, light green phase and stable phase were used as materials for protoplast isolation. The effects of some major factors such as enzymatic combination, digestion time and concentration of mannitol on the production and viability of protoplasts were analyzed in order to establish an efficient system for protoplast isolation and provide protoplasts for transient exogenous gene expression of and CRISPR genome editing of rubber tree. The results showed that the leaves at the color-changing phase under the same isolation conditions had the highest yield and viability of mesophyll protoplasts（14.8 ×107 protoplasts/g FW and97.3%, respectively）, followed by the leaves at the bronze phase（8.6×107 protoplasts/g FW and a viability of 95.2%）, while the leaves at the light green phase and stable phase had very low protoplast yield and relatively low viability（3.4 ×105 protoplasts/g FW and 89.5%; 7.2 ×104 protoplasts/g FW and 80.6%,respectively）. The single factor experimental results showed that the optimal conditions for protoplast isolation were 2% cellulase R-10＋0.6% macerozyme R-10 for the enzymatic combination, 6 h for the digestion time, and 0.6 mol/L for the concentration of mannitol. Under these optimal conditions the yield of protoplasts of rubber tree leaves could be up to 19.7×107 protoplasts/g FW with viability of 97.6%.

关 键 词：橡胶树 叶片发育阶段 原生质体分离 

分 类 号：S794.1[农业科学—林木遗传育种]