橡胶树叶片原生质体分离条件的优化

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 ×107个/g FW和97.3%；其次为古铜期叶片，可达到8.6×107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 ×105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19.7×107个/g FW，活力约为97.6%。

关键词 橡胶树 ；叶片发育阶段 ；原生质体分离

中图分类号 S794.1 文献标识码 A Doi：10.12008/j.issn.1009-2196.2018.03.011

Abstract 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 and 97.3%， 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%.

Keywords Hevea brasiliensis Mull. Arg ； leaf development stage ； protoplast isolation

巴西橡膠树（Hevea brasiliensis Müll. Arg.）是重要的热带经济作物，所产的天然橡胶是四大战略物资之一。随着分子生物学和功能基因组学的迅速发展，转基因育种已成为多种作物种质改良的有效途径。中国于2016年完成较高质量的橡胶树全基因组测序工作，并通过基因组的分析对橡胶树胶乳产量及物种适应性提出了新的认识[1]，对今后橡胶树分子生物学研究及基因工程育种起了极大的推动作用。但目前橡胶树转基因技术尚不成熟，转化效率偏低，且由于生长周期较长，转入的目标基因无法在橡胶树体内得到快速的验证。此外，橡胶树转基因起始材料主要为愈伤组织和体细胞胚等多细胞组织或器官[2-3]，再生株系易产生嵌合体，导入的目标基因在后代培育中易发生分离而难于稳定遗传。利用高效的原生质体瞬时表达体系可以快速对目标基因进行表达水平和部位分析及功能的初步验证，且转化后经原生质体培养获得的再生植株属于单细胞起源，不存在嵌合体现象，可节省大量繁琐的后续筛选工作，从而缩短培育周期、加快育种进程。

推荐访问： 橡胶树 质体 叶片 分离 优化

---