摘要
[目的]优化与构建云南松原生质体制备与纯化技术体系,为云南松原生质体培养、融合及瞬时表达体系的建立奠定基础.[方法]以云南松幼苗为试验材料,探索纤维素酶质量分数、离析酶质量分数、甘露醇浓度、真空处理时间、酶解条件和离心条件对云南松针叶原生质体制备的影响.[结果]云南松幼嫩针叶酶解前真空处理是必要的,且先在1.00％纤维素酶+0.80％离析酶+0.3 mol/L甘露醇的酶解液中抽真空10 min,然后在26 ℃、130 r/min条件下酶解4h,再以180 r/min离心5 min,原生质体产量达5.98×107个/g,活力为72.22％.此外,利用优化的原生质体制备程序,从云南松幼苗的根和茎中制备原生质体,其产量分别为4.35×107和5.27×107个/g.[结论]本研究以云南松幼苗为试验材料,建立了原生质体制备及优化技术体系,研究结果为云南松体细胞杂交、瞬时表达体系等研究的开展提供了技术支撑.
Abstract
[Purpose]To establish an optimized preparation and purification system of Pinus yun-nanensis protoplasts,laying a foundation for the establishment of protoplasts culture,fusion and tran-sient expression system of P.yunnanensis.[Methods]Using P.yunnanensis seedlings as the exper-imental material,the effects of cellulose enzyme mass fraction,macerozyme mass fraction,mannitol concentration,vacuum treatment time,enzymatic hydrolysis conditions,and centrifugation conditions on the preparation of protoplast from P.yunnanensis needles were explored.[Results]It was ne-cessary to vacuum the young P.yunnanensis needles before enzymolysis,and after being vacuumized in the enzymolysis solution of 1.00％cellulase enzyme+0.80％segregation enzyme+0.3 mol/L man-nitol for 10 minutes,the enzymolysis was carried out at 26 ℃,130 r/min for four hours,and then centrifugation at 180 r/min for five minutes,the yield of protoplasts reached 5.98× 107 per/g,and the vitality was 72.22％.In addition,the protoplasts were prepared from the roots and stems of P.yunnan-ensis seedlings by using the optimized protoplast preparation program,and the yields were 4.35× 107 and 5.27×107 per/g,respectively.[Conclusion]In this study,the preparation and purification sys-tem of the protoplasts of P.yunnanensis is established,which will provide technical support for the studies of somatic hybridization and transient expression system of P.yunnanensis.
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