玉米叶肉原生质体瞬时表达系统建立与优化

Establishment and Optimization of a Transient Expression System for Maize Mesophyll Protoplasts

以玉米B73黄化苗叶片为研究材料,采用酶解法分离并获得高活性的玉米叶肉原生质体细胞,优化PEG-Ca^(2+)介导的玉米叶肉原生质体细胞转化条件,探索电击介导玉米叶肉原生质体细胞瞬时转化方法。同时,以荧光蛋白为报告分子融合细胞器定位信号,建立玉米叶肉原生质体亚细胞定位系统,进一步验证该瞬时表达系统具备可用于基因功能研究的可能性。结果表明,通过酶解法分离获得的玉米叶肉原生质体细胞经过FDA染色统计发现,95%的细胞结构完整且活力较高。在PEG-Ca^(2+)介导瞬时转化方法中,当PEG浓度40%且质粒量增加至25μg时转化效率达到最高,为51.28%。在电击介导瞬时转化方法中,当电压为350 V且电击时间为5 ms的条件下电击转化效率最高,为32.66%。通过将核定位信号NLS与eGFP基因融合转入玉米叶肉原生质体细胞,eGFP成功定位于细胞核。通过将叶绿体定位基因ZmMSH1与DsRed2基因融合并转入,DsRed2蛋白成功定位于叶绿体中。在玉米叶肉原生质体细胞中过表达ZmDHDPS基因,通过qRT-PCR和Western Blot等技术确定了ZmDHDPS高表达活性,并显著提高原生质体细胞内游离赖氨酸含量。

The leaves of maize B73 etiolated seedling were isolated and obtained by enzymatic.The PEG-Ca2+mediated protoplast cell transformation was optimized,and the effects of PEG concentration and plasmid dosage on the transient conversion efficiency were analyzed.The method of electric shock transformation was explored,and then the influence of voltage and shock time on transformation efficiency was analyzed.Meanwhile,the subcellular localization system for maize mesophyll protoplasts was established using fluorescent protein to fusion localization signal.The possibility of this transient expression system was further verified by overexpressing the ZmDHDPS gene in maize mesophyll protoplast cells.The protoplast cells obtained by enzymatic were analyzed by FDA staining and the result show that about 95%cell was vitality.In the PEG-Ca^(2+)mediated transient transformation method,the con⁃version efficiency reached 51.28%when PEG concentration was 40%and plasmid increased to 25μg.In the elec⁃tric shock transformation method,the conversion efficiency is 32.66%when the voltage is 350 V and the shock time is 5 ms.By fusion the nuclear localization signal NLS with the eGFP gene into maize mesophyll protoplast cells,eGFP was successfully localized to the nucleus.The DsRed2 protein was successfully localized in the chloroplast by fusing the chloroplast localization gene ZmMSH1 with the DsRed2 gene.Overexpression ZmDHDPS samples were detected by the qRT-PCT and Western Blot technique,and cellular free lysine content in protoplasts was detected significantly increased.