摘要
以棉花幼嫩子叶为材料,分析影响棉花叶肉原生质体分离及目标基因转化的主要因素,以棉花叶肉原生质体为受体,建立了稳定、高效的目标基因瞬时表达与鉴定体系。技术体系包括,选择自然生长12 d的棉花幼嫩子叶为材料,混合1.5%纤维素酶、0.4%离析酶、0.5 mol L–1甘露醇、20 mmol L–1 KCl、20 mmol L–1 MES、0.1 mol L–1 CaCl2和1.0 g L–1 BSA,在28℃黑暗条件下振荡酶解8 h,可游离出浓度达1.0×106 mL–1以上的纯净棉花叶肉原生质体。利用该方法将棉花锌指蛋白基因GhZFP2整合到pJIT166-GFP质粒载体,构建了GhZFP2:GFP融合载体,采用40%PEG-4000介导转化,获得高转化率的棉花叶肉原生质体。对目标基因瞬时表达产物检测表明,GhZFP2蛋白清晰定位在细胞核上。
This paper aimed to set up a stable and efficient transient expression system based on cotton mesophyll protoplasts produced from cotyledon. We analyzed the key factors related to isolating effectively cotton mesophyll protoplasts and used it as a vehicle to express transfected genes for functional studies. The optimized techniques are as follows： （1） the healthy 12-day-old cotyledon grown naturally was selected to isolate cotton protoplasts; （2） the digestion system contained 1.5% cellulose, 0.4% macerozyme, 0.5 tool L ^-1 mannitol, 20 mmol L^-1 KC1, 20 mmol L^-1 MES, 0.1 mol L^-1 CaC12, and 1.0 g L^-1 BSA; and （3） the di- gestion was conducted in the dark under 28℃ for 8 h with gentle shaking. The protoplast yield was higher than 1.0 × 10^6 mL^-1. Using this system, we integrated a cotton zinc finger protein gene （GhZFP2） into pJIT166-GFP plasmid vector and constructed the GhZFP2：GFP fusion vector. The target gene was then transformed into purified cotton mesophyll protoplast mediated with 40% PEG-4000. As a result, cotton mesophyll protoplast with high transformation frequency was obtained. The transient expres- sion assay showed that the GhZFP2 protein was clearly located in the cell nucleus of cotton.
出处
《作物学报》
CAS
CSCD
北大核心
2014年第2期231-239,共9页
Acta Agronomica Sinica
基金
国家自然科学基金项目(31171590)资助
关键词
棉花
原生质体
PEG介导
转化
目标基因
瞬时表达
Cotton
Protoplast
PEG-mediated
Transformation
Target genes
Transient expression
