棉花叶肉原生质体分离及目标基因瞬时表达体系的建立

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。

Isolation of Mesophyll Protoplast and Establishment of Gene Transient Expression System in Cotton

In this paper, taking healthy cotyledon leaves as explants material, combining with the screening of key factors related to isolation and transformation of cotton mesophyll protoplasts, a stable and efficient transient expression and identification system for the target genes was established via transfoming the cotton mesophyll protoplast cells. The major technical system includes: the enzymatic method is used to isolate mesophyll protoplasts. In detail, healthy 12-day-old young cotton cotyledons in natural growth condition were selected as the explant, and were digested with enzyme solution including 1.5% cellulose, 0.4% macerozyme, 0.5 mol L^–1 mannitol, 20 mmol L^–1 KCl, 20 mmol L^–1 MES, 0.1 mol L^–1 CaCl₂, and 1.0 g L^–1 BSA for eight hours with gentle shaking at 28℃ and under dark conditions. The concentration of the pure cotton mesophyll protoplasts was more than 1.0×10⁶ mL^–1. Using the system, we integrated a cotton zinc finger protein gene GhZFP2 into pJIT166-GFP plasmid vector, constructed the GhZFP2:GFP fusion vectors, and transformed it into purified cotton mesophyll protoplast mediated with 40% PEG4000. As a result, cotton mesophyll protoplast with high transformation frequency was obtained. Transient expression detection for GhZFP2 protein showed that it was clearly located in the cell nucleus.