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作物学报 ›› 2014, Vol. 40 ›› Issue (02): 231-239.doi: 10.3724/SP.J.1006.2014.00231

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花叶肉原生质体分离及目标基因瞬时表达体系的建立

李妮娜,丁林云,张志远,郭旺珍*   

  1. 南京农业大学作物遗传与种质创新国家重点实验室 / 教育部杂交棉创制工程研究中心, 江苏南京210095
  • 收稿日期:2013-06-17 修回日期:2013-09-24 出版日期:2014-02-12 网络出版日期:2013-12-15
  • 通讯作者: 郭旺珍, E-mail: moelab@njau.edu.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31171590)资助。

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

LI Ni-Na,DING Lin-Yun,ZHANG Zhi-Yuan,GUO Wang-Zhen*   

  1. State Key Laboratory of Crop Genetics & Germplasm Enhancement, Hybrid Cotton Research & Development Engineering Research Center, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China
  • Received:2013-06-17 Revised:2013-09-24 Published:2014-02-12 Published online:2013-12-15
  • Contact: 郭旺珍, E-mail: moelab@njau.edu.cn

摘要:

以棉花幼嫩子叶为外植体材料,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的棉花幼嫩子叶为外植体材料,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等酶液,在28℃黑暗条件下振荡酶解8 h,可游离出浓度达1.0×106 m L–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。

关键词: 棉花, 原生质体, PEG介导, 转化, 目标基因, 瞬时表达

Abstract:

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.

Key words: Cotton, Protoplast, PEG-mediated, Transformation, Target genes, Transient expression

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