农杆菌介导的棕色棉胚珠离体培养遗传转化体系的建立  

Establishment of Agrobacterium-mediated Genetic Transformation System of In Vitro Culture Brown Cotton Ovules

作　　者：丁一 耿晓丽 何守朴[2] 潘兆娥[2] 贾银华[2] 王立如 吴立强[1] 杜雄明[2] Ding Yi;Geng Xiaoli;He Shoupu;Pan Zhaoe;Jia Yinhua;Wang Liru;Wu Liqiang;Du Xiongming(College of Agronomy,Agriculture University of Hebei,Baoding,071000;State Key Laboratory of Cotton Biology,Institute of Cotton Research of the Chinese Academy of Agricultural Sciences,Anyang,455000)

机构地区：[1]河北农业大学农学院,保定071000 [2]中国农业科学院棉花研究所,棉花生物学国家重点实验室,安阳455000

出　　处：《分子植物育种》2021年第1期218-223,共6页Molecular Plant Breeding

基　　金：转基因生物新品种培育科技重大专项子课题(2016ZX08005-005)资助。

摘　　要：本研究以棕色棉品种‘棕1-61’为受体材料,以茁-葡萄糖苷酸酶基因(GUS)为报告基因,探索农杆菌介导离体培养胚珠的遗传转化条件。结果表明最适合的遗传转化条件是:1 DPA胚珠预培养4 d后,用针头进行扎孔处理形成伤口,在OD600为0.6~0.8的农杆菌菌液中侵染3 min,22℃黑暗条件下共培养20 h后转移至含500 mg/L卡那霉素的BT培养基上进行暗培养。GUS染色结果发现,暗培养20 d的胚珠和30 d的纤维上均有染色,证明GUS报告基因转化成功。进一步将目的基因Gh UFGT构建RNAi载体并进行转基因,荧光定量PCR检测发现转基因阳性胚珠中Gh UFGT基因的表达量与对照相比显著下降。该实验为研究纤维发育和色泽相关基因的功能提供了一种快速高效的遗传转化方法。In this study,we used the beta-glucuronidase(GUS)gene as the reporter gene and the in vitro cultured ovules of cotton cultivar’Brown 1-61’as the explants to investigate the optimum conditions for Agrobacteriummediated transformation.We found that the optimum transformation system was:4 day pre-cultured ovules stabbed by syringe needle were infection with Agrobacterium(OD600=0.6~0.8)for 3 min,and after cocultivation at 22℃in darkness for 20 h the ovules were then transferred to the BT medium containing 500 mg/L kanamycin for growth in darkness.The GUS stainning results found that 20 d cultured ovules and the cotton fibers on the 30 d cultured ovules could been stained and proved that the transformation was success.We further constructed the RNAi vector of Gh UFGT and introduced into pre-cultured ovules via Agrobacterium-mediated transformation.And the q PCR result demonstrated that the Gh UFGT showed significant decreased expression levels in positive ovules when compared to the control.Our study provides a fast and efficient transformation method for studying the function of genes which involved in fiber development and pigment deposition.

关 键 词：棕色棉 胚珠 农杆菌介导法 遗传转化 离体培养 

分 类 号：S562[农业科学—作物学]