Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz)

文献类型： 外文期刊

第一作者： Wu, Jun-Zheng

作者： Wu, Jun-Zheng;Liu, Qin;Geng, Xiao-Shan;Luo, Li-Juan;Liu, Jin-Ping;Li, Kai-Mian

作者机构：

关键词： Cassava;Manihot esculenta;Protoplast isolation;Transient expression system;Subcellular localization

期刊名称：BMC BIOTECHNOLOGY （ 影响因子：2.563； 五年影响因子：3.292 ）

ISSN： 1472-6750

年卷期： 2017 年 17 卷

页码：

收录情况： SCI

摘要： Background: Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. Methods: Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. Results: In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 degrees C, resulting in the high yield (4.4 x 10(7) protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. Conclusion: We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

分类号：

  • 相关文献

[1]Isolation and Characterization of Ftsz Genes in Cassava. Geng, Meng-Ting,Yao, Yuan,Li, Rui-Mei,Fu, Shao-Ping,Duan, Rui-Jun,Liu, Jiao,Guo, Jian-Chun,Geng, Meng-Ting,Min, Yi,Chen, Xia,Fan, Jie,Yuan, Shuai,Wang, Lei,Zhang, Fan,Shang, Lu,Wang, Yun-Lin,Hu, Xin-Wen,Sun, Chong. 2017

[2]Two new terpenoids from the stems of Manihot esculenta. Zeng, Yan-Bo,Li, Shan-Shan,Mei, Wen-Li,Dong, Wen-Hua,Dai, Hao-Fu,Li, Kai-Mian. 2015

[3]A new diterpene from the stems of Manihot esculenta. Li, Shan-Shan,Hu, Li-Fei,Zhao, You-Xing,Zuo, Wen-Jian,Zeng, Yan-Bo,Li, Xiao-Na,Mei, Wen-Li,Dai, Hao-Fu,Zhao, You-Xing,Zuo, Wen-Jian,Zeng, Yan-Bo,Li, Xiao-Na,Mei, Wen-Li,Dai, Hao-Fu,Li, Shan-Shan. 2011

[4]Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava. Han, Bingying,Fu, Lili,Zhang, Dan,He, Xiuquan,Chen, Qiang,Peng, Ming,Zhang, Jiaming. 2016

[5]Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development. Guo, D.,Li, H. L.,Tang, X.,Peng, S. Q.,Tang, X.. 2014

[6]Expression of sucrose metabolism and transport genes in cassava petiole abscission zones in response to water stress. Liao, W. B.,Li, Y. Y.,Lu, C.,Peng, M.. 2017

[7]Antioxidant Phenolic Compounds of Cassava (Manihot esculenta) from Hainan. Yi, Bo,Wei, Xiaoyi,Hu, Lifei,Mei, Wenli,Zhou, Kaibing,Wang, Hui,Luo, Ying,Dai, Haofu,Yi, Bo,Yi, Bo. 2011

[8]Effects of tillage options on soil physical properties and cassava-dry-matter partitioning. Figueiredo, Priscila Gonzales,Bicudo, Silvio Jose,Tanamati, Fabio Yomei,Figueiredo, Priscila Gonzales,Bicudo, Silvio Jose,Tanamati, Fabio Yomei,Chen, Songbi,Fernandes, Adalton Mazetti,Djabou-Fondjo, Astride Stephanie Mouafi.

[9]Allelopathic potential and volatile compounds of Manihot esculenta Crantz against weeds. Li, J.,He, S. Y.,Qin, X. D..

[10]Cassava postharvest physiological deterioration: a complex phenomenon involving calcium signaling, reactive oxygen species and programmed cell death. Djabou, Astride S. M.,Chen, Songbi,Djabou, Astride S. M.,Niemenak, Nicolas,Carvalho, Luiz J. C. B.,Li, Qing X..

[11]Potential geographic distribution of two invasive cassava green mites. Parsa, Soroush,Herrera Campo, Beatriz Vanessa,Andres Vasquez-Ordonez, Aymer,Hazzi, Nicolas A.,Chen, Qing,Lu, Fuping,Yaninek, John Stephen.

[12]Physiological Investigation and Transcriptome Analysis of Polyethylene Glycol (PEG)-Induced Dehydration Stress in Cassava. Fu, Lili,Ding, Zehong,Han, Bingying,Hu, Wei,Li, Yajun,Zhang, Jiaming. 2016

[13]Transgenic rice expressing a cassava (Manihot esculenta Crantz) plasma membrane gene MePMP3-2 exhibits enhanced tolerance to salt and drought stresses. Yu, Y.,Cui, Y. C.,Ren, C.,Rocha, P. S. C. F.,Wang, M. L.,Xia, X. J.,Yu, Y.,Ren, C.,Peng, M.,Xu, G. Y.. 2016

[14]Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava. Geng, Meng-Ting,Wang, Yun-Lin,Hu, Xin-Wen,Yao, Yuan,Li, Rui-Mei,Fu, Shao-Ping,Duan, Rui-Jun,Liu, Jiao,Guo, Jian-Chun,Wu, Xiao-Hui,Sun, Chong. 2017

[15]Somatic Embryogenesis and Organogenesis of Biofuel Plant Cassava (Manihot esculenta Crantz) Chinese Cultivars. Li, Ruimei,Duan, Ruijun,Ji, Yimeng,Xi, Dujuan,Liu, Jiao,Guo, Jianchun,Fu, Shaoping,Li, Ruimei,Duan, Ruijun,Ji, Yimeng,Xi, Dujuan,Liu, Jiao,Guo, Jianchun,Fu, Shaoping,Ji, Yimeng,Xi, Dujuan,Liu, Jiao,Zhang, Peng. 2012

[16]Chilling acclimation provides immunity to stress by altering regulatory networks and inducing genes with protective functions in Cassava. Zeng, Changying,Chen, Xin,Zhou, Yufei,Bo, Weiping,Song, Shun,Deng, Deli,Guo, Xin,Wang, Bin,Wang, Wenquan,Peng, Ming,Chen, Zheng,Xia, Jing,Zhang, Kevin,Zhou, Junfei,Peng, Hai,Zhang, Weixiong,Chen, Zheng,Xia, Jing,Zhang, Kevin,Zhang, Weixiong,Zhang, Weixiong. 2014

[17]MeSAUR1, Encoded by a Small Auxin-Up RNA Gene, Acts as a Transcription Regulator to Positively Regulate ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava. Ma, Ping'an,Liu, Chen,Meng, Yuhong,Ma, Ping'an,Chen, Xin,Liu, Chen,Meng, Yuhong,Xia, Zhiqiang,Zeng, Changying,Lu, Cheng,Wang, Wenquan,Ma, Ping'an,Chen, Xin,Liu, Chen,Meng, Yuhong,Xia, Zhiqiang,Zeng, Changying,Lu, Cheng,Wang, Wenquan. 2017

[18]Production of calcium gluconate from cassava by Penicillium citrinum SCG-112. Sun, Hai-Yan,Zhao, Pingjuan,Li, Juanhua,Liu, Enshi,Peng, Ming,Sun, Hai-Yan,Zhao, Pingjuan,Peng, Ming. 2011

[19]Quantitative Trait Locus Analysis for Yield Traits of Cassava (Manihot esculenta Crantz). Yan, Qingxiang,Li, Kaimian,Zhang, Xueqin,Ye, Jianqiu,Chen, Songbi,Li, Qing X.,Huang, Dongyi. 2014

[20]Genome-Wide Identification and Expression Analysis of the WRKY Gene Family in Cassava. Wei, Yunxie,Shi, Haitao,Xia, Zhiqiang,Tie, Weiwei,Ding, Zehong,Yan, Yan,Wang, Wenquan,Hu, Wei,Li, Kaimian. 2016

作者其他论文 更多>>

微信小程序