Development of a rapid and efficient protoplast isolation and transfection method for chickpea (Cicer arietinum)
Development of a rapid and efficient protoplast isolation and transfection method for chickpea (Cicer arietinum)
复制标题
DOI：
10.1016/j.mex.2020.101025
复制
发表时间：
2020-01-01
期刊：
影响因子：
1.9
通讯作者：
Nakata, Paul A.
中科院分区：
文献类型：
--
作者：
Cheng, Ninghui;Nakata, Paul A.
鹰嘴豆（Cicer arietinum L.）是世界上第二重要的豆类作物。鹰嘴豆基因组测序的最新进展为基因发现和作物性状改良提供了新的有价值的资源。稳定的鹰嘴豆转基因技术的困难和缺乏一个瞬时表达系统的基因表达和功能的快速分析，但是，限制了这种基因组资源的有用性。作为减轻这种限制的一步，我们在这里报告的发展，一个简单而有效的瞬时基因表达协议。使用鹰嘴豆幼苗的叶子，我们已经建立了一个程序，使大量的重要鹰嘴豆原生质体的生成在短短几个小时内。此外，我们优化了PEG-钙介导的转染方法，以有效地将外源DNA导入鹰嘴豆原生质体。目前的研究是第一个提出了一个详细的一步一步的程序，原生质体分离，评估，转染，并在鹰嘴豆中的应用。此外，我们优化了转染效率，这是以前没有报道过的。我们的原生质体转染方法提供了一个平台，将允许快速高通量筛选和基因表达和功能的系统表征。通过这些研究获得的知识将有利于目前的努力，以提高鹰嘴豆生产和质量。改进酶消化液，以提高产量和活力。优化鹰嘴豆原生质体的转染。由爱思唯尔公司出版
Chickpea (Cicer arietinum L.) is the second most important grain legume worldwide. Recent advances in the sequencing of the chickpea genome has provided a new and valuable resource to aid effort s in gene discovery and crop trait improvement. Technical difficulties in stable chickpea transgenics and the lack of a transient expression system for rapid analysis of gene expression and function; however, has limited the usefulness of this genomic resource. As a step toward alleviating this limitation, we report here the development of a simple and efficient transient gene expression protocol. Using leaves from chickpea seedlings, we have established a procedure that enables the generation of large quantities of vital chickpea protoplasts within only a few hours. In addition, we have optimized a PEG-calcium-mediated transfection method to efficiently deliver exogenous DNA into the chickpea protoplast. The current study is the first to present a detailed step-by-step procedures for protoplast isolation, evaluation, transfection, and application in chickpea. In addition, we optimize the transfection efficiency which has not been previously reported. Our protoplast transfection approach provides a platform that will allow rapid high-throughput screening and systematic characterization of gene expression and function. Knowledge gained through such studies will benefit current effort s to improve chickpea production and quality.Modified enzymatic digestion solution for higher yield and viability.Optimize transfection of chickpea protoplasts. Published by Elsevier B.V.