Large-Scale Investigation of Soybean Gene Functions by Overexpressing a Full-Length Soybean cDNA Library in Arabidopsis.
Large-Scale Investigation of Soybean Gene Functions by Overexpressing a Full-Length Soybean cDNA Library in Arabidopsis.
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DOI：
10.3389/fpls.2018.00631
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发表时间：
2018
影响因子：
5.6
通讯作者：
Jiao Y
中科院分区：
文献类型：
--
作者：
Li X;Huang L;Lu J;Cheng Y;You Q;Wang L;Song X;Zhou X;Jiao Y
分子育种已成为作物改良的重要途径，而分子育种的前提是阐明遗传位点或基因的功能。大豆是世界上最重要的粮食和油料作物之一。然而，由于大豆遗传转化的困难，大豆功能基因组学的研究远远落后于水稻等其他作物，严重制约了大豆分子改良的进展。在这里，我们描述了一个有效的大规模的策略，通过在拟南芥中的全长大豆cDNA文库的过表达来研究大豆基因的功能。对过表达载体pJL 12进行修饰以用于构建标准化全长cDNA文库。构建的cDNA文库质量良好，重复克隆约占4%，插入片段约2.2kb，全长率约为98%。然后在拟南芥中过表达该cDNA文库，初步获得了约2000个转基因株系。表型分析表明，超过5%的T1代转基因株系表现出异常的发育表型，约1%的转基因株系表现出潜在的有利性状。我们随机扩增了4个具有明显表型（种子增大、叶片变黄、分枝增多和长角果密集）的基因，并在拟南芥中重复了转基因分析。随后的表型观察表明，这些表型确实是由于大豆基因的过度表达。我们相信我们的策略代表了一个有效的大规模的方法来研究大豆基因的功能，并进一步揭示有利于大豆分子改良的基因。
Molecular breeding has become an important approach for crop improvement, and a prerequisite for molecular breeding is elucidation of the functions of genetic loci or genes. Soybean is one of the most important food and oil crops worldwide. However, due to the difficulty of genetic transformation in soybean, studies of its functional genomics lag far behind those of other crops such as rice, which severely impairs the progress of molecular improvement in soybean. Here, we describe an effective large-scale strategy to investigate the functions of soybean genes via overexpression of a full-length soybean cDNA library in Arabidopsis. The overexpression vector pJL12 was modified for use in the construction of a normalized full-length cDNA library. The constructed cDNA library showed good quality; repetitive clones represented approximately 4%, insertion fragments were approximately 2.2 kb, and the full-length rate was approximately 98%. This cDNA library was then overexpressed in Arabidopsis, and approximately 2000 transgenic lines were preliminarily obtained. Phenotypic analyses of the positive T1 transgenic plants showed that more than 5% of the T1 transgenic lines displayed abnormal developmental phenotypes, and approximately 1% of the transgenic lines exhibited potentially favorable traits. We randomly amplified 4 genes with obvious phenotypes (enlarged seeds, yellowish leaves, more branches, and dense siliques) and repeated the transgenic analyses in Arabidopsis. Subsequent phenotypic observation demonstrated that these phenotypes were indeed due to the overexpression of soybean genes. We believe our strategy represents an effective large-scale approach to investigate the functions of soybean genes and further reveal genes favorable for molecular improvement in soybean.
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