Large-Scale Investigation of Soybean Gene Functions by Overexpressing a Full-Length Soybean cDNA Library in Arabidopsis

Abstract

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.

Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae

“…Also, in another Brassicaceae halophytic relative of Arabidopsis , Lepidium crassifolium , the cDNA library was screened with the FOX hunting system in Arbidopsis , and the proteins GDSL-like lipase and Acyl CoA binding protein 6 were supposed to be involved in salt tolerance and osmotic stress tolerance, respectively [ 67 ]. After a decade of development, this strategy has been identified as a powerful tool for elucidating functional genomics and facilitating the discovery of useful genes in model plants or crops [ 17 , 68 , 69 ] or digging useful candidate genes from some wild plants [ 65 , 66 , 67 ]. The functional screening and identification of genes by transgenic assays in plants is a time-consuming and costly process during the first course of cDNA library screening.…”

“…However, due to the lack of genomic information for non-model plant species, the changes in transcriptomic, proteomic, or metabolomic processes caused by stress do not provide sufficient information for the cloning and isolation of stress-related genes in non-model plant species. The F ull-length cDNA Over-eXpressor (FOX) gene hunting system is a functional screening method using the overexpression of full-length cDNA libraries in model plants ( Arabidopsis or rice) or microorganisms (mainly yeast) [ 13 , 14 , 15 , 16 , 17 ], by which some unknown stress-related genes have been cloned and preliminarily identified. Actually, in much earlier studies, the functional screening work with yeast mutants was widely used to separate salt tolerance genes from plant cDNA libraries due to the lack of any genomic sequence information, even in model plants such as Arabidopsis [ 18 , 19 ], tobacco [ 20 ], or rice [ 21 ].…”

Brassica rapa orphan genes largely affect soluble sugar metabolism

“…Functional analysis of BrOG s using A. thaliana is an efficient method, because it requires relatively little time to analyze gene function. In addition, the floral-dip method is the easiest way to perform highly efficient gene transformation 8 , 10 , 28 . These BrOG s do not contain any homologous sequences in A. thaliana and their ectopic expression would result in phenotypic variations, making it easier to understand the function of OG s 2 .…”

“…These results were similar to those of the visual phenotype of the overexpression and RNA interference mutant lines of Arabidopsis OG QQS , which are known to be indistinguishable from the WT at all stages—from seedling to senescence 30 , 31 . In a soybean full-length cDNA overexpression library, a low percentage of mutants exhibited abnormal or favorable phenotypes 10 . Moreover, the overexpression of the V. unguiculata OG UP12_8740 showed no visible phenotype under normal conditions compared with that of the control 4 .…”

“…Target genes can be introduced into a plant genome under the control of a suitable constitutive promoter and terminator sequence to generate specific gain-of-function mutants and, subsequently, the overexpression of these genes can result in corresponding distinct phenotypes, which would help in deducing gene function 9 . A previous study showed that a full-length soybean cDNA overexpression mutant library in A. thaliana , which was constructed to study the function of soybean genes, provided an abundance of mutants for the genetic development of soybean 10 . Approximately 6000 A. thaliana transgenic lines were generated by overexpressing full-length Brassica napus cDNAs under the inducible FOX-Hunting system and several lines showed visible phenotypes post induction 11 .…”