Introduction and Purpose

Soybean is used as a model for studies on seed physiology and biochemistry as well as plant-microbial interactions. RNA interference technology, simply called RNAi, has been shown to be an efficient tool to assign gene functions systematically in various organisms and has promise for soybean genomics. The RNAi approach allows large-scale, systematic functional assignment of genes in a high-throughput transformation system.

Project Specifics

This research project will seek to provide "proof of concept" to fully explore the RNAi technology for genome-wide functional assignment of soybean genes. RNAi is a universal mechanism to silence genes and is suitable for systematically assigning gene functions. The proposed research is also based on the researchers' recent break-through in developing a high-frequency transformation of soybean which enables them to generate large numbers of transgenic soybean lines in a short time at a minimal cost.

Specific Objectives

1. Design and construct RNAi constructs that allow efficient silencing of soybean genes.
2. Construct a plant transformation vector that enables RNAi technology and a high-throughput cloning process that is based on the Gateway Cloning Technology.
3. Develop transgenic soybean lines expressing RNAi.
4. Characterize RNAi soybean lines to evaluate silencing efficiency.

Methods

The researchers will test several working hypotheses including the impact of the sizes of functional introns, the expression status of RNAi as well as use four major representative and functional groups of soybean ESTs for RNAi on the RNAi efficacy. The sizes of functional introns will be from 80bp to over 1000bp. The matrix attachment regions (MARs) will be evaluated for a possible high level and stable expression of RNAi cassette. The average length of conserved regions suitable for reverted repeats of dsRNA are from 80 to 200 base pairs (Table). The researchers believe use of the shorter (80-200 bp) conserved sequences will decrease non-specific effects on non-target genes and reduce complications others have experiences using RNAi.

Four major representative and functional groups of soybean ESTs for RNAi*

Functional Groups	Soybean (Gm) EST name	Accession No.	Locations of conserved functional regions (bp) in EST	Locations of conserved regions for RNAi (bp) in EST	Lengths of Conserved region for reverted repeats (bp)
Transcriptional factor	Gm AGAMOUS	BU761215	160-300	180-280	100
	Gm nod factor binding protein gene	BQ253081	290-500	290-390	100
Receptor (like) kinase	Gm Serine/Threonine protein kinase gene	BU965024	160-250	160-250	90
	At SERK3	AF384970	1700-1950	1700-1900	200
Pathway enzyme	Gm raffinose synthase gene	BM887415	12-100	20-100	80
	Gm FAD3 gene	AB051215	600-700	600-700	100
Structural protein	Gm cytoskeleton-associated protein	BM525764	300-380	300-380	80

*: Four major representative and functional groups of soybean ESTs are selected as RNAi targets. They also represent major research interest in our research community. Only those ESTs showing multiple hits among at least three different organisms using BLAST (Stephen et al., 1997) are listed here. Each of these ESTs well represents a conserved functional sequence of a specific gene.

All the above critical designs will be incorporated into the high-throughput Gateway cloning technology. The resultant constructs will be used to transform soybean employing Agrobacterium-mediated transformation of soybean. All transgenic soybean lines and some of their progenies will be analyzed using leaf-painting, Southern blot analysis to determine transgene integration and copy numbers. Northern blot analysis will be used to detect transcript levels as well as siRNAi of transgens in transgenic soybean lines. Morphological observations, Gas chromatography (GC), root growth assay, and lethality estimates of cultures etc will be used to determine the phenotypes caused by the above RNAi expression cassettes.

Outcomes and Benefits

The completion of this project will lay a good foundation for verifying functions of numerous soybean genes and have a profound impact on engineering of this crop for genetic improvement to improve both research and the U.S. economy. Genome-wide functional assignment of soybean genes employing RNAi technology and ESTs will validate useful genes that will become invaluable resources immediately available for genetic improvement of soybean for value-added traits.