Two alkylresorcinol synthase genes from sorghum; cloning, expression, transformation and characterization
Inventors
Baerson, Scott R. • Pan, Zhiqiang • Rimando, Agnes M. • Dayan, Franck E. • Cook, Daniel
Assignees
US Department of Agriculture USDA
Publication Number
US-9248145-B2
Publication Date
2016-02-02
Expiration Date
2030-03-10
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Abstract
Sorghum is considered an allelopathic crop species and sorgoleone likely accounts for much of its allelopathic properties. Prior investigations into the biosynthesis of sorgoleone suggested the participation of one or more alkylresorcinol synthases (ARS), which are type III polyketide synthases (PKS) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. Quantitative real-time RT-PCR analysis of PKS-like sequences mined from isolated root hairs revealed that two sequences, designated ARS1 and ARS2, were preferentially expressed. Recombinant enzyme studies demonstrated that both sequences encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units. RNA interference (RNAi) experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 participate in the biosynthesis of sorgoleone in planta. ARS1 and ARS2 sequences were used to identify rice genes encoding alkylresorcinol synthases.
Core Innovation
This invention concerns two alkylresorcinol synthase genes cloned from sorghum, designated ARS1 and ARS2, which encode enzymes involved in the biosynthesis of 5-pentadecatrienyl resorcinol, a precursor to the allelochemical sorgoleone. The genes were cloned, expressed, and characterized using recombinant enzyme studies, showing their ability to accept a variety of fatty acyl-CoA starter units. Transgenic plants expressing these genes produce alkylresorcinol compounds in planta, and RNA interference (RNAi) constructs were developed to inhibit ARS1 and ARS2 expression, resulting in reduced sorgoleone levels in sorghum plants.
The problem being solved is the lack of detailed molecular understanding and genetic tools relating to the biosynthesis of sorgoleone, an important allelochemical produced by sorghum root hairs that contributes to its allelopathic properties by inhibiting the growth of competing plants and microbes. Previous to this invention, the genes and enzymes directly responsible for sorgoleone biosynthesis were not fully identified or characterized. Additionally, sorghum's allelopathic effects can inhibit certain crops grown in rotation, posing challenges in crop management.
The invention provides isolated nucleic acid molecules encoding ARS1 and ARS2, constructs and vectors for their expression, transgenic plants and cells expressing these genes that biosynthesize alkylresorcinol precursors of sorgoleone, and methods of transforming plants with these genes. It also encompasses RNAi-mediated repression vectors and methods to reduce sorgoleone accumulation by silencing ARS1 and ARS2, thereby enabling the generation of sorgoleone-deficient sorghum plants.
Claims Coverage
The patent includes five main inventive features based on independent claims relating to methods of increasing alkylresorcinol accumulation in plants and plant cells, and transgenic plants and cells comprising ARS1 cDNA.
Method for increasing alkylresorcinol accumulation in plants or plant cells
Introducing into a plant or plant cell at least one construct comprising a cDNA sequence operably linked to a promoter driving expression in the plant cell, wherein the cDNA encodes SEQ ID NO:2 or an amino acid sequence having at least 99% identity to SEQ ID NO:2, and wherein such expression increases alkylresorcinol accumulation compared to wild type.
Method for increasing phenolic lipid accumulation in plant cells
Transfecting a plant cell with the cDNA sequence encoding SEQ ID NO:2 or highly similar amino acid sequences, operatively linked to a regulatory sequence enabling expression, to increase alkylresorcinol accumulation, followed by selecting cells with increased alkylresorcinol levels relative to wild type.
Use of regulatory sequences to control expression
Employing regulatory sequences such as enhancers or tissue-specific promoters to control expression of the ARS1 cDNA for enhanced accumulation of alkylresorcinols.
Transgenic plants comprising the ARS1 cDNA
Generating transgenic plants or their progeny containing the ARS1 cDNA, selected for increased alkylresorcinol accumulation compared to wild type plants of the same variety.
Transgenic plant cells comprising the ARS1 cDNA
Generating transgenic plant cells or their progeny containing the ARS1 cDNA, selected for increased alkylresorcinol accumulation compared to wild type plant cells of the same variety.
The claims cover nucleic acid constructs encoding ARS1 for enhancing alkylresorcinol production in plants and plant cells via transformation and expression, including specific regulatory sequences, and the resulting transgenic plants and cells selected for elevated alkylresorcinol levels relative to wild type.
Stated Advantages
Enables production of alkylresorcinol precursors to sorgoleone in transgenic plants, potentially increasing allelopathy and disease resistance.
Provides a method to generate sorgoleone-deficient sorghum plants which can facilitate cultivation of sensitive crops in rotation systems.
Offers new genetic engineering tools for altering phenolic lipid content in plants for agricultural and industrial applications.
Uses RNA interference to specifically inhibit sorgoleone biosynthesis, minimizing adverse allelopathic effects.
Allows production of phenolic lipids in specific tissues or in response to elicitors via use of alternative promoters, enhancing functional versatility.
Documented Applications
Production of transgenic plants expressing ARS1 or ARS2 to accumulate alkylresorcinols such as 5-pentadecatrienyl resorcinol for enhanced allelopathic and antimicrobial properties.
Generation of sorgoleone-deficient sorghum plants via RNA interference targeting ARS1 and ARS2 to prevent allelopathic inhibition of succeeding crops in rotation.
Use of plants as bioreactors for large scale production of phenolic lipids useful in industry and agriculture.
Identification and cloning of related alkylresorcinol synthase genes in other crop species, e.g., rice, for modification of phenolic lipid biosynthesis.
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