CN110938615A

CN110938615A - Oxalate metabolism related enzyme and application thereof in oxalate degradation

Info

Publication number: CN110938615A
Application number: CN201811109369.0A
Authority: CN
Inventors: 巫永睿; 杨俊�
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2018-09-21
Filing date: 2018-09-21
Publication date: 2020-03-31

Abstract

The invention provides an oxalate metabolism related enzyme and application thereof in oxalate degradation, and particularly can obviously regulate and control the oxalate content in plants by regulating and controlling the expression or activity of oxalate metabolism related enzyme genes or proteins thereof in the plants.

Description

Oxalate metabolism related enzyme and application thereof in oxalate degradation

Technical Field

The invention relates to the field of biotechnology, in particular to oxalate metabolism related enzyme and application thereof in oxalate degradation.

Background

Oxalic acid is the simplest dicarboxylic acid and can be found in most organisms, such as microorganisms, plants, and animals. Oxalic acid in plants exists mainly in two different forms, soluble free oxalic acid and insoluble metal oxalate crystals. The content of oxalic acid in the plant can reach 3-10% of dry weight, and the oxalic acid plays various roles in the growth, development and metabolic processes of the plant, such as metal tolerance, ion balance, insect defense and the like.

Oxalic acid in the plant body helps to improve its adaptability, but excessive accumulation of oxalic acid may also adversely affect the growth of the plant. For example, in Arabidopsis, overexpression of bacterial oxalate synthase can cause excessive accumulation of oxalate leading to stunting of the plant growth. Evidence also suggests that pathogens can increase their infectivity by promoting host oxalate secretion, stimulating stomatal opening and cell wall permeability, ultimately leading to programmed death of infected plant cells. Oxalic acid acts as a nutritional antagonist, impairing the absorption of calcium and other minerals. For example, spinach has a high level of oxalic acid, which can interfere with calcium absorption and utilization if eaten with a high calcium containing soy product. In addition, excessive intake of metal oxalate crystals in human bodies may induce breast cancer, kidney stones and other related serious diseases. Therefore, the content of oxalic acid in plants needs to be accurately regulated, and the biosynthesis and degradation pathways of oxalic acid are key steps for regulating the content of oxalic acid.

To date, a variety of (or potential) pathways for oxalate biosynthesis have been reported. Glyoxylic acid can be used to produce oxalic acid from ester oxidase or lactate dehydrogenase. In oxalate secreting fungi, oxaloacetate can be catalytically cleaved by oxaloacetate acetylhydrolase to produce oxalate. Ascorbic acid may also serve as a precursor for oxalate synthesis, but the enzymes responsible for this pathway have not been reported.

Therefore, there is an urgent need in the art to develop an enzyme capable of regulating the oxalate content in plants and a novel approach thereof.

Disclosure of Invention

The invention provides an enzyme capable of regulating and controlling the oxalic acid content in plants and a new way thereof.

In a first aspect the present invention provides the use of a substance selected from the group consisting of: an oxalate metabolism-related enzyme or a coding sequence thereof, or an enhancer or inhibitor thereof, for use in modulating an agronomic trait in a plant, the agronomic trait comprising oxalate content in the plant, wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

In another preferred embodiment, when the substance is an oxalate metabolism-related enzyme or a coding sequence thereof, or an enhancer thereof, the controlling of the agronomic trait of the plant comprises reducing the oxalate content in the plant.

In another preferred embodiment, when the substance is an inhibitor of an enzyme associated with oxalate metabolism, the modulating an agronomic trait in a plant comprises increasing oxalate content in the plant.

In another preferred embodiment, the oxalate metabolism-related enzyme gene includes a cDNA sequence, a genomic sequence, or a combination thereof.

In another preferred example, the oxalate metabolism-related enzyme is derived from a plant of the family Poaceae.

In another preferred example, the oxalate metabolism-related enzyme is derived from one or more plants selected from the group consisting of: corn, arabidopsis thaliana, rice bean, and alfalfa.

In another preferred example, the oxalate metabolism-related enzyme is a wild-type or mutant-type enzyme.

In another preferred embodiment, the amino acid sequence of the oxalate metabolism-related enzyme is selected from the group consisting of:

(i) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 1 or 2;

(ii) (ii) a polypeptide derived from (i) having the function of reducing and/or increasing oxalic acid content in plants, which is formed by substituting, deleting or adding one or more (e.g. 1-10) amino acid residues in the amino acid sequence shown in SEQ ID NO. 1 or 2; or

(iii) The polypeptide with the function of reducing and/or increasing the oxalic acid content in plants has homology of more than or equal to 90 percent (preferably more than or equal to 95 percent, and more preferably more than or equal to 98 percent) with the amino acid sequence shown in SEQ ID NO. 1 or 2.

In another preferred embodiment, the amino acid sequence of the oxalate metabolism-related enzyme is shown in SEQ ID No. 1 or 2.

In another preferred embodiment, the nucleotide sequence of the gene encoding the oxalate metabolism-related enzyme is selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 1 or 2;

(b) a polynucleotide having a sequence shown in SEQ ID NO.3 or 4;

(c) a polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more, more preferably 99% or more) to a sequence represented by SEQ ID No.3 or 4;

(d) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO.3 or 4;

(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In another preferred embodiment, the oxalic acid content is reduced by more than or equal to 10%, preferably more than or equal to 20%, more preferably more than or equal to 50% in said plant compared to the control plant;

wherein the control plant is the same plant into which the coding sequence of the exogenous oxalate metabolism-related enzyme has not been introduced and to which the promoter or inhibitor of the oxalate metabolism-related enzyme has not been applied.

In another preferred embodiment, the plant includes a monocotyledon and a dicotyledon.

In another preferred embodiment, the plant includes herbaceous plants and woody plants.

In another preferred embodiment, the plant is selected from the group consisting of: cruciferous plants, gramineae, leguminous plants, solanaceae, actinidiaceae, malvaceae, paeoniaceae, rosaceae, liliaceae, or combinations thereof.

In another preferred embodiment, the plant is selected from the group consisting of: corn, rice, spinach, cabbage, soybean, tomato, tobacco, wheat, sorghum, barley, oat, millet, peanut, kiwi, cotton, strawberry, peony, lilium brownii, tulip, mulberry, apple, pear, peach, cherry, pomegranate, or a combination thereof.

In another preferred embodiment, the composition comprises an agricultural composition.

In another preferred embodiment, the promoter refers to a substance that promotes the expression of an oxalate metabolism-related enzyme or a coding sequence thereof.

In another preferred embodiment, the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof.

In another preferred embodiment, the inhibitor is selected from the group consisting of: an antisense nucleic acid, an antibody, a small molecule compound, a Crispr agent, a small molecule ligand, or a combination thereof.

In another preferred embodiment, the antisense nucleic acid comprises siRNA, shRNA, and/or miRNA.

In a second aspect, the present invention provides the use of an oxalate metabolism-related enzyme, or a coding sequence thereof, or a promoter thereof, for (a) reducing oxalate content in a plant; and/or (b) preparing a composition or formulation for reducing oxalic acid content in a plant; wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

In a third aspect, the present invention provides the use of an inhibitor of an oxalate metabolism-related enzyme, or a coding sequence thereof, for (a) increasing oxalate content in a plant; and/or (b) preparing a composition or formulation that increases the total oxalic acid content of the plant; wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

The fourth aspect of the invention provides a method for regulating and controlling the oxalic acid content in plants, which comprises the following steps:

(a) providing a plant;

(b) identifying an oxalate metabolism-related enzyme in the plant;

(c) changing the expression amount and/or activity of the oxalate metabolism-related enzyme so as to regulate the oxalate content in the plant;

wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

In another preferred example, the alteration of the expression amount and/or activity of the oxalate metabolism-related enzyme includes increasing the expression amount and/or activity of the oxalate metabolism-related enzyme; and/or reducing or inhibiting the expression level and/or activity of the oxalate metabolism-related enzyme.

In another preferred example, when the modification of the expression amount and/or activity of the oxalate metabolism-related enzyme is to increase the expression amount and/or activity of the oxalate metabolism-related enzyme, the oxalate content in the plant is decreased.

In another preferred embodiment, the improvement of the expression level and/or activity of the oxalate metabolism-related enzyme is an improvement of the expression level and/or activity of the oxalate metabolism-related enzyme by 50% or more, preferably 100% or more, more preferably 200% or more.

In another preferred example, when the alteration of the expression or activity of the oxalate metabolism-related enzyme is a decrease or inhibition of the expression or activity of the oxalate metabolism-related enzyme, the oxalate content in the plant is increased.

In another preferred embodiment, the expression or activity of the oxalate metabolism-related enzyme is decreased by RNAi interference, miRNA interference, or nucleic acid editing (e.g., criprpr method).

In another preferred embodiment, said reducing or inhibiting the expression or activity of said oxalate metabolism-related enzyme means reducing the expression or activity of said oxalate metabolism-related enzyme to 50% or less, preferably 30% or less, preferably 20% or less, more preferably 10% or 5% or less or to 0%.

In a fifth aspect, the present invention provides a method of improving a trait in a plant, comprising the steps of:

increasing or decreasing the expression level or activity of an oxalate metabolism-related enzyme or a gene encoding the same in a plant, thereby improving an agronomic trait of the plant, wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

In another preferred example, the agronomic trait of the improved plant comprises modulating oxalic acid content in the plant.

In another preferred example, the regulating the oxalic acid content in the plant comprises decreasing or increasing the oxalic acid content in the plant.

In another preferred example, when the expression level or activity of an oxalate metabolism-related enzyme or a gene encoding the same is increased in a plant, the "improving an agronomic trait of a plant" includes: reducing the oxalic acid content in the plant.

In another preferred example, when the expression level or activity of an oxalate metabolism-related enzyme or a gene encoding the same is decreased in the plant, the "improving an agronomic trait of a plant" includes: increasing the oxalic acid content in the plant.

In another preferred embodiment, the method comprises administering to the plant an promoter or inhibitor of an oxalate metabolism-related enzyme or a gene encoding the same.

In another preferred example, the method comprises the steps of:

(i) providing a plant or plant cell; and

(ii) introducing an oxalate metabolism-related enzyme or an promoter or inhibitor of a gene encoding the same into the plant or plant cell, thereby obtaining a transgenic plant or plant cell, wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

In another preferred embodiment, the inhibitor is selected from the group consisting of: an antisense nucleic acid, an antibody, a small molecule compound, a Crispr agent, or a combination thereof.

In another preferred embodiment, the promoter refers to a substance that promotes the expression of the oxalate metabolism-related enzyme or a gene encoding the same.

In a sixth aspect, the present invention provides a method for producing a transgenic plant having reduced oxalate content in the plant, comprising the steps of:

introducing a gene encoding an oxalate metabolism-related enzyme into a plant cell, culturing the plant cell, and regenerating a plant, wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

In another preferred example, the method comprises the steps of:

(s1) providing Agrobacterium carrying an expression vector containing a gene encoding said oxalate metabolism-related enzyme;

(s2) contacting the plant cell or tissue or organ with the Agrobacterium of step (s1) thereby transferring the gene encoding the enzyme involved in oxalate metabolism into the plant cell, tissue or organ;

(s3) screening plant cells or tissues or organs into which genes encoding enzymes involved in oxalate metabolism have been transferred; and

(s4) regenerating the plant cell or tissue or organ of step (s3) into a plant.

The seventh aspect of the present invention provides a transgenic plant into which a gene encoding an oxalate metabolism-related enzyme or an promoter or inhibitor thereof has been introduced.

The eighth aspect of the present invention provides an in vitro enzymatic reaction method, comprising the steps of:

(a) enzymatically reacting a compound of formula Ia in the presence of an oxalate metabolism-related enzyme, thereby forming a compound of formula Ib;

wherein the oxalate metabolism-related enzyme comprises O7.

In another preferred example, the method further comprises the steps of:

(b) enzymatically reacting a compound of formula Ib in the presence of an oxalate metabolism-related enzyme, thereby forming a compound of formula Ic;

wherein the oxalate metabolism-related enzyme comprises OCD 1.

The ninth aspect of the present invention provides an in vitro enzymatic reaction method, comprising the steps of:

wherein the oxalate metabolism-related enzyme comprises OCD 1.

In another preferred embodiment, the compound of formula Ib is prepared by the following process:

wherein the oxalate metabolism-related enzyme comprises O7.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the grain phenotype of the ocd1-1 mutant.

(A) A cluster for self-separating the powdery grains. Bar is 1 cm.

(B) And (C) mature grain of wild type (B) and ocd1-1(C) are viewed on a light box. Bars is 0.5 cm.

(D) And (E) cross-sectional views of wild type (D) and ocd1-1(E) mature kernels. Bars is 0.5 cm.

(F) And (G) scanning electron microscopy images of the peripheral region of mature (F) and ocd1-1(G) endosperm. Bars 20 μm.

FIG. 2 shows the map-based cloning and identification of the Ocd1 gene.

(A) Ocd 1. The numbers below the vertical line indicate the number of recombined individuals at the position of the corresponding molecular marker.

(B) Ocd1 schematic representation of the gene structure is shown in relation to mutant alleles. Black bold and grey lines indicate exons and introns, respectively. Triangles indicate transposon insertions. Primers indicated by arrows were used for mutant identification.

(C) Identification of transposon insertion in ocd 1-1.

(D) ocd1-2 and ocd1-3 transposon insertion identification.

(E) RT-PCR analyzed Ocd1 expression in three ocd mutants. Ubi is an internal reference gene.

FIG. 3 shows allelic testing and validation of the Ocd1 gene.

(A) Ear after ocd1-2/+ (left) and ocd1-3/+ (right) selfing. Bar 1 cm.

(B) The ocd1-2/+ (left) and ocd1-3/+ (right) pollen crosses were conferred to ocd1-1/+ ears to harvest the ears. Bars 1 cm.

FIG. 4 shows the expression pattern analysis of Ocd1 gene.

(A) Quantitative PCR was performed to determine the expression of Ocd1 gene in different W22 tissues.

(B) Quantitative PCR determined the expression of the Ocd1 gene in seed and endosperm at different days after pollination. The Z1C gene was used as a marker for endosperm-specific expression. All expression levels were calculated using the Ubi gene as an internal reference.

FIG. 5 shows the domains, sequence alignment and subcellular localization of ZmOCD 1.

(A) Schematic representation and conserved domains of ZmOCD1 protein. aa, an amino acid.

(B) Alignment of the ZmOCD1 protein sequence with the Arabidopsis and oxalic acid bacterium formigenes homologous protein (AtOCD1 and OfOCD1) sequences the key amino acid points of the OfOCD1 protein are labeled with different shapes. ■: ThDP binding site; ●: ADP binding site color: ◆: magnesium ion binding site, ▲: enzyme active site.

(C) Subcellular localization of 35S-GFP and 35S: ZmOCD 1-GFP. The left is the laser confocal picture of transiently transformed arabidopsis mesophyll protoplasts. The right side is an agrobacterium tumefaciens-mediated tobacco transient transformation laser confocal picture. Bar 25 μm.

FIG. 6 shows the purification of the protein used for enzymatic activity and the preparation of the substrate.

(A) Oxalate degradation pathway. O7 and ZmOCD1 catalyze the first two steps of oxalic acid degradation, respectively.

(B) After purification, the protein is used for SDS-PAGE analysis of in vitro enzyme activity experimental protein. The purified protein was separated by electrophoresis on a 4-20% gradient gel and stained with Coomassie blue after electrophoresis. M, protein molecular weight standards.

(C) LC-MS/MS analysis of the synthesized product showed a peak of ions with a nuclear to cytoplasmic ratio of 840.1 corresponding to oxalyl-CoA. The LC-MS/MS was run in positive ionization mode.

(D) The secondary mass spectrum peak diagram of LC-MS/MS analysis of the synthesized product, oxalyl coenzyme A with nuclear-to-cytoplasmic ratio 840.1 is marked by structural formula and chemical name. The LC-MS/MS is performed in the positive mode.

FIG. 7 shows the in vitro enzyme activity assay of ZmOCD1 protein.

(A) LC-MS/MS chromatograms in a ZmOCD1-His reaction system. The red and blue curves represent the peaks at a nuclear to cytoplasmic ratio of 840.1 (oxalyl-coa) and 796.1 (formyl-coa), respectively. The LC-MS/MS is performed in the positive mode.

(B) LC-MS/MS chromatograms in a control protein reaction system. The red and blue curves represent the peaks at a nuclear to cytoplasmic ratio of 840.1 (oxalyl-coa) and 796.1 (formyl-coa), respectively. The LC-MS/MS is performed in the positive mode. No formyl-CoA was formed in the reaction.

(C) The secondary mass spectrum peak diagram of LC-MS/MS analysis of the reaction product, the structural formula and the chemical name of formyl coenzyme A with the nuclear-to-cytoplasmic ratio of 796.1 are labeled. The LC-MS/MS is performed in the positive mode.

FIG. 8 shows the verification of the enzyme activity of maize O7.

(A) And performing enzyme activity reaction on the LC-MS/MS chromatogram by taking oxalic acid with different concentrations as substrates. The nuclear to cytoplasmic ratio at the peak position was 838.1(m/z), i.e., the oxalyl-CoA nuclear to cytoplasmic ratio. LC-MS/MS was performed in negative ion mode.

(B) LC-MS/MS ion peak fragment pattern of oxalyl-CoA. LC-MS/MS was performed in negative ion mode.

Detailed Description

As a result of extensive and intensive studies, the present inventors have screened an oxalate metabolism-related enzyme gene or a protein thereof by a large number of screens, and have remarkably controlled the oxalate content in plants by controlling the expression or activity of the oxalate metabolism-related enzyme gene or the protein thereof in plants, and have found for the first time that the oxalate metabolism-related enzyme gene or the protein thereof of the present invention catalyzes oxalic acid to produce formyl CoA, and the oxalate content can be reduced by at least 10% (more preferably, 20%, still more preferably, 50%). On this basis, the present inventors have completed the present invention.

The oxalate metabolism-related enzyme of the present invention and a gene encoding the same

As used herein, the terms "oxalate metabolism-related enzyme", "polypeptide of the present invention", "oxalate metabolism-related enzyme of the present invention" are used interchangeably and refer to an oxalate metabolism-related enzyme derived from a plant, such as gramineae, preferably corn, and variants thereof, which specifically hydrolyze oxalate to formyl-CoA. Preferably, said polypeptide of the invention refers to an enzyme as defined in the first aspect of the invention.

In the present invention, the oxalate metabolism-related enzyme includes OCD1 and/or O7.

Wherein OCD1 is a protein encoded by gene GRMZM2G175171, 575 amino acids in full length, catalyzing oxalyl CoA (formula Ib) to produce formyl CoA (formula Ic);

o7 is a protein encoded by the gene GRMZM2G074759, 527 amino acids in length, catalyzing the production of oxalyl CoA (formula Ib) from oxalate (formula Ia);

in a preferred embodiment, the amino acid sequence of the wild-type OCD1 is shown in SEQ ID No. 1, and the coding gene sequence of the wild-type OCD1 is shown in SEQ ID No. 3. MASDSTAAVTVDGSALAGRALAAAGTRHMFGVVGIPVTSLASRAAAAGVRFLAFRNEQSAGYAAAAYGFLTGSPGALLTVSGPGCVHGLAGLSHATANAWPLLMISGSCDQADAGRGDFQELDQIAATKPFVKLAVKATTIADIPRLVFQALAAAVSGRPGGCYLDIPSDVLHQTLPESEAAALIAAAAANSAASDPSPSKHKTLDEGIAKAADLLRCAERPLVVIGKGAAYARAEEAIRKLVDTTGIPFLPTPMGKGVVPDSHPLSATAARSLAIGQCDVALVIGARLNWLLHFGEPPKWSKDVKFILVDVSEEEIELRKPHVGIVGDAKRVTELINREIKDNPFCLARSHPWVEAITKKANDNVLKMEAQLVKDVVPFNFMTPLRIIRDAILAEGSPAPIVVSEGANTMDVGRAVLVQNEPRTRLDAGTWGTMGVGLGYCIAAAAAEPERLVVAVEGDSGFGFSAMEVETLVRYQLPVVVIVFNNNGVYGGDRRSPDEITGPYKGDPAPTSFVPAAGYHKMMEAFGGKGYLVETPDELKSALSESFRARKPAVINVIIDPYAGAESGRMQHKN (SEQ ID NO: 1).

ATGGCATCTGACTCCACGGCCGCCGTGACGGTGGACGGCAGCGCGCTTGCGGGGCGCGCGCTGGCCGCGGCGGGCACGCGGCACATGTTCGGGGTGGTGGGCATCCCCGTCACCTCCCTCGCGTCCCGTGCCGCCGCGGCCGGGGTCCGCTTCCTCGCCTTCCGCAACGAGCAGTCCGCGGGGTACGCCGCCGCCGCCTACGGCTTCCTCACGGGCTCCCCGGGCGCCCTACTCACCGTCTCCGGCCCAGGATGCGTCCACGGGCTCGCGGGCCTCTCCCACGCCACCGCCAACGCCTGGCCGCTCCTCATGATCTCCGGATCCTGCGACCAGGCTGACGCCGGCAGGGGCGACTTCCAGGAGCTCGACCAGATCGCCGCCACTAAGCCCTTCGTCAAGCTCGCCGTCAAGGCAACAACCATCGCCGACATCCCGCGCCTCGTCTTCCAGGCCCTCGCCGCCGCCGTGTCCGGCCGTCCCGGAGGGTGCTACCTCGATATCCCGTCCGATGTCCTCCACCAAACCCTCCCTGAATCCGAGGCCGCGGCTCTCATAGCAGCAGCAGCCGCCAATTCGGCCGCATCCGATCCTTCCCCGTCAAAGCACAAGACTCTTGACGAGGGAATCGCGAAAGCTGCGGACCTGCTCCGGTGTGCGGAGCGGCCTCTTGTTGTGATTGGGAAGGGTGCGGCGTATGCGCGCGCGGAGGAGGCGATTCGGAAGCTGGTGGACACCACGGGCATCCCCTTCCTCCCGACGCCGATGGGGAAAGGGGTCGTGCCTGACTCGCATCCACTCTCCGCCACGGCGGCACGCTCACTCGCCATCGGGCAGTGTGATGTGGCTCTGGTCATCGGTGCTAGGCTTAATTGGTTGCTTCACTTTGGCGAGCCACCCAAGTGGTCCAAGGATGTTAAGTTCATTCTTGTTGACGTCTCCGAGGAGGAGATTGAGCTCCGGAAGCCACATGTGGGGATTGTTGGGGACGCGAAGAGAGTAACTGAGCTGATCAACCGGGAGATCAAGGACAATCCATTCTGCCTGGCAAGGTCGCACCCATGGGTCGAAGCGATCACTAAGAAGGCCAATGACAATGTTCTTAAGATGGAGGCGCAGCTAGTGAAGGATGTTGTGCCATTCAATTTCATGACACCATTGCGGATCATCCGGGATGCGATCCTTGCCGAGGGGAGTCCTGCTCCAATAGTGGTCTCAGAAGGGGCAAATACCATGGATGTTGGCAGGGCTGTGCTTGTGCAGAATGAACCAAGGACAAGACTGGATGCAGGGACATGGGGGACGATGGGGGTTGGATTGGGGTACTGTATTGCGGCTGCAGCAGCTGAACCTGAACGACTCGTGGTTGCTGTGGAGGGCGATTCTGGATTTGGCTTCAGTGCAATGGAGGTTGAGACGTTGGTGAGGTACCAGCTGCCTGTTGTTGTAATCGTTTTCAACAACAACGGTGTCTACGGCGGGGACCGGAGAAGCCCTGACGAGATAACCGGACCCTACAAAGGTGACCCAGCCCCAACCTCATTTGTTCCGGCAGCAGGGTACCATAAGATGATGGAGGCTTTTGGAGGGAAAGGGTATCTTGTGGAGACACCAGACGAGCTCAAATCTGCCCTTTCAGAATCGTTCCGCGCCAGGAAACCGGCGGTGATTAATGTTATCATTGATCCTTACGCCGGCGCAGAGAGCGGTCGAATGCAGCACAAGAACTGA(SEQ ID NO.:3)。

In a preferred embodiment, the amino acid sequence of the wild-type O7 is shown in SEQ ID No. 2, and the coding gene sequence of the wild-type O7 is shown in SEQ ID No. 4.

MAMATTEATTLTALLKEAAAAFPTRRAVAVPGRLELTHAALDALVDAAAARLAADAGVLPGHVVALSFPNTVELVIMFLAVIRARGVAAPLNPAYTQEEFEFYLSDSEARLLVTNAEGNAAAQAAAAKLGLAHAAASLHDAAGPVHLAGLPAHAPENGSHGGGAAGSLSPNDPSDVALFLHTSGTTSRPKGVPLKQRNLAASVRNIRSVYRLAETDATVVVLPLFHVHGLLCALLSSLASGASVALPAAGRFSASTFWADMRASGATWYTAVPTIHQIILDRHASRPEAYPALRFIRSCSASLAPAILERLEAAFSAPVLEAYAMTEASHLMTSNPLPEDGPRKPGSVGRAVGQELAVLDEEGRLVAAGSPGEVCVRGDNVTAGYKGNPEANEAAFRFGWFHTGDIGVVDDQGYVRLVGRIKELINRGGEKISPIEVDAVLLGLPGVAQAVSFGVPDDKYGEEINCAVIPRDGSALREEEVLAHCRRNLASFKVPKKVFITDDLPKTATGKIQRRIVAQHFVQPTSA(SEQ ID NO.:2)

ATGGCTATGGCGACGACGGAGGCCACCACGCTGACGGCGCTGCTCAAGGAGGCGGCGGCCGCCTTCCCGACCCGCCGCGCAGTTGCCGTACCGGGCAGGCTGGAGCTCACGCACGCCGCCCTCGACGCTCTCGTCGACGCGGCGGCCGCACGACTCGCCGCCGACGCCGGAGTTCTCCCGGGCCACGTCGTCGCGCTTTCCTTCCCCAACACCGTCGAGCTGGTGATCATGTTCCTGGCGGTAATCCGCGCGCGCGGCGTGGCGGCGCCGCTCAACCCGGCCTACACGCAGGAGGAGTTCGAGTTCTACCTCTCCGACTCGGAGGCGCGCCTCCTCGTCACCAACGCGGAGGGCAACGCGGCGGCGCAGGCGGCCGCTGCCAAGCTCGGCCTCGCCCACGCCGCCGCCAGCCTCCACGACGCGGCCGGCCCTGTCCACCTCGCCGGCCTCCCGGCCCATGCTCCGGAGAACGGGTCCCACGGCGGCGGCGCGGCGGGCTCTCTCTCCCCCAACGACCCGTCGGACGTGGCCCTGTTCCTGCACACCTCCGGCACGACGAGCCGGCCCAAGGGCGTGCCGCTGAAGCAGCGCAACCTGGCGGCGTCGGTGCGGAACATCCGGTCCGTGTACCGCCTCGCCGAGACGGACGCCACGGTGGTGGTGCTGCCGCTGTTCCACGTGCACGGGCTCCTCTGCGCCCTGCTGAGCTCGCTGGCTTCGGGCGCGTCCGTGGCGCTGCCGGCGGCGGGGCGGTTCTCGGCGTCCACGTTCTGGGCCGACATGCGGGCGTCGGGCGCCACCTGCTGCAGCGCGTCGCTGGCGCCGGCGATCCTGGAGCGGCTGGAGGCGGCGTTCAGCGCGCCGGTGCTGGAGGCGTACGCGATGACGGAGGCGTCGCACCTGATGACGTCAAACCCGCTGCCGGAGGACGGGCCACGGAAGCCCGGGTCAGTGGGGCGCGCGGTGGGGCAGGAGCTGGCGGTGCTGGACGAGGAGGGGCGGCTCGTGGCGGCGGGGAGCCCCGGGGAGGTGTGCGTCCGCGGGGACAACGTGACGGCGGGTTACAAGGGGAACCCAGAGGCGAACGAGGCGGCGTTCCGGTTCGGGTGGTTCCACACGGGGGACATCGGCGTGGTGGATGACCAAGGGTACGTCCGGCTGGTGGGGCGCATCAAGGAGCTCATCAACCGCGGCGGGGAGAAGATCTCCCCGATCGAGGTGGACGCCGTGCTGCTGGGCCTCCCCGGTGTGGCGCAGGCGGTGTCGTTCGGGGTGCCTGACGACAAGTACGGGGAGGAGATCAACTGTGCGGTGATCCCGCGGGACGGGTCGGCGCTGCGGGAGGAGGAGGTGCTGGCGCACTGCCGGCGGAACCTGGCGTCGTTCAAGGTGCCCAAGAAGGTGTTCATCACGGACGACCTGCCCAAGACGGCCACCGGAAAGATCCAGCGCCGCATCGTGGCGCAGCACTTCGTGCAGCCGACCAGTGCCTGA(SEQ ID NO.:4)。

The present invention also includes polypeptides or proteins having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, e.g., 99%) homology to the sequences shown in SEQ ID NO. 1 or 2 of the present invention and having the same or similar functions.

The "same or similar functions" mainly refer to: "modulating agronomic traits in crops (such as maize), such as reducing or increasing oxalic acid content in plants".

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The present invention also includes oxalate metabolism-related enzyme protein fragments and analogs having oxalate metabolism-related enzyme protein activity. As used herein, the terms "fragment" and "analog" refer to a polypeptide that retains substantially the same biological function or activity of the native SPL7 protein of the invention.

The polypeptide fragment, derivative or analogue of the invention may be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

In the present invention, the polypeptide variant is an amino acid sequence shown in SEQ ID NO. 1 or 2, a derivative sequence obtained by several (usually 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-10) substitutions, deletions or additions of at least one amino acid, and one or several (usually less than 20, preferably less than 10, and more preferably less than 5) amino acids added at the C-terminal and/or N-terminal. For example, in the protein, when the performance similar or similar amino acid substitution, usually does not change the protein function, C terminal and/or \ terminal addition of one or several amino acids usually does not change the protein function. These conservative changes are preferably made by making substitutions according to Table A.

TABLE A

In a preferred embodiment, the polypeptide of the invention refers to a polypeptide having the sequence of SEQ ID No. 1 or 2 that modulates an agronomic trait (e.g., reducing or increasing oxalic acid content in a plant) of a crop (e.g., maize). Also included are variants of the sequence of SEQ ID No. 1 or 2 having the same function as the oxalate metabolism-related enzyme. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of oxalate metabolism-related enzymes.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA of an oxalate metabolism-related enzyme, and polypeptides or proteins obtained using antisera raised against oxalate metabolism-related enzymes. The invention also provides other polypeptides, such as fusion proteins comprising an oxalate metabolism-related enzyme or fragment thereof. In addition to almost full-length polypeptides, the invention also includes soluble fragments of oxalate metabolism-related enzymes. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the sequence of the oxalate metabolism-related enzyme.

The invention also includes analogs of the claimed proteins that differ from the native SEQ ID NO. 1 or 2 by amino acid sequence differences, by modifications that do not affect the sequence, or by both, analogs of these proteins include natural or induced genetic variants, induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to a mutagen, by site-directed mutagenesis, or other known biological techniques.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetoxylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those performed during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

The present invention also provides a polynucleotide sequence encoding an oxalate metabolism-related enzyme or a variant thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or artificially synthesized DNA, the DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID NO.3 or 4 or may be a degenerate variant.

The term "polynucleotide encoding a mutein" may be a polynucleotide comprising a polynucleotide encoding a protein of the invention, or may also comprise additional coding and/or non-coding sequences. In the present invention, a preferred polynucleotide sequence encoding an oxalate metabolism-related enzyme is shown in SEQ ID No.3 or 4.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention, fragments, analogues and derivatives thereof. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially changing the function of the oxalate metabolism-related enzyme encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) with the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.

The proteins and polynucleotides of the invention are preferably provided in isolated form, more preferably, purified to homogeneity.

It is to be understood that while the genes encoding the oxalate metabolism-related enzymes of the invention are preferably from maize, other genes from other plants that are highly homologous (e.g., have greater than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) to the genes encoding the oxalate metabolism-related enzymes of maize are also within the contemplation of the invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The full-length sequence of the polynucleotide of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

Methods for amplifying DNA/RNA using PCR techniques are preferably used to obtain the polynucleotides of the invention. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The method of the invention

As described above, based on the above findings concerning oxalate metabolism-related enzymes or genes encoding the same (e.g., OCD1, O7), the present inventors further provide a method for modulating agronomic traits in plants, such as reducing or increasing the content of oxalate in plants.

The methods of the invention achieve regulation of an agronomic trait, such as oxalate content in a plant, by increasing or decreasing expression of an oxalate metabolism-related enzyme or a gene encoding the same, and/or increasing or decreasing activity of an oxalate metabolism-related enzyme.

In a preferred embodiment, the method comprises the steps of:

(a) providing a plant;

(b) identifying an oxalate metabolism-related enzyme in the plant;

Method for improving plant traits

The invention also provides a method for improving plant traits, which comprises the following steps:

The main advantages of the invention include:

(1) the invention discovers a key enzyme (oxalate metabolism related enzyme, OCD1 and O7) of an oxalate degradation pathway capable of obviously regulating and controlling the oxalate content in plants for the first time.

(2) The invention clones the coding gene of oxalyl coenzyme A decarboxylase in the corn oxalate metabolism process and verifies the biochemical function of the oxalate decarboxylase in detail; the invention also verifies the biochemical function of the corn oxalyl coenzyme A synthetase. The research of the invention clearly clarifies the first two steps of reactions of the maize oxalic acid degradation pathway, increases the understanding of the maize oxalic acid degradation pathway, and provides an important theoretical basis for accurately regulating and controlling the oxalic acid level of maize and other crop vegetables in the future.

(3) The invention firstly discovers that the OCD1 gene can catalyze oxalyl CoA to generate formyl CoA, and the O7 gene can catalyze oxalyl CoA to generate oxalyl CoA.

(4) The invention discovers for the first time that the oxalate metabolism-related enzyme can significantly reduce the oxalate content in plants (at least 10%, preferably 20%, more preferably 50%).

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, reagents and materials in the examples of the present invention are commercially available products.

EXAMPLE 1 acquisition of maize ocd1 mutant

One seed, numbered UFMu06493, was obtained from the international corn genetic improvement center during the course of the study (W22 background). After sub-species selfing, the grain on one ear exhibited a clear floury endosperm phenotype (fig. 1A) with a 1: 3, separating. The identification found that UFMu06493 seed produced a mutant phenotype that is not linked to the predicted insertion site, suggesting that the phenotype is caused by another gene mutation. We named this powdery mutant ocd1-1 (see below). The mutant seeds were observed on a light box to be opaque (FIGS. 1B and 1C). Cross-sections of the grain showed that the ocd1-1 endosperm was completely loose white (FIG. 1E) while the wild-type control grain was tightly transparent around and slightly white in the central region (FIG. 1D). Scanning electron microscopy showed that the starch granules in the peripheral region of endosperm were tightly packed with proteosome (Wu and Messing, 2010), and the ocd1-1 kernel starch granules were naked and loose (FIGS. 1F and 1G).

Example 2 cloning and characterization of the maize Ocd1 Gene

The Ocd1 gene was located on the long arm of chromosome eight using the F2 population obtained by crossing with the B73 inbred line and SNP (single nucleotide polymorphism) markers (fig. 2A). SSR and Indel markers were then used for fine localization. By 597 single DNA samples, the Ocd1 gene was mapped to a region of 210kb between the markers SSR17.80 and SSR18.21 (FIG. 2A). Based on the B73 genome sequence in the MaizeGDB database, this interval contains four protein-encoding genes GRMZM2G080722, GRMZM2G080521, GRMZM2G175253, and GRMZM2G175171 (fig. 3A). PCR amplification and sequencing showed a 1.4kb transposon (Mutat) element (Mu8) in the first exon (+297-bp relative to the start codon) of GRMZM2G175171 (FIG. 2B), with no changes in the other three genes. To confirm that the ocd1-1 phenotype is linked to GRMZM2G175171, four primers were designed. GRMZM2G175171 specific primers P1 and P2, Mu8

specific primers

810F and 810R. The results show that in homozygous inserted seeds P1+810R and P2+801F can be amplified, P1+ P2 amplification produces bands containing the inserts. Whereas control wild type seeds did not have a Mu8 insertion (fig. 2C), indicating that the Mu8 insertion on GRMZM2G175171 is linked to the floury endosperm phenotype. To further confirm that GRMZM2G175171 is a gene causing a mutation, we obtained the strains UFmu-06550 and UFmu-02221 from the International maize genetic improvement center with an additional transposon insertion on this gene. We named these two mutants ocd1-2 and ocd 1-3. PCR amplification and sequencing show that the two mutants have transposon insertions in +94bp and +540bp of the first exon of GRMZM2G175171 gene. The transposon insertions at the two loci were closely linked to the phenotype of the floury endosperm of the selfed progeny of the heterozygous plants (fig. 2D). Semi-quantitative RT-PCR showed that expression of GRMZM2G175171 could not be detected in none of the ocd1-1, ocd1-2 and ocd1-3 mutants, indicating that all three mutants were knock-out mutants (FIG. 2E).

Example 3 validation of maize Ocd1 Gene allelic hybridization

To further verify that the GRMZM2G175171 mutation is responsible for the pink nature of the ocd1 mutant. We identified heterozygous backgrounds for ocd mutants. We conferred pollen from ocd1-2/+ and ocd1-3/+ on the ear of ocd1-1/+ while selfing. At 45 days post pollination harvest, both the ears after ocd1-2/+ and ocd1-3/+ selfing (FIG. 3A) and after crossing to ocd1-1/+ ear were found to separate the floury grains (FIG. 3B), with normal and floury seeds separated at a 3:1 ratio. Allelic hybridization experiments further confirmed that the GRMZM2G175171 mutation is the gene that causes flourishing in the ocd1 endosperm.

Example 4 maize Ocd1 Gene is widely expressed in maize

The spatiotemporal expression pattern of Ocd1 was analyzed by quantitative PCR. Ocd1 was found to be expressed in different tissues of maize and was higher in roots, cusps, filaments and seeds 10(10DAP) days after pollination (FIG. 4A). The expression of the Ocd1 gene during seed development was further investigated. We extracted seed and endosperm RNA at different times after pollination. Quantitative PCR analysis showed that Ocd1 expression increased gradually after pollination, peaking at 10DAP (fig. 4B). Thereafter from 12DAP to 32DAP, Ocd1 slowly decreased in endosperm and at 36DAP, expression of Ocd1 appeared to increase again (fig. 4B).

Example 5 Domain, sequence and subcellular localization analysis of ZmOCD1 protein

Ocd1 contains two exons and an intron that encodes a protein consisting of 575 amino acid residues. Search of Pfam database using the full-Length amino acid sequence of ZmOCD 1: (http://pfam.xfam.org) The protein is shown to contain three conserved domains, the N-terminal thiamine pyrophosphate (ThDP) binding domain, the middle ThDP and magnesium ion binding domain and the C-terminal ThDP binding and catalytic active domain (fig. 5A). To gain more insight into the function of the ZmOCD1 protein, we also used the ZmOCD1 protein sequence to perform a query at the NCBI database, identifying the homologous proteins of ZmOCD1 in Arabidopsis and oxalic acid producing Bacillus (Oxalobacter formigenes) (AtOCD1 and OfOCD1, respectively). The alignment found that ZmOCD1 and AtOCD1 and OfOCD1 were in sequenceThe similarity was very high (83% and 62%, respectively). Structural biology analysis identified ThDP of OfOCD1, key amino acid tastes of Adenosine Diphosphate (ADP) and magnesium ion binding, and the active center of the enzyme (Berthold et al, 2005). We compared these key amino acid sites and found that the ThDP binding site and the ADP binding site were identical in the three proteins, OfOCD1, AtOCD1 and ZmOCD1 (FIG. 5B). Both magnesium ion binding sites and active site amino acids of ZmOCD1 and OfOCD1 were the same or similar (fig. 5B). The high similarity of ZmOCD1 to the sequence of OfOCD1 suggests that ZmOCD1 may also be involved in oxalate degradation as an Oxalyl-CoA Decarboxylase (Oxalyl-CoA decarbonylase 1). Therefore, we named its coding gene GRMZM2G175171 as ZmOcd1 and its mutant as ocd1 mutant. Next, the subcellular localization of ZmOCD1 protein was studied and, as found by transient transformation of arabidopsis protoplasts and tobacco epidermal cells by fusion of GFP fluorescent protein, GFP fluorescence was predominantly localized in the cytoplasm (fig. 5C), indicating that ZmOCD1 is a cytoplasmic-localized protein that fits the subcellular localization pattern of most enzymes.

Example 6 purification of ZmOCD1 protein expression and chemical Synthesis of oxalyl-CoA as substrate

The oxalate degradation process is divided into four steps, the first two steps are reaction of catalyzing and connecting oxalic acid and coenzyme A by oxalyl coenzyme A synthetase to form oxalyl coenzyme A, and ZmOCD1 protein catalyzes and degrades oxalyl coenzyme A to generate formyl coenzyme A and carbon dioxide (figure 6A). To investigate whether ZmOCD1 indeed functions as an oxalyl decarboxylase in the oxalate degradation pathway, we constructed and purified a 6 × histidine (ZmOCD1-His) tagged protein. Empty vector containing no recombinant sequence was purified for use as an experimental control (FIG. 6B). Another key element of the enzymatic activity reaction is the substrate, and we searched for oxalyl-coa that was not commercially available from many companies. By searching the literature, a method for chemically synthesizing oxalyl-CoA was found (Quayle, 1962). We bought relevant reagents from the literature and performed chemical synthesis reactions. The LC-MS/MS detection of the product shows that the synthesized oxalyl-CoA has a specific peak (FIG. 6C), and the peak pattern of the secondary mass spectrum also accords with the mode of oxalyl-CoA fragment (FIG. 6D), which indicates that the enzyme activity experiment can be carried out after the substrate of ZmOCD1 protein, oxalyl-CoA, is successfully synthesized.

Example 7 ZmOCD1 degradation of oxalyl-CoA to formyl-CoA

Purified ZmOCD1-His protein or control protein (vector control), synthetic oxalyl-CoA and other cofactors were mixed well and incubated at 37 ℃. After 15 min incubation the reaction was stopped with methanol and the product was detected by LC-MS/MS. The results showed that in the reaction system to which ZmOCD1-His protein was added, oxalyl-CoA (m/z) as a substrate⁺840.1) a new peak (m/z) was generated in addition to the corresponding peak at the nuclear to cytoplasmic ratio 796.1 (formyl-CoA)⁺796.1) (fig. 7A). Further secondary mass spectrometry ion fragmentation showed that the product was similar to the formyl-CoA fragmentation pattern, indicating that formyl-CoA was produced in the reaction system for ZmOCD1-His protein (FIG. 7C). Whereas the control protein incubated with substrate was only at oxalyl-CoA (m/z)⁺840.1) one peak at position and no peak corresponding to formyl-CoA was generated (FIG. 7B). At the same time we found that the difference between the nuclear to cytoplasmic ratio of the two peaks is equal to 44.0, which is exactly the nuclear to cytoplasmic ratio of carbon dioxide (m/z 44.0). In addition, we performed in vivo enzyme activity assays, which showed that wild-type (WT) endosperm could catalyze the production of formyl-CoA from oxalyl-CoA (FIG. 7D), whereas ocd1-1 mutant did not produce formyl-CoA (FIG. 7E). The above in vitro and in vivo enzyme activity experiments show that ZmOCD1 can degrade oxalyl-CoA to formyl-CoA and carbon dioxide.

Example 8 corn O7 degradation of oxalate to oxalyl-CoA

Corn ZmO7 is a homologous protein of Arabidopsis thaliana oxalyl-CoA synthetase (Miclaus et al, 2011; Wangt al, 2011), however, to date, the function of ZmO7 in corn has not been validated, nor has maize oxalyl-CoA synthetase been reported. To demonstrate that ZmO7 in maize is an oxalyl-coa synthetase, capable of linking oxalate to coa and thereby degrading oxalate to produce oxalyl-coa. ZmO7 protein was expressed and purified (FIG. 6A), and enzymatic activity was carried out using oxalic acid and coenzyme A as substrates. LC-MS/MS analysis of the reaction product shows that ZmO7 protein can be in nuclear-to-cytoplasmic ratio (m/z)^-838.1) generated a new peak (fig. 8A) corresponding to oxalyl-coa. ZmO7 ProteinThe amount of oxalyl-coa was directly proportional to the oxalate concentration (fig. 8A). The secondary mass spectrometric ion fragmentation showed that the product was in a similar pattern to the oxalyl-coa fragmentation (fig. 8B), indicating that ZmO7 is indeed an oxalyl-coa synthetase that degrades oxalic acid to produce oxalyl-coa.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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gtggctctgg tcatcggtgc taggcttaat tggttgcttc actttggcga gccacccaag 900

tggtccaagg atgttaagtt cattcttgtt gacgtctccg aggaggagat tgagctccgg 960

aagccacatg tggggattgt tggggacgcg aagagagtaa ctgagctgat caaccgggag 1020

atcaaggaca atccattctg cctggcaagg tcgcacccat gggtcgaagc gatcactaag 1080

aaggccaatg acaatgttct taagatggag gcgcagctag tgaaggatgt tgtgccattc 1140

aatttcatga caccattgcg gatcatccgg gatgcgatcc ttgccgaggg gagtcctgct 1200

ccaatagtgg tctcagaagg ggcaaatacc atggatgttg gcagggctgt gcttgtgcag 1260

aatgaaccaa ggacaagact ggatgcaggg acatggggga cgatgggggt tggattgggg 1320

tactgtattg cggctgcagc agctgaacct gaacgactcg tggttgctgt ggagggcgat 1380

tctggatttg gcttcagtgc aatggaggtt gagacgttgg tgaggtacca gctgcctgtt 1440

gttgtaatcg ttttcaacaa caacggtgtc tacggcgggg accggagaag ccctgacgag 1500

ataaccggac cctacaaagg tgacccagcc ccaacctcat ttgttccggc agcagggtac 1560

cataagatga tggaggcttt tggagggaaa gggtatcttg tggagacacc agacgagctc 1620

aaatctgccc tttcagaatc gttccgcgcc aggaaaccgg cggtgattaa tgttatcatt 1680

gatccttacg ccggcgcaga gagcggtcga atgcagcaca agaactga 1728

<210>4

<211>1494

<212>DNA

<213> corn (Zea mays)

<400>4

atggctatgg cgacgacgga ggccaccacg ctgacggcgc tgctcaagga ggcggcggcc 60

gccttcccga cccgccgcgc agttgccgta ccgggcaggc tggagctcac gcacgccgcc 120

ctcgacgctc tcgtcgacgc ggcggccgca cgactcgccg ccgacgccgg agttctcccg 180

ggccacgtcg tcgcgctttc cttccccaac accgtcgagc tggtgatcat gttcctggcg 240

gtaatccgcg cgcgcggcgt ggcggcgccg ctcaacccgg cctacacgca ggaggagttc 300

gagttctacc tctccgactc ggaggcgcgc ctcctcgtca ccaacgcgga gggcaacgcg 360

gcggcgcagg cggccgctgc caagctcggc ctcgcccacg ccgccgccag cctccacgac 420

gcggccggcc ctgtccacct cgccggcctc ccggcccatg ctccggagaa cgggtcccac 480

ggcggcggcg cggcgggctc tctctccccc aacgacccgt cggacgtggc cctgttcctg 540

cacacctccg gcacgacgag ccggcccaag ggcgtgccgc tgaagcagcg caacctggcg 600

gcgtcggtgc ggaacatccg gtccgtgtac cgcctcgccg agacggacgc cacggtggtg 660

gtgctgccgc tgttccacgt gcacgggctc ctctgcgccc tgctgagctc gctggcttcg 720

ggcgcgtccg tggcgctgcc ggcggcgggg cggttctcgg cgtccacgtt ctgggccgac 780

atgcgggcgt cgggcgccac ctgctgcagc gcgtcgctgg cgccggcgat cctggagcgg 840

ctggaggcgg cgttcagcgc gccggtgctg gaggcgtacg cgatgacgga ggcgtcgcac 900

ctgatgacgt caaacccgct gccggaggac gggccacgga agcccgggtc agtggggcgc 960

gcggtggggc aggagctggc ggtgctggac gaggaggggc ggctcgtggc ggcggggagc 1020

cccggggagg tgtgcgtccg cggggacaac gtgacggcgg gttacaaggg gaacccagag 1080

gcgaacgagg cggcgttccg gttcgggtgg ttccacacgg gggacatcgg cgtggtggat 1140

gaccaagggt acgtccggct ggtggggcgc atcaaggagc tcatcaaccg cggcggggag 1200

aagatctccc cgatcgaggt ggacgccgtg ctgctgggcc tccccggtgt ggcgcaggcg 1260

gtgtcgttcg gggtgcctga cgacaagtac ggggaggaga tcaactgtgc ggtgatcccg 1320

cgggacgggt cggcgctgcg ggaggaggag gtgctggcgc actgccggcg gaacctggcg 1380

tcgttcaagg tgcccaagaa ggtgttcatc acggacgacc tgcccaagac ggccaccgga 1440

aagatccagc gccgcatcgt ggcgcagcac ttcgtgcagc cgaccagtgc ctga 1494

Claims

1. Use of a substance selected from the group consisting of: an oxalate metabolism-related enzyme or a coding sequence thereof, or an enhancer or inhibitor thereof, for use in modulating an agronomic trait in a plant, the agronomic trait comprising oxalate content in the plant, wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

2. Use of an oxalate metabolism-related enzyme or a coding sequence thereof, or a promoter thereof, for (a) reducing oxalate content in a plant; and/or (b) preparing a composition or formulation for reducing oxalic acid content in a plant; wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

3. Use of an inhibitor of an oxalate metabolism-related enzyme or a coding sequence thereof for (a) increasing oxalate content in a plant; and/or (b) preparing a composition or formulation that increases the total oxalic acid content of the plant; wherein the oxalate metabolism-related enzyme is selected from the group consisting of: OCD1, O7, or a combination thereof.

4. A method of regulating oxalic acid content in a plant, comprising the steps of:

(a) providing a plant;

(b) identifying an oxalate metabolism-related enzyme in the plant;

5. A method of improving a trait in a plant comprising the steps of:

6. The method of claim 5, wherein the agronomic trait of the improved plant comprises modulating oxalate content in the plant.

7. A method of making a transgenic plant having reduced oxalate content in the plant, comprising the steps of:

8. An in vitro enzymatic reaction method comprising the steps of:

wherein the oxalate metabolism-related enzyme comprises O7.

9. The method of claim 8, wherein the method further comprises the steps of:

wherein the oxalate metabolism-related enzyme comprises OCD 1.

10. An in vitro enzymatic reaction method comprising the steps of:

wherein the oxalate metabolism-related enzyme comprises OCD 1.