Leguminous molybdenum transporter gene and application thereof

Abstract

The invention provides a leguminous molybdenum transporter gene and application thereof. Molybdenum transporter genes obtained through identification from leguminous plants are named as MOT1.1 and MOT1.2 for the first time, participate in regulation and control of natural variation of the molybdenum content of soybeans, and influence agronomic traits of the soybeans by influencing synthesis of auxin. Targeted regulation of the gene is beneficial to improvement of characters of leguminous plants, and analysis of different haplotypes of the gene can guide optimized planting of the leguminous plants and application of molybdenum fertilizer. The new discovery of the invention provides a new way for improvement and species identification of leguminous plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and more specifically, to a leguminous molybdenum transporter gene and application thereof. Background Art

[0002] Molybdenum (Mo) is a very important trace element for plants. In plants, molybdenum can participate in important biological processes such as nitrogen assimilation, purine metabolism, nitrite detoxification and plant hormone synthesis as a molybdenum cofactor (Moco). However, for most plants, very little molybdenum is needed to maintain normal growth, and molybdenum is basically not a growth-limiting nutrient. Therefore, for most plants, except for extreme soil environmental conditions, molybdenum deficiency is not a common problem.

[0003] As an important source of plant protein for humans and animals, legumes are significantly different from most plants. Studies have shown that the high demand for molybdenum in legumes is due to the involvement of molybdenum in the biological nitrogen fixation process. Compared with other molybdenum enzymes, the nitrogenase activity of rhizobia is particularly dependent on the molybdenum content in the nodules. Molybdenum deficiency is very common for most legumes, especially in acidic soil environments. Therefore, the application of molybdenum foliar fertilizer is a universal and very effective agricultural production measure to increase the yield of legume crops. A more common explanation for this is that legumes need molybdenum to meet their biological nitrogen fixation needs. However, since the existing evidence has not proved that there is molybdenum transport from the aboveground to the roots in legumes, this explanation is not completely consistent with the fact that soybean yield can be increased by applying molybdenum foliar fertilizer in agricultural production. In addition, whether there is natural variation in molybdenum absorption and utilization in legumes, and the genetic mechanism that regulates natural variation, remains unresolved. More importantly, whether there is natural variation in molybdenum utilization efficiency and how this variation affects the agronomic traits of legumes is still unknown.

[0004] As one of the most important leguminous crops, soybeans require Mo fertilizers to maximize their production potential. Therefore, the natural variation in Mo use efficiency is particularly important for soybeans. Summary of the invention

[0005] The purpose of the present invention is to provide a leguminous molybdenum transporter gene and application thereof.

[0006] In the first aspect of the present invention, a method for improving the traits of legume plants is provided, comprising increasing the expression or activity of MOT1.1 and / or MOT1.2 in legume plants; wherein the improved traits include those selected from the following groups: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

[0007] In one or more embodiments, in (i), increasing the molybdenum content includes: increasing the molybdenum content in the aboveground part; preferably, the aboveground part includes: grains or leaves.

[0008] In one or more embodiments, in (i), the improved molybdenum transport capacity includes: promoting the transport of molybdenum from the roots to the aboveground parts (including improving the transport of xylem flow), and promoting the absorption of molybdenum from the soil to the roots.

[0009] In one or more embodiments, in (ii), the yield improvement includes: increasing the size of grains, increasing the number of branches, increasing the 100-grain weight, or increasing the number of pods.

[0010] In one or more embodiments, in (iii), increasing biomass includes: increasing plant height, increasing leaf size, or increasing epidermal cell volume.

[0011] In one or more embodiments, the method of increasing the expression or activity of MOT1.1 and / or MOT1.2 in plants includes: using MOT1.1 and / or MOT1.2 genes or expression constructs or vectors containing the genes to transfer into plants; expressing an enhanced promoter or tissue-specific promoter to increase the expression efficiency of MOT1.1 and / or MOT1.2 genes in plants; using enhancers to increase the expression efficiency of MOT1.1 and / or MOT1.2 genes in plants.

[0012] In one or more embodiments, increasing the expression or activity of MOT1.1 and / or MOT1.2 in the plant comprises increasing its expression in the plant root stele or nodule vascular system.

[0013] In one or more embodiments, the increase or improvement indicates a significant increase or improvement, such as an increase or improvement of 20%, 40%, 60%, 80%, 90% or more.

[0014] In one or more embodiments, the plant is a plant that low-expresses (significantly lower than the average value of gene expression of the species) or does not express the MOT1.1 and / or MOT1.2 gene.

[0015] In one or more embodiments, the yield is increased by about 25% or more.

[0016] In another aspect of the present invention, the application of MOT1.1 and / or MOT1.2 up-regulators is provided for improving the traits of leguminous plants; wherein the improved traits include those selected from the following groups: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

[0017] In one or more embodiments, the up-regulator includes but is not limited to: an expression construct or vector for recombinant expression of MOT1.1 and / or MOT1.2, a chemical up-regulator of MOT1.1 and / or MOT1.2, and an up-regulator that promotes the gene promoter driving ability of MOT1.1 and / or MOT1.2.

[0018] In another aspect of the present invention, a method for screening substances that improve the traits of legumes is provided, comprising: (1) adding a candidate substance to a system containing MOT1.1 and / or MOT1.2; (2) detecting the system and observing the expression or activity of MOT1.1 and / or MOT1.2 in the system of (1); if the candidate substance promotes the expression or activity of MOT1.1 and / or MOT1.2, the candidate substance is a substance that improves the traits of legumes; wherein the improved traits include those selected from the following groups: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

[0019] In one or more embodiments, a control group is further included to clearly distinguish the difference between the expression or activity of MOT1.1 and / or MOT1.2 protein in the test group and that in the control group.

[0020] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules designed for MOT1.1 and / or MOT1.2 proteins or their encoding genes or their upstream or downstream proteins or genes (such as upregulators, small molecule compounds, gene editing constructs, etc.).

[0021] In another aspect of the present invention, the application of MOT1.1 and / or MOT1.2 is provided, for use as markers for identifying the traits of legumes; or for targeted selection of legumes for improved traits; wherein the traits include molybdenum content, molybdenum transport capacity, yield, biomass or IAA content; the improved traits include: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

[0022] In one or more embodiments, the identification of the traits of legumes includes: analyzing the expression of MOT1.1 and / or MOT1.2 in the plant; if the expression is normal or highly expressed (significantly higher than the average value of gene expression of the species), the legumes are normal or have improved traits; if the expression is reduced (significantly lower than the average value of gene expression of the species), the legumes do not have normal or improved traits.

[0023] In one or more embodiments, the targeted selection of legumes with improved traits includes: analyzing the expression of MOT1.1 and / or MOT1.2 in the plants. If the expression is high (significantly higher than the average value of gene expression of the species), the legumes have improved traits.

[0024] In one or more embodiments, the GmMOT1.1 and GmMOT1.2 are expressed in the soybean root stele and nodule vascular system.

[0025] In one or more embodiments, the GmMOT1.1 and GmMOT1.2 are localized in vesicles and / or cell membranes.

[0026] In one or more embodiments, the MOT1.1 and / or MOT1.2 is MOT1.1 from the legume family; preferably, the amino acid sequence of the MOT1.1 includes proteins selected from the following groups: (a) proteins selected from the amino acid sequences shown in SEQ ID NOs: 8-11; (b) proteins derived from (a) that have the protein function of (a) and are formed by replacing, deleting or adding one or more (such as 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequences of SEQ ID NOs: 8-11; (c) proteins derived from (a) that have more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology with the protein sequence defined in (a) and have the protein function of (a); or (d) protein active fragments defined in (a), or proteins formed by adding tag sequences, enzyme cleavage sequences, and reporter proteins to both ends of the protein.

[0027] In one or more embodiments, the amino acid sequence of the MOT1.2 protein includes proteins selected from the following groups: (a') proteins such as SEQ ID NO:12-14 amino acid sequences; (b') proteins selected from SEQ ID NO:12-14 amino acid sequences that are formed by substitution, deletion or addition of one or more (such as 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues, and derived from (a) and having the function of (a) protein; (c') proteins derived from (a) that have more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology with the protein sequence defined by (a') and have the function of (a) protein; or (d') protein active fragments defined by (a'), or proteins formed by adding tag sequences, enzyme cleavage sequences, and reporter proteins to both ends of the fragments.

[0028] In another aspect of the present invention, a method for identifying the traits of leguminous plants is provided, comprising: analyzing the haplotype of the MOT1.1 and / or MOT1.2 genes in the plants; if the protein encoding the amino acid sequence of SEQ ID NO:8, or the nucleotide sequence is such as SEQ ID NO:1, it is the GmMOT1.1-Hap1 haplotype; if the protein encoding the amino acid sequence of SEQ ID NO:9, or the nucleotide sequence is such as SEQ ID NO:2, it is the GmMOT1.1-Hap2 haplotype; if the protein encoding the amino acid sequence of SEQ ID NO:10, or the nucleotide sequence is such as SEQ ID NO:3, it is the GmMOT1.1-Hap3 / 4 haplotype; if the protein encoding the amino acid sequence of SEQ ID NO:11, or the nucleotide sequence is such as SEQ ID NO:4, it is the GmMOT1.1-Hap5 haplotype; if the protein encoding the amino acid sequence of SEQ ID NO:12, or the nucleotide sequence is such as SEQ ID NO:5, it is the GmMOT1.2-Hap1 haplotype; if the protein encoding the amino acid sequence of SEQ ID NO:13, or the nucleotide sequence is such as SEQ ID NO:6, it is the GmMOT1.2-Hap1 haplotype; NO:13 amino acid sequence of the protein, or nucleotide sequence such as SEQ ID NO:6, is GmMOT1.2-Hap2 / 3 / 5 haplotype; if the protein encoding SEQ ID NO:14 amino acid sequence, or the nucleotide sequence such as SEQ ID NO:7, is GmMOT1.2-Hap4 haplotype; if identified as Hap5 haplotype, the plant is a high-molybdenum haplotype, which is a plant with high molybdenum content in the aboveground part, easy to absorb molybdenum, and high yield; if identified as Hap4 haplotype, the plant is a low-molybdenum haplotype, which is a plant with low molybdenum content in the aboveground part, not easy to absorb molybdenum, and low yield; if identified as Hap1, Hap2 or Hap3 haplotype, the plant is a plant with aboveground molybdenum content, yield or molybdenum fertilizer application amount between Hap5 haplotype and Hap4 haplotype.

[0029] In one or more embodiments, the nucleic acid sequence is identified using methods including (but not limited to): sequencing, PCR amplification, restriction enzyme analysis, probe method, hybridization method, chip method, and allele polymorphism analysis.

[0030] In one or more embodiments, after identifying the characteristics of the legume plant, the process also includes analyzing the soil suitable for planting the legume plant; wherein, if it is identified as the Hap5 haplotype (high molybdenum haplotype), it is relatively suitable for planting in acidic soil (pH <6.5); if it is identified as the Hap4 haplotype, it is relatively suitable for planting in alkaline soil (pH>7.5); if it is identified as the Hap1, Hap2 or Hap3 haplotype, the plant is relatively suitable for planting in soil close to neutral (pH≈7).

[0031] In one or more embodiments, after identifying the characteristics of the legume plant, it also includes: analyzing whether the legume plant needs to be applied with molybdenum fertilizer; wherein, if it is identified as the Hap5 haplotype (high molybdenum haplotype), it does not need to be applied with molybdenum fertilizer (preferably, it does not need to be applied in soils that are not extremely molybdenum-deficient or extremely acidic); if it is identified as the Hap4 haplotype, it needs to be applied with molybdenum fertilizer (preferably molybdenum foliar fertilizer); if it is identified as the Hap1, Hap2 or Hap3 haplotype, the amount of molybdenum fertilizer required for the plant is between Hap5 and Hap4.

[0032] In one or more embodiments, the legume includes legumes selected from the group consisting of legumes expressing MOT1.1 and / or MOT1.2 genes; more preferably, including (but not limited to): soybeans, peas, lupines, beans, chickpeas, Medicago truncatula, Lotus japonica, Trifolium repens, and Ephedra sinica.

[0033] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Results of genome-wide association analysis of molybdenum content in soybean seeds.

[0035] A is the frequency distribution histogram of molybdenum content in seeds of 597 natural soybean varieties.

[0036] B is the Manhattan plot of genome-wide association analysis.

[0037] C is the quantile map of genome-wide association analysis.

[0038] D is a detailed enlarged view of the red box area in Figure B. SNPs exceeding the significance threshold are marked in blue.

[0039] E is the linkage disequilibrium analysis of the candidate interval 32.27kb-32.52kb on chromosome 17. The candidate genes GmMOT1.1 and GmMOT1.2 are shown as red vertical lines above the figure. Different degrees of red represent different linkage disequilibrium values ​​(r 2 ).

[0040] F indicates the polymorphic sites that cause promoter variation and protein variation of GmMOT1.1 and GmMOT1.2; arrows with different fillings represent different types of promoters.

[0041] G is the molybdenum content of seeds of natural soybean varieties with different GmMOT1.1 and GmMOT1.2 haplotypes.

[0042] H is the molybdenum transport capacity of different haplotypes of GmMOT1.1 and GmMOT1.2 in Saccharomyces cerevisiae.

[0043] I and J represent the expression levels of GmMOT1.1 (I) and GmMOT1.2 (J) in different natural varieties; GmELF1b is used as the internal reference gene.

[0044] Figure 2 To verify the experimental results that GmMOT1.1 and GmMOT1.2 are involved in the absorption of molybdenum and its transport from roots to aboveground parts.

[0045] A and B represent the molybdenum contents in leaves (A) and seeds (B) of the wild type (DN50) and mot1.1 / 1.2 double mutant, respectively.

[0046] C and D represent the molybdenum content in leaves (C) and seeds (D) of wild-type (DN50) and overexpression plants

[0047] E represents the content of molybdenum in the xylem flow of the wild type and the double mutant.

[0048] F and G represent the molybdenum content in leaves (F) and seeds (G) of grafted soybeans; SG: self-grafting; WT / mot1.1mot1.2: wild type as the aboveground part and the double mutant as the root; mot1.1mot1.2 / WT: double mutant as the aboveground part and the wild type as the root.

[0049] H represents the amount of molybdenum absorbed by the wild type (DN50) and the double mutant mot1.1 / 1.2.

[0050] I represents the ratio of molybdenum content in the roots and shoots of the wild type (DN50) and the double mutant mot1.1 / 1.2.

[0051] Figure 3 The tissue expression and subcellular localization results of GmMOT1.1 and GmMOT1.2 are shown.

[0052] AF represent the GUS staining results of the roots of pGmMOT1.1-GUS and pGmMOT1.2-GUS soybean transgenic plants. A and D are roots; B and E are cross sections of roots; E and F are nodules.

[0053] G is the result of observation of Arabidopsis leaf protoplasts expressing different haplotypes GmMOT1.1-GFP or GmMOT1.2-GFP under confocal microscope. Mitochondria were stained with mitochondrial specific dye MitoTracker TM For dyeing.

[0054] H shows the result of aggregation of GmMOT1.1-GFP and GmMOT1.2-GFP fluorescence signals after 1 h of BFA treatment.

[0055] I shows the results of localization of GmMOT1.1-GFP and GmMOT1.2-GFP fluorescence signals on the cell membrane after treatment with TyrA23 for 1 h.

[0056] Figure 4 The experimental results on the involvement of GmMOT1.1 and GmMOT1.2 in soybean yield regulation are shown.

[0057] A represents the 100-grain weight of natural soybean varieties with different haplotypes GmMOT1.1 and GmMOT1.2 under the same planting conditions. The figure shows the haplotypes of mot1.1 and mot1.2 combined (mot1.1 and mot1.2 are linked, based on the sequence segment analysis containing the two genes) grouped together (reference Figure 1 F).

[0058] B is the growth phenotype of the wild type and mutant grown in Shanghai area (pH 5.5) for eight weeks.

[0059] C represents the growth phenotype of the plant with inverted three leaves in G.

[0060] D and E show the phenotypes of pod number and yield per plant of wild type and double mutant, respectively.

[0061] FJ represents the plant height, branch number, yield per plant, pod number and 100-grain weight of the wild type (DN50) and the mot1.1 / 1.2 double mutant.

[0062] K is the growth phenotype diagram of the wild type (DN50) and GmMOT1.1-Hap5 overexpressing plants grown in Shanghai area (pH 5.5) for eight weeks.

[0063] L and M represent the phenotypes of pod number (l) and yield per plant (m) of wild type and overexpression plants, respectively.

[0064] NQ represents the yield per plant, plant height, number of pods and 100-grain weight of the wild type (DN50) and overexpression plants.

[0065] Figure 5 The experimental results show that GmMOT1.1 and GmMOT1.2 affect the synthesis of IAA (they themselves increase IAA synthesis, while the mutant plants reduce IAA synthesis) without affecting ABA and biological nitrogen fixation.

[0066] A is an optical microscope image of nail polish blots of epidermal cells of the wild type and double mutant. The scale bar is 50 μm.

[0067] B is the statistical results of epidermal cell size quantification of wild type and double mutant.

[0068] C shows the microscopic observation results of semi-thin sections of the second lobe of the wild type and double mutants. The scale bar is 50 μm.

[0069] D is the quantitative statistical results of the cross-sectional area of ​​mesophyll cells of the wild type and double mutant.

[0070] E is the results of IAA content detection in the shoot tips of the wild type and double mutants at four weeks old.

[0071] F is the rate of IAA synthesis catalyzed by aldehyde oxidase (AO) in wild type and double mutant within 4 weeks.

[0072] G is a staining band diagram of aldehyde oxidase activity in 14-day-old wild-type and double mutant soybean seedlings. The figure contains two groups of experiments, one group with indole-3-acetaldehyde (IAAld) added as a substrate, and the other group without IAAld added.

[0073] H is the histone H3 content to normalize the staining intensity.

[0074] I is the quantitative statistical results of aldehyde oxidase activity staining in wild-type and overexpression plants.

[0075] J is the rate of IAA synthesis catalyzed by aldehyde oxidase (AO) in wild-type and overexpression plants within 4 weeks.

[0076] K is the staining band of aldehyde oxidase activity in wild-type and overexpressed soybean seedlings at 14 days old. The staining intensity was normalized by histone H3 content.

[0077] L is the quantitative statistical results of aldehyde oxidase activity staining in wild-type and overexpression plants.

[0078] Figure 6 The correlation between the haplotype distribution of GmMOT1.1 and GmMOT1.2 and soil pH is shown, and the chi-square analysis of the distribution of different haplotypes in different soil pH regions is shown. NER: northern China; HHH: Huanghuaihai region; SR: southern China.

[0081] Figure 7 , sequence structure of the mot1.1 / 1.2 double mutant. DETAILED DESCRIPTION

[0082] Leguminous plants, such as soybeans, are high-molybdenum-demanding crops and usually require the application of molybdenum fertilizers to maximize their production potential; the genetic mechanism of natural variation in molybdenum content in leguminous plants and its effects on agronomic traits are still unknown in the art. After in-depth research, the inventors revealed for the first time newly identified molybdenum transporter genes from leguminous plants, named MOT1.1 and MOT1.2, which are involved in the regulation of natural variation in molybdenum content in soybeans and affect soybean agronomic traits by affecting auxin synthesis. Targeted regulation of the genes is beneficial to improving the traits of leguminous plants, and analysis of different haplotypes of the genes can guide the optimized planting of leguminous plants and the application of molybdenum fertilizers. The new findings of the present invention provide a new way to improve leguminous plants and identify species.

[0083] the term

[0084] As used in the present invention, the terms "up-regulate", "enhance", "improve", "increase", "promote", "enhance" and the like are interchangeable and should be used to mean an increase of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100% or more compared to a control such as a "control plant" or "control gene" or "control protein" as defined herein.

[0085] About "control plant", it is a routine part of experimental design to select suitable control plant, and can comprise corresponding wild-type plant or corresponding transgenic plant without target gene.Control plant is generally the same plant species or even the variety that is the same as plant to be evaluated or belongs to the same class.Control plant can also be the individual that lost transgenic plant because of separation.Control plant as used in the present invention refers not only to complete plant, but also to plant part, including seed and seed part.

[0086] As used in the present invention, the high expression or high activity refers to a statistically significant increase in expression or activity compared to the average value of the expression or activity of the same species or the same plant, such as an increase of 10%, 20%, 40%, 60%, 80%, 90% or more.

[0087] As used in the present invention, the low expression or low activity refers to a statistically significant decrease in expression or activity compared to the average value of the expression or activity of the same species or the same plant, such as a decrease of 10%, 20%, 40%, 60%, 80%, 90% or less.

[0088] Genes and plants

[0089] As used in the present invention, the "MOT1.1 gene" or "MOT1.1 protein (polypeptide)" refers to a gene or protein from soybean, a gene or polypeptide that is homologous to a gene or polypeptide derived from soybean, contains substantially the same structural domain, and has substantially the same function.

[0090] As used in the present invention, the "MOT1.2 gene" or "MOT1.2 protein (polypeptide)" refers to a gene or protein from soybean, a gene or polypeptide that is homologous to a gene or polypeptide derived from soybean, contains substantially the same structural domain, and has substantially the same function.

[0091] In the present invention, the MOT1.1 protein or MOT1.2 protein also includes their fragments, derivatives and analogs. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to protein fragments that substantially maintain the same biological function or activity of the polypeptide, which may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein with a substitution group in one or more amino acid residues, or (iii) a protein formed by the fusion of an additional amino acid sequence to this protein sequence, etc. According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. The biologically active fragments of the MOT1.1 protein and MOT1.2 protein can be applied to the present invention.

[0092] In the present invention, the term "MOT1.1 protein" refers to a protein selected from the sequence of SEQ ID NO: 8-11 having the activity of MOT1.1 protein. The term also includes variant forms of the sequence of SEQ ID NO: 8-11 having the same function as the MOT1.1 protein. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, the addition or deletion of one or several amino acids at the C-terminus and / or N-terminus generally does not change the function of the protein.

[0093] In the present invention, the term "MOT1.2 protein" refers to a protein of SEQ ID NO: 12-14 sequence having MOT1.2 protein activity. The term also includes variant forms of SEQ ID NO: 12-14 sequence having the same function as MOT1.2 protein. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus.

[0094] The present invention also includes polynucleotides (genes) encoding the polypeptides, for example, polynucleotides of the nucleotide sequences shown in any one of SEQ ID NOs: 1-4 or their degenerate sequences, which can encode the MOT1.1 proteins of SEQ ID NOs: 8-11; polynucleotides of the nucleotide sequences shown in SEQ ID NOs: 5-7 or their degenerate sequences, which can encode the MOT1.2 proteins of SEQ ID NOs: 12-14.

[0095] It should be understood that although the MOT1.1 gene and MOT1.2 gene of the present invention are preferably obtained from leguminous plants, especially soybeans, other genes obtained from other plants that are highly homologous to the soybean MOT1.1 gene and MOT1.2 gene (such as having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) or genes with degeneracy with the genes are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0096] The vector containing the coding sequence, and the host cell produced by genetic engineering using the vector or the polypeptide coding sequence are also included in the present invention. Methods well known to those skilled in the art can be used to construct a suitable expression vector.

[0097] Host cells are usually plant cells. Plants can generally be transformed using methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method, rice embryo transformation method, etc.; Agrobacterium method is preferred. Transformed plant cells, tissues or organs can be regenerated into plants using conventional methods to obtain plants with altered traits relative to the wild type.

[0098] As used in the present invention, the plants include but are not limited to plants selected from the following group: leguminous plants expressing MOT1.1 gene and MOT1.2 gene; preferably leguminous root nodule plants.

[0099] The legumes include (but are not limited to): edible plants such as soybean, broad bean, pea, mung bean, red bean, cowpea, kidney bean, broad bean, pigeon pea, peanut, etc.; feed such as alfalfa, astragalus, broad bean, yarrow, etc.; wood such as albizzia, Dalbergia, Gleditsia sinensis, euphorbia cerasifera, red bean, Sophora japonica, etc.; dyes such as horsethorn, Sophora japonica, indigo, sappanwood, etc.; gums, etc.; resins such as gum arabic, gum tragacanth, gum kohlrabi, etc.; fibers such as Indian hemp, kudzu vine, etc.; oils such as soybean, peanut, etc. It should be understood that, under the guidance of the technical solution of the present invention, it is easy for people in this field to think of changing the types of various legume crops to achieve the same or similar technical effects, and these changes are also included in the present invention.

[0100] MOT1.1, MOT1.2 haplotypes and their applications

[0101] The present invention also reveals that the molybdenum transporter gene exists in a variety of haplotypes in the soybean population. After analysis, the inventors divide them into: GmMOT1.1-Hap1 haplotype, GmMOT1.1-Hap2 haplotype, GmMOT1.1-Hap3 / 4 haplotype, GmMOT1.1-Hap5 haplotype; GmMOT1.2-Hap1 haplotype, GmMOT1.2-Hap2 / 3 / 5 haplotype, GmMOT1.2-Hap4 haplotype.

[0102] The inventors found that soybean varieties with the Hap5 genotype have the highest expression levels of MOT1.1 and MOT1.2 and the strongest molybdenum transport activity of the corresponding proteins, which further makes this type of soybean variety have the largest 100-grain weight and high yield. The present invention also reveals that the distribution of different haplotype soybean varieties in China has a significant correlation with soil pH.

[0103] The distribution of natural soybean varieties with different haplotypes of GmMOT1.1 and GmMOT1.2 in China is closely related to soil pH. Hap5 is mainly distributed in the southern acidic soil planting areas, while haplotype four (Hap4) is mainly distributed in the northern alkaline soil planting areas. They can be used as molecular markers to screen soybean varieties suitable for planting in different pH soil environments.

[0104] Based on the above new discovery of the inventors, the molecular characteristics of MOT1 can be analyzed to analyze the type and yield of plants and guide the planting of plants.

[0105] On the one hand, the molybdenum absorption / utilization of plants can be identified by analyzing the molecular characteristics of MOT1: if it is identified as the Hap5 haplotype, the plant is a high-molybdenum haplotype, which has a high molybdenum content in the aboveground part, is easy to absorb molybdenum, and has a high yield; if it is identified as the Hap4 haplotype, the plant is a low-molybdenum haplotype, which has a low molybdenum content in the aboveground part, is not easy to absorb molybdenum, and has a low yield; if it is identified as the Hap1, Hap2 or Hap3 haplotype, the plant has an aboveground molybdenum content, yield or molybdenum fertilizer application amount between the Hap5 haplotype and the Hap4 haplotype.

[0106] As used in the present invention, the "aerial part" is also called the "aerial part" and refers to part of the tissue of the plant, which is located above the ground or the surface of the culture solution when the plant is planted in the soil or cultured in the culture solution.

[0107] On the other hand, the soil suitable for planting the legume plant can be analyzed by analyzing the molecular characteristics of MOT1; if it is identified as the Hap5 haplotype (high molybdenum haplotype), it is relatively suitable for planting in acidic soil (pH<6.5); if it is identified as the Hap4 haplotype, it is relatively suitable for planting in alkaline soil (pH>7.5); if it is identified as the Hap1, Hap2 or Hap3 haplotype, the plant is relatively suitable for planting in soil close to neutral (pH≈7).

[0108] On the other hand, whether the legume plant needs to be fertilized with molybdenum can be analyzed by analyzing the molecular characteristics of MOT1; if it is identified as the Hap5 haplotype (high molybdenum haplotype), it does not need to be fertilized with molybdenum (preferably, it does not need to be fertilized in soils that are not extremely molybdenum-deficient or extremely acidic); if it is identified as the Hap4 haplotype, it needs to be fertilized with molybdenum (preferably molybdenum foliar fertilizer); if it is identified as the Hap1, Hap2 or Hap3 haplotype, the amount of molybdenum fertilizer required by the plant is between Hap5 and Hap4.

[0109] The method of obtaining DNA of the sample to be tested is a technique well known to those skilled in the art, for example, the conventional phenol / chloroform / isoamyl alcohol method can be adopted, or some commercially available DNA extraction kits can be adopted, and such kits are well known to those skilled in the art. Various methods known in the art can be used for analysis of nucleic acid sequences, including (but not limited to): sequencing, PCR amplification, restriction enzyme analysis, probe method, hybridization method, chip method, and allele polymorphism analysis method for identification of nucleic acid sequences.

[0110] Therefore, the polymorphic sites of GmMOT1.1 and GmMOT1.2 can be used as good molecular markers for soybean breeding in different pH agricultural production areas and for evaluating whether soybean varieties need additional molybdenum fertilizer to increase their yield. The molecular characteristics of MOT1 disclosed in the present invention play a very important role and application potential in the process of breeding high-nutrition and soil pH-adaptive soybean varieties.

[0111] Plant improvement applications

[0112] In an embodiment of the present invention, the molybdenum content of 597 naturally variable soybean varieties distributed worldwide was analyzed by the GWAS method. The results showed that there was a large variation between the molybdenum content of soybean seeds distributed worldwide, and the natural variation of soybean molybdenum content was very likely to be regulated by GmMOT1.1 and GmMOT1.2. The present invention points out that the changes in gene expression and protein function caused by gene polymorphism are the root causes of the natural variation of soybean molybdenum content. Weakly functional GmMOT1.1 and GmMOT1.2 lead to a significant reduction in the molybdenum element transported from the roots to the aboveground parts, which in turn leads to a significant reduction in yield during normal planting, which has not been observed in other species outside the Leguminosae. In addition, the present invention further reveals that the damage to growth and yield caused by these two gene defects is not through affecting nitrogen assimilation and biological nitrogen fixation processes, but through reducing aldehyde oxidase activity and IAA content. GmMOT1.1 and GmMOT1.2 can participate in the regulation of soybean yield by affecting the molybdenum content required in the IAA synthesis process.

[0113] Expression and localization analysis showed that GmMOT1.1 and GmMOT1.2 were expressed in the stele and nodule vascular system of soybean roots. GmMOT1.1 and GmMOT1.2 were localized in vesicles. Both of them were involved in the transport of molybdenum from roots to the aerial parts.

[0114] Mutation analysis showed that compared with the wild type, the molybdenum content in leaves and grains of the GmMOT1.1 and GmMOT1.2 double mutants was significantly reduced, and the plant height, number of branches, yield per plant, number of pods, and 100-grain weight of the mutants were significantly reduced. The aldehyde oxidase activity of the GmMOT1.1 and GmMOT1.2 double mutants was significantly reduced compared with the wild type, and the auxin content was reduced, which led to smaller cells and reduced yield of the mutants.

[0115] Overexpression analysis was also carried out in the examples of the present invention. By using the CaMV35S promoter to drive the overexpression of GmMOT1.1 and GmMOT1.2 in soybean, the molybdenum content in the aboveground part and grains of the transgenic strains can be significantly increased, and the 100-grain weight of the overexpressing plants is increased by about 30% compared with the wild type, further demonstrating that GmMOT1.1 and GmMOT1.2 have great application potential in agriculture.

[0116] Based on the new discovery of the present inventors, a method for improving plants is provided, the method comprising: increasing the expression or activity of MOT1.1 and MOT1.2 in plants, or promoting the interaction between MOT1.1 and MOT1.2; wherein the improved traits include those selected from the following group: increasing molybdenum content or molybdenum transport capacity, increasing yield, increasing biomass, and increasing IAA content.

[0117] It should be understood that according to the experimental data and regulatory mechanisms provided by the present invention, a variety of methods well known to those skilled in the art can be used to regulate the expression of the MOT1.1 and MOT1.2, and these methods are all included in the present invention.

[0118] In the present invention, substances that increase the expression or activity of MOT1.1 and MOT1.2 in plants, or promote the interaction between MOT1.1 and MOT1.2 include promoters, agonists, and activators. The "upregulation", "improvement", and "promotion" described herein include "upregulation", "promotion" of protein activity or "upregulation", "improvement", and "promotion" of protein expression. Any substance that can increase the activity of MOT1.1 and / or MOT1.2 proteins, increase the stability of MOT1.1 and / or MOT1.2 genes or proteins encoded therein, upregulate the expression of MOT1.1 and / or MOT1.2 genes, and increase the effective action time of MOT1.1 and / or MOT1.2 proteins can be used in the present invention as substances useful for upregulating MOT1.1 and / or MOT1.2 genes or proteins encoded therein. They can be compounds, small chemical molecules, and biological molecules. The biological molecules described herein can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0119] After knowing the functions of MOT1.1 and / or MOT1.2, they can be used as molecular markers for targeted screening of plants. Based on this new discovery, it is also possible to screen for substances or potential substances that regulate the molybdenum content, yield, biomass or IAA content of plants by regulating this mechanism.

[0120] Therefore, the present invention provides a method for screening substances that improve the traits of legumes, comprising: (1) adding a candidate substance to a system containing MOT1.1 and / or MOT1.2; (2) detecting the system and observing the expression or activity of MOT1.1 and / or MOT1.2 in the system of (1); if the candidate substance promotes the expression or activity of MOT1.1 and / or MOT1.2, then the candidate substance is a substance that improves the traits of legumes; wherein the improved traits include those selected from the following groups: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

[0121] Methods for screening substances acting on a protein or gene or a specific region thereof as a target are well known to those skilled in the art, and all of these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0122] After large-scale screening, a class of potential substances that specifically act on MOT1.1 and / or MOT1.2 and have regulatory effects on plant traits can be obtained.

[0123] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0124] Materials and methods

[0125] 1. Genome-wide association analysis method

[0126] The ion groups of 597 soybean natural varieties collected from all over the world were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). A genome-wide association study (GWAS) was conducted using a single nucleotide polymorphism (SNP) dataset obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The analysis included more than four million markers, with a homozygous frequency (MAF) greater than or equal to 5% and a missing rate less than 10%.

[0127] Association analysis was performed using a mixed model (MLM) using the Efficient MixedModel Association eXpedited (EMMAX) software package. EMMAX also calculated the pairwise genetic distance matrix, which was used as the variance-covariance matrix for the random effects. The significance threshold was calibrated using the whole genome significance cutoff method.

[0128] 2. Analysis of Molybdenum Transport Function

[0129] Yeast heterologous expression experiments verify the molybdenum transport function of different haplotypes of GmMOT1.1 and GmMOT1.2 proteins

[0130] (1) Construction of GmMOT1.1 and GmMOT1.2 yeast expression vectors

[0131] Primers were designed based on the CDS sequences of GmMOT1.1 and GmMOT1.2, and the CDS of the two genes corresponding to the five haplotypes of Hap1-Hap5 were cloned into the pDR196 yeast expression vector by homologous recombination. The correctly sequenced plasmid was transformed into the INVSc1 yeast strain by lithium acetate.

[0132] (2) Molybdenum transport capacity test

[0133] Inoculate the Saccharomyces cerevisiae strain containing the target plasmid into 5 mL of SD medium lacking molybdenum and Ura, and culture it in a shaker at 30°C and 220 rpm for 20 hours. Add 2 mM sodium molybdate mother solution to the culture medium until the molybdenum concentration in the culture medium reaches 200 nM, and culture it again in a shaker at 30°C and 220 rpm for 30 minutes. Centrifuge at 1500g for 3 minutes, discard the upper culture medium, and resuspend it in ice water. Centrifuge again at 1500g for 3 minutes, discard the supernatant, and repeat the washing 4 times. Dry the washed bacteria in a 65°C oven overnight. Detect the content of ions in yeast using ICP-MS.

[0134] 3. ICP-MS detection of sample ion group content

[0135] Take 2-3 mg of wild type (DN50), GmMOT1.1 and GmMOT1.2 double knockout mutants or grafted plant leaves, seeds, and nodules inoculated with rhizobia for 28 days, wash them once in 1mM EDTA solution, and then wash them 4 times in 18.2Ω deionized water, or directly take 30μl of xylem flow and put them into a clean glass test tube. Place the glass test tube containing the sample in a 65℃ oven to dry overnight. Take the glass tube that has been dried overnight out of the oven, and after cooling to room temperature, accurately weigh the dry weight of the sample to be tested with a precision balance. Add 1mL of 70% nitric acid containing internal standard In to the glass test tube. In a fume hood, place the glass test tube with nitric acid in a graphite digestion furnace and digest at 115℃ for 4 hours. After digestion, cool it at room temperature. Add 18Ω deionized water to the cooled digested sample to adjust the total volume to 10mL. After the sample is thoroughly mixed, 1700 μL of the sample is taken into a clean disposable sample plate. The sample ion group is detected by inductively coupled plasma mass spectrometry (ICP-MS).

[0136] 4. Tissue localization of GmMOT1.1 and GmMOT1.2

[0137] By homologous recombination, the sequence of 2kb upstream of the start codon of GmMOT1.1 and GmMOT1.2 was cloned into the pCAMBIA1301 vector. The plasmid with the correct sequence was transformed into Agrobacterium rhizogenes k599 by click transformation. The positive strain with successful plasmid transfer was identified by PCR. The soybean seedlings grown for 10 days were cut with a clean single-sided blade at a distance of about 1cm from the soil, and the cut was infected with the positive strain obtained by propagation. The infected plants were placed in FP medium supplemented with 1mM potassium chloride and calcium chloride and cultured for about 6 weeks, and transformed roots were observed at the bottom swelling.

[0138] The transformed roots were removed, immersed in a 20 μM GUS staining solution, placed on ice and vacuumed for 30 minutes, and then placed at 37°C for staining for 12 hours. The stained transformed roots were observed with a stereo microscope. Some of the stained transformed roots were prepared by resin embedding to prepare semi-thin sections, and the staining results of cells at different levels inside the transformed roots were observed. The remaining transformed plants were planted in perlite vermiculite and inoculated with the slow-growing rhizobium USDA110. 28 days after inoculation, the nodules were stained in the same way, and the staining results were observed with a stereo microscope after staining.

[0139] 5. Subcellular localization of GmMOT1.1 and GmMOT1.2

[0140] The CDSs corresponding to the five haplotypes of GmMOT1.1 and GmMOT1.2 were cloned into the pA7 expression vector by homologous recombination. Protoplasts were hydrolyzed from 4-week-old Arabidopsis leaves using an enzymatic solution containing cellulase, macerate, mannitol, potassium chloride, and MES. 2+ Transformation method: The recombinant plasmid containing the correct haploid sequence was transformed into protoplasts. After washing with W5 and resuspending with WI, the protoplasts were placed at 22°C in the dark for 20 hours.

[0141] Before observation, add The mother solution is added to make the concentration in the solution reach 50nM, and incubated at 37℃ in the dark for 30min. Alternatively, an appropriate amount of BFA mother solution is added to make the concentration reach 50μM, and incubated at room temperature for one hour in the dark. After the staining is completed, the centrifuge tube is placed vertically on the test tube rack. After the protoplasts settle, the protoplasts below are aspirated and the fluorescence signal is observed using fluorescence confocal and other equipment. The excitation wavelength is 561nM, and the emission wavelength is 600-650nM. After BFA treatment, the fluorescence signal aggregated, indicating that GmMOT1.1 and GmMOT1.2 were localized in BFA-sensitive vesicles. After 1 hour of TyrA23 treatment, GmMOT1.1 and GmMOT1.2 were more localized on the cell membrane, indicating that the two genes are simultaneously localized on the cell membrane and vesicles, and their localization on the cell membrane is strictly regulated.

[0142] 6. Detection of plant hormone (IAA and ABA) content and aldehyde oxidase activity in GmMOT1.1 and GmMOT1.2 double knockout mutants

[0143] (1) Detection of plant hormone content

[0144] Remove the second leaf from a four-week-old soybean plant and grind it into powder with liquid nitrogen. Place the powder in a freeze dryer and freeze-dry it at -80°C for 6 hours. Take about 30-60 mg of the dried powder into a clean 2mL centrifuge tube, accurately weigh and record the weight of the powder. Add 100μL of deionized water to the centrifuge tube and grind it at 60Hz for 60s to fully homogenize the sample. Add 400μL of ethyl acetate to the centrifuge tube and vortex for 20min to fully extract the plant hormones in the sample. Centrifuge at 12,000rpm and 4°C for 10min, and transfer the supernatant to a new centrifuge tube. Repeat the extraction with new ethyl acetate 3 times, and combine the collected ethyl acetate into the same centrifuge tube. Dry the ethyl acetate with a nitrogen blow dryer in a fume hood. Add 500μL of 30% methanol solution to the centrifuge tube and vortex for 10min to fully dissolve the plant hormones in the precipitate into the methanol solution. Centrifuge at 12,000 rpm at 4°C for 15 min, transfer the supernatant to a new centrifuge tube. Centrifuge again at 12,000 rpm at 4°C for 10 min, take the supernatant for detection or store at -20°C for a short time.

[0145] Dissolve indole-3-acetic acid and abscisic acid standards in 30% methanol solution to prepare gradient concentration standards, use 30% methanol solution as a blank control, and load it on the machine together with the samples and standards. Use liquid chromatography to detect the auxin and abscisic acid content in the sample, and the detection liquid phase is methanol solution.

[0146] (2) Aldehyde oxidase activity detection

[0147] Preparation of Salkowski reagent: Prepare 0.5M ferric chloride and 35% perchloric acid solution by mass volume. Add 1mL of 0.5M ferric chloride solution to 50mL of 35% perchloric acid solution in a fume hood, mix thoroughly by inversion, and store in a brown bottle after the solution cools down in a cool and dry place. Preparation of indole-3-acetaldehyde Prepare a saturated sodium carbonate aqueous solution. Weigh 5mg of indole-3-acetaldehyde sulfite and dissolve it in 2ml of water, adjust the pH to 10 with saturated Na2CO3 solution, and leave it at room temperature for 5min. Add an equal volume of anhydrous ether, mix thoroughly by inversion, and transfer the aqueous phase to another centrifuge tube after the solution is separated. Repeat the extraction with ether three times, and transfer the extracted ether to the same centrifuge tube. Add 10mL of deionized water to the ether and mix thoroughly by inversion. Dry the ether with a nitrogen blow dryer so that the free indole-3-acetaldehyde is redissolved in water.

[0148] The absorbance of the solution at 280 nm was measured using an enzyme reader, and the concentration of the prepared indole-3-acetaldehyde was calculated according to Beer's law.

[0149] Crude extraction of aldehyde oxidase: Take 200 mg of plant tissue sample from the target part into a centrifuge tube, and add 50 mM Tris (pH 7.3) buffer containing 1 mM magnesium chloride in ice to the centrifuge tube at a weight: volume ratio of 1:1. Homogenize with a grinder at 50 Hz for 90 seconds, taking out the sample every 30 seconds and cooling it in an ice bath to prevent overheating of the sample due to grinding. Filter the homogenized sample with a 100 μm cell sieve. Centrifuge at 10,000 rpm and 4 ° C for 20 minutes, and the supernatant is the crude extract of aldehyde oxidase. Take the supernatant to detect aldehyde oxidase activity.

[0150] Aldehyde oxidase activity detection: Add equal volumes of 0.2mL 0.8mM indole-3-acetaldehyde aqueous solution and 0.2mL aldehyde oxidase crude extract to the same centrifuge tube, and mix thoroughly by inversion. Place the centrifuge tube in a 30℃ water bath for 30min. Add a quarter of the total volume of 10% (v / v) trichloroacetic acid solution, and mix thoroughly by inversion to terminate the previous step. Centrifuge at 4000rpm for 5min, transfer the supernatant to a new centrifuge tube, and discard the precipitate. Add the two solutions to the same brown centrifuge tube in a ratio of supernatant: Salkowski reagent = 1:1, and mix thoroughly by pipetting. React in the dark for 25min, and detect the absorbance at 530nm. According to the standard curve of indoleacetic acid concentration and absorbance. Substitute the absorbance value at 530nm measured by the sample into the standard curve to calculate the concentration of indole-3-acetic acid produced after catalysis by aldehyde oxidase.

[0151] 7. Establishment of mutants

[0152] The mot1.1 / 1.2 double mutant Figure 7 .

[0153] WT / mot1.1mot1.2 grafting: the wild type was used as the aboveground part, and the double mutant was used as the root; among them, the wild type was the DN50 strain.

[0154] mot1.1mot1.2 / WT grafting: the double mutant was used as the aboveground part, and the wild type was used as the root; among them, the wild type was the DN50 strain.

[0155] 8. Establishment of overexpression strain (OE)

[0156] To construct the GmMOT1.1 overexpression plasmid, the CDS of GmMOT1.1-Hap (such as Hap5) was ligated into the plasmid PFGC5941 via the Noc I and XmaI restriction sites, and then the construct was introduced into Agrobacterium EHA101 and transformed into DN50, with the CaMV35S promoter driving the expression.

[0157] Example 1: Natural variation in molybdenum content in soybeans

[0158] The inventors collected and analyzed the frequency distribution of molybdenum content in the seeds of 597 natural soybean varieties, proving for the first time that there is natural variation in molybdenum content in soybean, a crop with high molybdenum requirements, as shown in Figure A.

[0159] Furthermore, the inventors used the whole genome association analysis method, and after in-depth screening and analysis, identified two genes that regulate the natural variation of soybean molybdenum content, named GmMOT1.1 and GmMOT1.2. Figure 1 BE.

[0160] The inventors conducted sequence analysis on the two genes GmMOT1.1 and GmMOT1.2. The results showed that there were at least five haplotypes Hap1-Hap5 of GmMOT1.1 and GmMOT1.2 in the natural soybean population. Three non-synonymous mutations in the coding region of GmMOT1.1 and two non-synonymous mutations in GmMOT1.2 divided the two genes into five haplotypes. Among them, Hap5 has the strongest molybdenum transport ability and Hap4 has the weakest molybdenum transport ability. Figure 1 F.

[0161] The haplotypes are as follows:

[0162] The nucleotide sequence of GmMOT1.1-Hap1 is as shown in SEQ ID NO:1, encoding GmMOT1.1-Hap1 protein, and the amino acid sequence is as shown in SEQ ID NO:8.

[0163] The nucleotide sequence of GmMOT1.1-Hap2 is as shown in SEQ ID NO:2, encoding GmMOT1.1-Hap2 protein, and the amino acid sequence is as shown in SEQ ID NO:9.

[0164] The nucleotide sequence of GmMOT1.1-Hap3 / 4 is as shown in SEQ ID NO:3, encoding GmMOT1.1-Hap3 / 4 protein, and the amino acid sequence is as shown in SEQ ID NO:10.

[0165] The nucleotide sequence of GmMOT1.1-Hap5 is as shown in SEQ ID NO:4, encoding the GmMOT1.1-Hap5 protein, and the amino acid sequence is as shown in SEQ ID NO:11.

[0166] The nucleotide sequence of GmMOT1.2-Hap1 is as shown in SEQ ID NO:5, encoding the GmMOT1.2-Hap1 protein, and the amino acid sequence is as shown in SEQ ID NO:12.

[0167] The nucleotide sequence of GmMOT1.2-Hap2 / 3 / 5 is as shown in SEQ ID NO:6, encoding the GmMOT1.2-Hap2 / 3 / 5 protein, and the amino acid sequence is as shown in SEQ ID NO:13.

[0168] The nucleotide sequence of GmMOT1.2-Hap4 is as shown in SEQ ID NO:7, encoding the GmMOT1.2-Hap4 protein, and the amino acid sequence is as shown in SEQ ID NO:14.

[0169] The molybdenum content in seeds of natural soybean varieties with different GmMOT1.1 and GmMOT1.2 haplotypes is different, with the highest content in Hap5 haplotype and the lowest in Hap4 haplotype. The molybdenum content of other haplotypes is between the two, such as Figure 1 G.

[0170] Different haplotypes of GmMOT1.1 and GmMOT1.2 have different molybdenum transport capacities in Saccharomyces cerevisiae. The Hap5 haplotype has the highest molybdenum content, while the Hap4 haplotype has the lowest molybdenum content. The molybdenum content of other haplotypes is between the two, such as Figure 1 H.

[0171] The expression levels of different haplotypes of GmMOT1.1 and GmMOT1.2 in different natural varieties are also different, such as Figure 1 I and Figure 1 J.

[0172] Therefore, the natural variation in soybean molybdenum content is caused by changes in the expression levels of GmMOT1.1 and GmMOT1.2 and their protein transport capacity.

[0173] Example 2: Effects of GmMOT1.1 and GmMOT1.2 on the transport of molybdenum in soybeans

[0174] Using DN50 as the background, the inventors double-mutated GmMOT1.1 and GmMOT1.2 to obtain the double mutant mot1.1 / 1.2.

[0175] Molybdenum content in leaves and seeds of wild type (DN50) and mot1.1 / 1.2 double mutant Figure 2 A and Figure 2 B. It can be seen that after GmMOT1.1 and GmMOT1.2 were mutated (downregulated), the molybdenum content in leaves and seeds decreased significantly, indicating that GmMOT1.1 and GmMOT1.2 are key genes affecting the molybdenum content in soybean leaves and seeds.

[0176] Molybdenum contents in leaves and seeds of wild-type and overexpressing plants Figure 2 C and Figure 2D. Therefore, when GmMOT1.1 is overexpressed, the molybdenum content in leaves and seeds increases significantly. This further indicates that GmMOT1.1 affects the molybdenum content in soybean leaves and seeds, and overexpression of this gene can improve seed molybdenum nutrition.

[0177] Analysis of the molybdenum content in the xylem flow of the wild type and the mot1.1 / 1.2 double mutant showed that after GmMOT1.1 and GmMOT1.2 were mutated (downregulated), the molybdenum content in the xylem flow decreased significantly, as shown in Figure 2. Figure 2 E. It showed that GmMOT1.1 and GmMOT1.2 were the key genes affecting the molybdenum content in soybean xylem flow.

[0178] The molybdenum content in the leaves and seeds of grafted soybeans was analyzed. The molybdenum content in the leaves of the mutant (mot1.1mot1.2 / WT) with the double mutant as the aerial part and the wild type as the root was high and close to that of the wild type, but the molybdenum content in the leaves of the self-grafted mutant and the mutant (WT / mot1.1mot1.2) with the wild type as the aerial part and the double mutant as the root was significantly reduced, such as Figure 2 F and Figure 2 G.

[0179] The ability of the wild type and double mutants to absorb molybdenum in the nutrient solution was analyzed, and it was found that the ability of the mutants to absorb molybdenum was significantly reduced compared with the wild type. Figure 2 H. This indicates that GmMOT1.1 and GmMOT1.2 solutions are involved in the process of soybean absorbing molybdenum from the soil.

[0180] Therefore, GmMOT1.1 and GmMOT1.2 affect (promote) the long-distance transport of molybdenum from the soil to the roots and from the aboveground parts to the roots in soybeans, and then affect (promote) the accumulation of molybdenum in the aboveground parts and grains of soybeans, which provides a further theoretical basis for further research on the molecular function of MOT1.

[0181] Example 3: Subcellular and tissue localization of GmMOT1.1 and GmMOT1.2

[0182] The inventors studied the expression locations of GmMOT1.1 and GmMOT1.2 in roots and nodules and the subcellular localization of their proteins and found that these two genes were mainly expressed in the root stele, such as Figure 3 AF.

[0183] The inventors studied the subcellular localization of GmMOT1.1 and GmMOT1.2 proteins. The results showed that GmMOT1.1 and GmMOT1.2 proteins were localized in BFA-sensitive vesicles and on the cell membrane ( Figure 3 GI).

[0184] Example 4: GmMOT1.1 and GmMOT1.2 regulate soybean agronomic traits

[0185] Planting conditions: Shanghai area, outdoor planting conditions with soil pH of about 5.5.

[0186] Under the same planting conditions, the 100-grain weight of soybean natural varieties with different haplotypes GmMOT1.1 and GmMOT1.2 (the two gene combinations are considered as a whole in the figure) was analyzed. Figure 4 A, The results showed that there were significant differences in 100-grain weight among some haplotypes, with Hap5 having the highest 100-grain weight.

[0187] The growth phenotypes of the wild type and mutants grown for eight weeks in Shanghai, and the growth phenotypes of the inverted clover Figure 4 B and Figure 4 C, Compared with the wild type, the double mutant has a significantly smaller plant and the leaves of the inverted trifoliate leaf are very significantly reduced.

[0188] The phenotypes of yield per plant and pod number of wild type and double mutant are shown in Figure 4 D and Figure 4 E, Compared with the wild type, the yield per plant and the number of pods in the double mutant were significantly reduced.

[0189] From planting to harvest, the plant height, branch number, yield per plant, pod number, and 100-grain weight of the wild type (DN50) and the mot1.1 / 1.2 double mutant were analyzed. The results are shown in 4F-J. Compared with the wild type, the plant height, branch number, yield per plant, pod number, and 100-grain weight of the double mutant were significantly reduced.

[0190] Growth phenotypes of wild-type and overexpressing plants grown for six weeks in Shanghai Figure 4 K, Compared with the wild type, the plant height of the overexpressing plants was significantly increased.

[0191] The phenotypes of pod number and yield per plant of wild type and double mutant are shown in Figure 4 L and Figure 4 M, Compared with the wild type, the yield per plant and the number of pods in the overexpressing plants were significantly increased.

[0192] From planting to harvest, the plant height, yield per plant, number of pods, and 100-grain weight of the wild type (DN50) and overexpression plants were analyzed. The results are shown in 4N-Q. Compared with the wild type, the plant height, yield per plant, number of pods, and 100-grain weight of the overexpression plants increased significantly.

[0193] Therefore, GmMOT1.1 and GmMOT1.2 are involved in the regulation of agronomic traits of soybean under normal planting conditions and are positive regulatory factors for traits such as yield and plant type.

[0194] Example 5: Analysis of the mechanism of GmMOT1.1 and GmMOT1.2 regulating yield traits

[0195] The images of the nail polish blots of epidermal cells of the wild type and double mutants observed under an optical microscope are shown in Figure 5 A, shows that the mutant cells are significantly smaller than the wild type.

[0196] The statistical results of the quantification of epidermal cell size between wild type and double mutant are shown in Figure 5 B, Epidermal cells of the double mutant were significantly smaller than those of the wild type.

[0197] The results of semi-thin section microscopy of wild-type and double mutant leaves are as follows Figure 5 C, The mesophyll cells of the double mutant were significantly smaller than those of the wild type.

[0198] The quantitative statistical results of the cross-sectional area of ​​mesophyll cells of wild type and double mutant are shown in Figure 5 D, The mesophyll cells of the double mutant were significantly reduced compared with those of the wild type.

[0199] The results of the analysis of IAA content and aldehyde oxidase activity in young leaves and stem tips of soybean wild type and mutants are as follows Figure 5 E and Figure 5 F, Compared with the wild type, the IAA content and aldehyde oxidase activity in young leaves and shoot tips of the double mutant were significantly reduced.

[0200] The results of aldehyde oxidase activity staining and quantitative statistics of soybean wild-type and double mutant seedlings are shown in Figure 2. Figure 5 G and 5H, The ability of the double mutant aldehyde oxidase to oxidize IAAld was significantly reduced compared to the wild type.

[0201] The results of the analysis of IAA content and aldehyde oxidase activity in young leaves and stem tips of wild-type and overexpressing soybean plants are shown in Figure 2. Figure 5 I and Figure 5 J, Compared with the wild type, the IAA content and aldehyde oxidase activity in the young leaves and shoot tips of the overexpressing plants were significantly increased.

[0202] The results of aldehyde oxidase activity staining and quantitative statistics of soybean wild-type and overexpression plant seedlings are shown in Figure 2. Figure 5 K and 5L, the ability of overexpressing aldehyde oxidase to oxidize IAAld was significantly increased compared with the wild type.

[0203] Therefore, GmMOT1.1 and GmMOT1.2 affect soybean yield by affecting (increasing) the accumulation of molybdenum in the aboveground parts and thus affecting IAA synthesis, rather than nitrogen assimilation and symbiotic nitrogen fixation, which provides a theoretical basis for applying molybdenum foliar fertilizer in agricultural production to increase soybean yield.

[0204] Example 6: Overexpression of GmMOT1.1 and GmMOT1.2 can increase soybean yield

[0205] The present inventors overexpressed GmMOT1.1 and GmMOT1.2 in the wild soybean variety Dongnong 50 using the CaMV35S promoter.

[0206] The results showed that the seeds of the overexpression (OE) transgenic lines were significantly larger than those of the wild-type plants. Figure 4 M.

[0207] ICP-MS detection showed that the molybdenum content accumulated in the aboveground parts and grains of transgenic plants was higher, increased by about 25%.

[0208] Moreover, the transformed plants were significantly superior to the wild type in terms of plant height, number of branches, 100-grain weight and number of pods. The 100-grain weight increased by 25%. Figure 4 NQ. According to statistics, the yield of the overexpression plant strains increased by about 30% compared with the wild type. Figure 4 LM.

[0209] Therefore, GmMOT1.1 and GmMOT1.2 can be used as good genes for breeding high-molybdenum nutritional soybean varieties and increasing soybean yield, which has very important application value. It also shows that the two genes have great application value in breeding high-nutrition soybean varieties and increasing soybean yield.

[0210] Example 7: The haplotypes of GmMOT1.1 and GmMOT1.2 are regional and can be used as molecular markers in agriculture

[0211] The inventors analyzed the distribution of different haplotypes Hap1-5 in different regions and their correlation with soil acidity and alkalinity based on 597 natural soybean varieties, their geographical distribution and soil pH values. The distribution of the five haplotype varieties GmMOT1.1 and GmMOT1.2 in China is as follows: Figure 6 .

[0212] 1. As a molecular marker for analyzing soybean varieties suitable for different acid-base soil environments

[0213] The results show that natural soybean varieties containing GmMOT1.1 and GmMOT1.2 haplotype Hap5 are mainly distributed in the acidic soil areas of southern China, while natural varieties containing Hap4 are mainly distributed in the alkaline planting areas of northern China. This indicates that under acidic molybdenum-deficient conditions, varieties containing Hap5 can absorb more molybdenum than other varieties. According to the results of the present invention, the higher the molybdenum content in soybeans, the higher the yield.

[0214] Before planting soybeans, the pH value of the local soil extract can be tested with pH test paper or pH meter. Soybean varieties with different haplotypes can be selected based on the results. For example, soybean varieties with seeds containing the Hap5 haplotype can be selected in acidic soils to obtain greater yield benefits in the same soil planting area. In alkaline soils, molybdenum is not a limiting condition. If the variety is determined to have the Hap4 haplotype, it is more suitable for planting in alkaline soils.

[0215] Therefore, the present invention reveals the distribution of five haplotype varieties of GmMOT1.1 and GmMOT1.2 in China, which not only provides molecular markers for screening soybean varieties suitable for different acid-base soil environments, but also plays a guiding role in the application of molybdenum fertilizers to different soybean varieties.

[0216] 2. As a molecular marker to determine whether additional molybdenum fertilizer is needed

[0217] The present invention shows that the soybean molybdenum content accumulated in soybean varieties containing Hap5 is the highest compared to soybean varieties containing other haplotypes, so the accumulated molybdenum content can fully meet or even exceed the molybdenum content required for soybean growth. In soils that are not extremely molybdenum-deficient or extremely acidic, soybean varieties containing the Hap5 haplotype basically do not need to apply additional molybdenum foliar fertilizer.

[0218] Therefore, during the soybean planting process, the GmMOT1.1 and GmMOT1.2 sequences contained in the selected soybean varieties are first identified to determine the haplotype to which the variety belongs. If the haplotype belongs to a high-molybdenum haplotype, no molybdenum fertilizer is required during the planting process, thereby reducing the economic and time costs of planting and providing convenience for the planting process.

[0219] Conclusion of the Example

[0220] A major QTL on chromosome 17 was identified and two candidate genes were nominated by genome-wide association analysis of molybdenum content in grains of soybean varieties collected worldwide.

[0221] Through the analysis of population genotype data, it was found that there are a large number of polymorphic sites in the two genes, which can be mainly divided into five haplotypes Hap1-Hap5. The nucleotide sequences of the coding regions of the five haplotypes are SEQ ID NO.1-SEQ ID NO.7, and the corresponding protein amino acid sequences are SEQ ID NO.8-SEQ ID NO.14.

[0222] Through the yeast heterologous expression system, it was found that GmMOT1.1 and GmMOT1.2 have the ability to transport molybdate, and Hap5 has the strongest molybdenum transport ability, while Hap4 has the weakest transport ability. Therefore, the change in the molybdenum transport ability of GmMOT1.1 and GmMOT1.2 is one of the reasons for the natural variation of soybean molybdenum content.

[0223] The results of fluorescence quantitative PCR experiments showed that the expression levels of GmMOT1.1 and GmMOT1.2 were the highest in Hap5, while the lowest in Hap4. Therefore, the difference in the expression levels of GmMOT1.1 and GmMOT1.2 also contributed to the natural variation of soybean molybdenum content.

[0224] The molybdenum content in leaves and grains of three independent double mutants of GmMOT1.1 and GmMOT1.2 was detected by ICP-MS (inductively coupled plasma-mass spectrometry). The molybdenum content in the mutants was significantly reduced.

[0225] Through ICP-MS detection of molybdenum content in xylem flow and grafting experiments, it was found that the molybdenum content in xylem flow of the mutant was significantly reduced, and the molybdenum content in the aboveground part and grains was mainly regulated by the roots.

[0226] Using the promoter 2kb upstream of GmMOT1.1 and GmMOT1.2 to drive the expression of β-glucuronidase in soybean roots and nodules, it was found that GmMOT1.1 and GmMOT1.2 were mainly expressed in the root stele and nodule vascular system, and may be involved in the absorption of molybdenum from the soil to the roots, the long-distance transport from the roots to the aboveground parts, and the transport from the roots to the nodules.

[0227] The subcellular localization of GmMOT1.1 and GmMOT1.2 was studied using a protoplast transient transfection system and found that the five haplotypes of the protein were all localized in BFA-sensitive vesicles and cell membranes.

[0228] By quantifying the agronomic traits of natural populations under the same planting conditions, it was found that natural soybean varieties with the Hap5 haplotype had the highest 100-grain weight.

[0229] Quantitative statistics of the agronomic traits of the wild-type DN50 and the GmMOT1.1 and GmMOT1.2 double mutants revealed that the plant height, number of branches, single plant yield, number of pods per plant, and 100-grain weight of the double mutants were significantly reduced.

[0230] Optical microscopy revealed that the leaf epidermal cells of the GmMOT1.1 and GmMOT1.2 double mutants were significantly smaller than those of the wild type.

[0231] The contents of indole-3-acetic acid (IAA) and abscisic acid (ABA) in young leaves and stem tips were detected by liquid chromatography, and it was found that the content of IAA in the GmMOT1.1 and GmMOT1.2 double mutants was significantly reduced compared with the wild type, while there was no significant difference in the content of ABA.

[0232] In vitro detection experiments found that the ability of aldehyde oxidase (AO) in the GmMOT1.1 and GmMOT1.2 double mutants to oxidize indole-3-acetaldehyde to IAA was significantly reduced.

[0233] By analyzing the distribution of natural soybean populations with five GmMOT1.1 and GmMOT1.2 haplotypes in China, it was found that the strong haplotype Hap5 was mainly distributed in the low-pH soil environment in southern China, while the weak haplotype Hap4 was mainly distributed in the high-pH soil environment in the north.

[0234] Sequence information

[0235] GmMOT1.1-Hap1 (SEQ ID NO: 1)

[0236] ATGGCATACCAAAACCCTCCTTCAATTCCAATCTCAGATCCTGAAGCACCGGAAATCACTCCTACTCATACTCCTTCAACCACCACCCACTAGCCAATGGATTTTCA ACC AAAGGAGTGGTGGAAAAAATAAAAAACAACTTGGTTTTTCACTCAAAATGGGGTGAGCTCAATGGCGCCATGGGTGACCTCGGCACTTATGTACCAATTGTTTTGGCCTTGACCCTTGCTAGGGACCTCAACCTTGGCACCACGCTGATATTTACCGGTGTGTACAATATCATC ACT GAT AAACCACTTTCTACAATTTGGACGATGAAAAGCCCCGAGCAAGTTTGA

[0237] GmMOT1.1-Hap2(SEQ ID NO:2)

[0238] ATGGCATACCAAAACCCTCCTTCAATTCCAATCTCAGATCCTGAAGCACCGGAAATCACTCCTACTCATACTCCTTCAACCACCACCCCACTAGCCAATGGATTTTCA GCC AAAGGAGTGGTGGAAAAAATAAAAAACAACTTGGTTTTTCACTCAAAATGGGGTGAGCTCAATGGCGCCATGGGTGAC CTT GGCACTTATGTACCAATTGTTTTGGCCTTGACCCTTGCTAGGGACCTCAACCTTGGCACCACGCTGATATTTACCGGTGTGTACAATATCATC ACT

[0239] GmMOT1.1-Hap3 / 4(SEQ ID NO:3)

[0240]

[0241] GmMOT1.1-Hap5(SEQ ID NO:4)

[0242]

[0243] GmMOT1.2-Hap1(SEQ ID NO:5)

[0244]

[0245] GmMOT1.2-Hap2 / 3 / 5(SEQ ID NO:6)

[0246]

[0247] GmMOT1.2-Hap4(SEQ ID NO:7)

[0248]

[0249] GmMOT1.1-Hap1(SEQ ID NO:8)

[0250] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFS T KGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNLGTTLIFTGVYNII T GVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLMQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHE IKFGPSSIEVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLLRRNWTR D KPLSTIWTMKSPEQV.

[0251] GmMOT1.1-Hap2(SEQ ID NO:9)

[0252] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFS A KGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNLGTTLIFTGVYNII TGVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLMQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHE IKFGPSSIEVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLLRRNWTR D KPLSTIWTMKSPEQV.

[0253] GmMOT1.1-Hap3 / 4(SEQ ID NO:10)

[0254] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFS A KGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNLGTTLIFTGVYNII A GVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLMQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHE IKFGPSSIEVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLLRRNWTR DKPLSTIWTMKSPEQV.

[0255] GmMOT1.1-Hap5(SEQ ID NO:11)

[0256] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFS A KGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNLGTTLIFTGVYNII T GVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLMQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKSLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHE IKFGPSSIEVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLLRNWTR G KPLSTIWTMKSPEQV.

[0257] GmMOT1.2-Hap1(SEQ ID NO:12)

[0258] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFS T KGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNLGTTLIFTGVYNII TGVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLMQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKSLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHEIKFGPSSIEVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCLKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGLVGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLLRRNWTRGKPLSTIWMQKSPEQV.

[0259] GmMOT1.2-Hap2 / 3 / 5(SEQ ID NO:13)

[0260] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFSAKGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNGTTLIFTGVYNIITGVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKSLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHEIKFGPSSIEVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCLKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGVGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLLRRNWTRGKPLSTIWMQKSPEQV.

[0261] GmMOT1.2-Hap4 (SEQ ID NO: 14)

[0262] MAYQNPPSIPISDPEAPEITPTHTPSTTTPLANGFS A KGVVEKIKNNLVFHSKWGELNGAMGDLGTYVPIVLALTLARDLNLGTTLIFTGVYNII A GVIYGVPMPVQPMKSIAAQALSDTDFGVPEIMTAGILTGGVLFVLGVTGLMQLVYMLIPLCVVRGIQLAQGLSFALTAVKYVRKIQDLPKSKSLGERHWFGLDGLVLAIVCLCFIVVVNGAGEKSRGCCDVVESGGGDDDLGGQKRNNEVVARSRTSRVRKVIFSLPSAFMVFVLGVVLAFIRRHEVVHEIKFGPSSI EVVKFSKHAWKKGFVKGAIPQLPLSILNSVVAVCKLSSDLFPGKDFSATSLSVTVGLMNLIGSWFGAMPSCHGAGGLAGQYKFGGRSGGCVALLGVAKLVLGLVLGTSLAHILKQFPVGILGVLLLFAGIELAMCARDMNTKEDSFVTLVITAVSLVGSSAALGFLCGMVVYVLLRLRNWTRGKPLSTIWMQKSPEQV.

[0263] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for improving the traits of legumes, comprising increasing the expression or activity of MOT1.1 and / or MOT1.2 in the legumes; wherein: The improved traits include those selected from the group consisting of: (i) increased molybdenum content or molybdenum transport capacity, (ii) increased yield, (iii) increased biomass, and (iv) increased IAA content.

2. The method according to claim 1, characterized in that In (i), the increasing the molybdenum content comprises: increasing the molybdenum content of the aboveground part; preferably, the aboveground part comprises: grains or leaves; In (i), the improving the molybdenum transport capacity comprises: promoting the transport of molybdenum from the roots to the aboveground parts, and promoting the absorption of molybdenum from the soil to the roots; (ii), the yield increase comprises: increasing the size of the grains, increasing the number of branches, increasing the 100-grain weight or increasing the number of pods; or In (iii), the increasing of biomass comprises: increasing plant height, enlarging leaves or increasing epidermal cell volume.

3. The method according to claim 1, characterized in that The method of increasing the expression or activity of MOT1.1 and / or MOT1.2 in plants comprises: using MOT1.1 and / or MOT1.2 genes or expression constructs or vectors containing the genes to transfer into plants; increasing the expression efficiency of MOT1.1 and / or MOT1.2 genes in plants by expressing enhanced promoters or tissue-specific promoters; increasing the expression efficiency of MOT1.1 and / or MOT1.2 genes in plants by using enhancers; Preferably, said increasing the expression or activity of MOT1.1 and / or MOT1.2 in the plant comprises increasing its expression in the plant root stele or nodule vascular system.

4. Application of MOT1.1 and / or MOT1.2 up-regulators for improving the traits of leguminous plants; wherein: The improved traits include those selected from the group consisting of: (i) increased molybdenum content or molybdenum transport capacity, (ii) increased yield, (iii) increased biomass, and (iv) increased IAA content.

5. The use according to claim 4, characterized in that The up-regulator includes one selected from: an expression construct or vector for recombinantly expressing MOT1.1 and / or MOT1.2, a chemical up-regulator of MOT1.1 and / or MOT1.2, and an up-regulator that promotes the gene promoter driving ability of MOT1.1 and / or MOT1.

2.

6. A method for screening a substance for improving the properties of leguminous plants, comprising: (1) Adding the candidate substance to a system containing MOT1.1 and / or MOT1.2; (2) detecting the system, observing the expression or activity of MOT1.1 and / or MOT1.2 in the system of (1); if the candidate substance promotes the expression or activity of MOT1.1 and / or MOT1.2, the candidate substance is a substance that improves the traits of legumes; wherein the improved traits include those selected from the following groups: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

7. Application of MOT1.1 and / or MOT1.2 for: As a marker for identifying traits of leguminous plants; or Directed selection of improved traits in legumes; in, The traits include: molybdenum content, molybdenum transport capacity, yield, biomass or IAA content; the trait improvement includes: (i) increasing molybdenum content or molybdenum transport capacity, (ii) increasing yield, (iii) increasing biomass, and (iv) increasing IAA content.

8. The method or use according to any one of claims 1 to 7, characterized in that: The MOT1.1 and / or MOT1.2 is MOT1.1 from the leguminous plant; preferably, the amino acid sequence of the MOT1.1 includes the following groups: (a) a protein selected from the amino acid sequence shown in SEQ ID NO: 8-11; (b) a protein derived from (a) formed by replacing, deleting or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO: 8-11, and having the protein function of (a); (c) a protein derived from (a) that has more than 80% homology with the protein sequence defined in (a) and has the protein function of (a); or (d) a protein active fragment defined in (a), or a protein formed by adding a tag sequence, an enzyme cleavage sequence, or a reporter protein to both ends of the protein; The amino acid sequence of the MOT1.2 protein includes proteins selected from the following groups: (a') proteins such as SEQ ID NO: 12-14 amino acid sequences; (b') proteins selected from SEQ ID NO: 12-14 amino acid sequences formed by substitution, deletion or addition of one or more amino acid residues, and derived from (a) and having the function of (a) protein; (c') proteins derived from (a) that have more than 80% homology with the protein sequence defined by (a') and have the function of (a) protein; or (d') protein active fragments defined by (a'), or proteins formed by adding tag sequences, enzyme cleavage sequences, and reporter proteins to both ends of the protein.

9. A method for identifying the properties of leguminous plants, comprising: Analyzing the haplotype of the MOT1.1 and / or MOT1.2 gene in the plant; If it encodes a protein with the amino acid sequence of SEQ ID NO:8, or a nucleotide sequence such as SEQ ID NO:1, it is the GmMOT1.1-Hap1 haplotype; If it encodes a protein with the amino acid sequence of SEQ ID NO:9, or a nucleotide sequence such as SEQ ID NO:2, it is the GmMOT1.1-Hap2 haplotype; If the protein encoding the amino acid sequence of SEQ ID NO: 10, or the nucleotide sequence is SEQ ID NO: 3, then it is the GmMOT1.1-Hap3 / 4 haplotype; If the protein encoding the amino acid sequence of SEQ ID NO: 11, or the nucleotide sequence is SEQ ID NO: 4, then it is the GmMOT1.1-Hap5 haplotype; If it encodes a protein with the amino acid sequence of SEQ ID NO: 12, or a nucleotide sequence such as SEQ ID NO: 5, it is the GmMOT1.2-Hap1 haplotype; If the protein encoding the amino acid sequence of SEQ ID NO: 13, or the nucleotide sequence is SEQ ID NO: 6, then it is the GmMOT1.2-Hap2 / 3 / 5 haplotype; If the protein encoding the amino acid sequence of SEQ ID NO: 14, or the nucleotide sequence is SEQ ID NO: 7, then it is the GmMOT1.2-Hap4 haplotype; If the plant is identified as the Hap5 haplotype, the plant is a high-molybdenum haplotype, which is a plant with high aboveground molybdenum content, easy molybdenum absorption and high yield; if the plant is identified as the Hap4 haplotype, the plant is a low-molybdenum haplotype, which is a plant with low aboveground molybdenum content, not easy to absorb molybdenum and low yield; if the plant is identified as the Hap1, Hap2 or Hap3 haplotype, the plant has an aboveground molybdenum content, yield or molybdenum fertilizer application amount between the Hap5 haplotype and the Hap4 haplotype.

10. The method according to claim 9, characterized in that After identifying the characteristics of the legume plant, the method further includes analyzing the soil suitable for planting the legume plant; wherein, if the legume plant is identified as the Hap5 haplotype, it is relatively suitable for planting in acidic soil; if the legume plant is identified as the Hap4 haplotype, it is relatively suitable for planting in alkaline soil; if the legume plant is identified as the Hap1, Hap2 or Hap3 haplotype, the plant is relatively suitable for planting in soil close to neutral.

11. The method according to claim 9, characterized in that After identifying the characteristics of the legume plant, it also includes: analyzing whether the legume plant needs to be applied with molybdenum fertilizer; if it is identified as the Hap5 haplotype, it does not need to be applied with molybdenum fertilizer; if it is identified as the Hap4 haplotype, it needs to be applied with molybdenum fertilizer; if it is identified as the Hap1, Hap2 or Hap3 haplotype, the amount of molybdenum fertilizer required for the plant is between Hap5 and Hap4.

12. The method or use according to any one of claims 1 to 11, characterized in that: The legumes include legumes selected from the group consisting of legumes expressing MOT1.1 and / or MOT1.2 genes; more preferably, legumes including soybeans, peas, lupines, beans, chickpeas, Medicago truncatula, Lotus japonica, Trifolium repens, and Ephedra sinica.

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