CN116640781B

CN116640781B - Application of MtAHA5 gene and MtAHA5 protein in alfalfa plants

Info

Publication number: CN116640781B
Application number: CN202310897596.9A
Authority: CN
Inventors: 江文波; 庞永珍; 魏解冰; 闫依诺
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2023-07-21
Filing date: 2023-07-21
Publication date: 2023-11-14
Anticipated expiration: 2043-07-21
Also published as: CN116640781A

Abstract

The application provides an application of MtAHA5 gene and MtAHA5 protein in alfalfa plants, relates to the application fields of biotechnology and genetic engineering, and is specifically applied to S1) regulating and controlling a proton electrochemical potential gradient crossing a vacuole membrane in alfalfa plants, and promoting a proanthocyanidin precursor to be transported into vacuoles of the alfalfa plants; s2) regulating and controlling proanthocyanidins of alfalfa to increase the content of the proanthocyanidins in the alfalfa; s3) regulating and controlling proanthocyanidins of alfalfa to reduce the content of the proanthocyanidins in the alfalfa; s4) application in preparing alfalfa products with high content of proanthocyanidins; s5) cultivating transgenic alfalfa with high proanthocyanidin content; s6) cultivating alfalfa for preventing the ruminant hoove from happening, wherein the alfalfa has high content of proanthocyanidins. The application provides a feasible way for regulating and controlling the content of proanthocyanidins in alfalfa plants, has simple method and great significance for animal husbandry.

Description

Application of MtAHA5 gene and MtAHA5 protein in alfalfa plants

Technical Field

The application relates to the application fields of biotechnology and genetic engineering, in particular to a MtAHA5 gene, a MtAHA5 protein and application thereof.

Background

Proanthocyanidin (PA), also known as condensed tannin, whose basic structural unit is flavan-3-ol, is a polyphenolic compound, which is oxidized and dehydrated under the action of acid, base or enzyme to produce a water-insoluble macromolecular compound (see "phytotannin chemistry", published by chinese forestry press, sun Dawang). The proanthocyanidins in plants are mostly present in the form of polymers. Proanthocyanidins are mainly present in leaves, stems, fruits, seeds, flowers and husks of plants, and are usually present in vacuoles of cells. The vegetables, fruits, forage grass, trees, shrubs, leguminous plants, cereals and seeds are all rich in proanthocyanidins. Not only plays an important role in the regulation of seed dormancy, longevity and germination, but also is involved in regulating biotic and abiotic stress in plants (Debeaujon et al, 2003). In addition, it has antioxidant, anti-inflammatory and anticancer effects, and is beneficial to human health (Dixon et al 2005).

The alfalfa is known as the pasture king, and has the characteristics of high yield, stable yield, easy cultivation, good feeding value, high protein content and the like. However, the ruminants, such as cows, can develop hooves after feeding alfalfa, thereby severely limiting the utilization and nutritional potential of alfalfa in animal husbandry. When the content of the proanthocyanidin is higher than 2% of dry weight in alfalfa, the occurrence of the hoove of ruminants can be effectively prevented (Verdier et al 2012), because the proanthocyanidin is combined with soluble protein in rumen of a weak acid environment (pH 5.5-7.0) of ruminants to form a relatively stable tannin protein conjugate, namely rumen bypass protein, and when the proanthocyanidin enters into the true stomach (pH 2.5-3.5) and small intestine (pH 8.0-8.5), the pH value is changed, the protein is released and is absorbed and utilized in the small intestine, so that the occurrence of the hoove is avoided, the degradation of the soluble protein in the rumen and the foam generated in the rumen are reduced, the hoove is prevented, the effect of protecting the rumen protein is achieved, and a proper amount of the proanthocyanidin is added into feed of ruminants, so that the feed has a certain nutrition physiological effect. The proanthocyanidin is easy to combine with protein in organism to form a stable complex, reduces the solubility and surface activity of protein in rumen, and plays a role in protecting protein. Meanwhile, the proanthocyanidin can inhibit rumen protein decomposing bacteria, and finally improve the utilization rate of protein. In addition, degradation products of proanthocyanidins do not produce toxic effects on ruminants. However, too high (over 3%) proanthocyanidin content in pasture is very likely to affect the feed intake of ruminants and the degradation rate of the forage in the rumen, and reduces the overall digestibility of the forage. Because the proanthocyanidin has astringency and astringency, the excessive ingestion of the animal can cause uncomfortable feeling to the oral cavity and the forestomach epithelium, thus affecting the palatability of the animal and further reducing the feed intake of ruminants. Excessive feeding of pasture rich of proanthocyanidins by ruminants can cause poisoning symptoms, damage of organs such as liver and kidney and functions thereof (Niu Julan and the like, and protection research of tannin in red bean grass on rumen bypass proteins).

Some current studies have attempted to increase the proanthocyanidin content of alfalfa and the like (Verdier et al 2012; escaray et al 2014; yuan and Grotewold, 2015; li et al 2016), with results that are not ideal, far from a level of 2% dry weight, and therefore do not effectively prevent the occurrence of ruminant hooves such as cattle, sheep and the like. The biggest obstacle to the inability of Zhao et al and Li et al to achieve a better proanthocyanidin modification is the very complex biosynthesis and regulation of proanthocyanidins, and there are still many problems to be solved (Zhao et al, 2010; li et al, 2016).

Disclosure of Invention

In order to realize the regulation of the content of proanthocyanidins in alfalfa plants from the gene level and thus obtain alfalfa with high content of proanthocyanidins for reducing the occurrence of ruminant hooves, the application provides an alfalfa plant with high content of proanthocyanidins by changing the biosynthesis of the proanthocyanidins in alfalfa through applying the MtAHA5 gene or the MtAHA5 protein so as to enable the content of the proanthocyanidins in the plants to be adjustable and controllable.

To achieve the technical object of the present application, a first aspect of the present application provides an application of MtAHA5 gene, which is S1) or S2) or S3) or S4) or S5) or S6):

s1) regulating and controlling proton electrochemical potential gradient across a vacuole membrane in the alfalfa plant, and promoting the transportation of proanthocyanidin precursors into vacuoles of the alfalfa plant;

s2) regulating and controlling proanthocyanidins of alfalfa to increase the content of the proanthocyanidins in the alfalfa;

s3) regulating and controlling proanthocyanidins of alfalfa to reduce the content of the proanthocyanidins in the alfalfa;

s4) application in preparing alfalfa products with high content of proanthocyanidins;

s5) cultivating transgenic alfalfa with high proanthocyanidin content;

s6) cultivating alfalfa for preventing the ruminant hoove from happening, wherein the alfalfa has high proanthocyanidin content.

In particular, the nucleotide sequence of the MtAHA5 gene is shown as SEQ ID NO. 1.

In particular, the MtAHA5 gene is obtained by a primer pair with a nucleotide sequence shown as SEQ ID NO. 3-4.

In particular, the expression level of the MtAHA5 gene can be detected by a primer set having a nucleotide sequence shown in SEQ ID NO. 5-6.

In a second aspect, the application provides the use of a MtAHA5 protein, K1) or K2) or K3) or K4) or K5) or K6):

k1 Regulating and controlling the proton electrochemical potential gradient across the vacuole membrane in the alfalfa plant, and promoting the transportation of the proanthocyanidin precursor into the vacuoles of the alfalfa plant;

k2 Regulating and controlling proanthocyanidin of alfalfa to increase the content of proanthocyanidin in alfalfa;

k3 Regulating and controlling proanthocyanidin of alfalfa to reduce the content of proanthocyanidin in alfalfa;

k4 Use of alfalfa products having a high proanthocyanidin content;

k5 The application of the transgenic alfalfa with high content of proanthocyanidins is cultivated;

k6 Cultivation of alfalfa with high proanthocyanidin content for preventing the occurrence of hoove diseases in ruminants.

In particular, the amino acid sequence of the MtAHA5 protein is shown as SEQ ID NO. 2.

In particular, the MtAHA5 protein may be obtained from a biological material, which is any one of the following B1) to B3):

b1 A nucleic acid molecule encoding a MtAHA5 protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

in particular, the nucleotide sequence of the nucleic acid molecule B1) is shown as SEQ ID NO. 3.

In a third aspect, the present application provides a regulator of alfalfa proanthocyanidin content, which has the above MtAHA5 gene, or the above MtAHA5 protein, or the above B1) -B3) biological material.

The fourth aspect of the present application provides a method for cultivating a transgenic plant by increasing the expression level of MtAHA5 protein in a recipient plant to obtain a transgenic plant; the transgenic alfalfa has an increased proanthocyanidin content as compared to the recipient plant;

wherein, the improvement of the expression level of the MtAHA5 protein in the receptor plant is realized by introducing a nucleic acid molecule encoding the MtAHA5 protein into alfalfa.

Drawings

FIG. 1 is a graph showing the results of analysis of the identification of the insertion mutant Tnt of the MtAHA5 gene provided in examples 1 and 3, wherein FIG. 1A shows the insertion positions Tnt of NF0707, NF19445 and NF7687, FIG. 1B shows the result of PCR identification of the insertion homozygous mutant Tnt1 of NF0707, FIG. 1C shows the result of PCR identification of the insertion homozygous mutant Tnt of NF7687, and FIG. 1D shows the result of PCR identification of the insertion homozygous mutant Tnt of NF 19445; FIG. 1E shows the results of analysis of the expression level of the MtAHA5 gene in the NF0707 homozygous mutant, FIG. 1F shows the results of analysis of the expression level of the MtAHA5 gene in the NF7687 homozygous mutant, and FIG. 1G shows the results of analysis of the expression level of the MtAHA5 gene in the NF19445 homozygous mutant;

FIG. 2 is a graph showing the results of analysis of the identification of the insertion mutants Tnt of the MtAHA3, mtAHA4 and MtAHA9 genes provided in example 1 of the present application, wherein FIG. 2A shows the result of PCR identification of the Tnt insertion homozygous mutant of NF 12901; FIG. 2B shows the PCR identification of Tnt1 insert homozygous mutant for NF 10457; FIG. 2C shows the PCR identification of the Tnt1 insert homozygous mutant of NF 3114; FIG. 2D shows the results of analysis of the expression level of the MtAHA3 gene in the NF12901 homozygous mutant; FIG. 2E shows the results of analysis of the expression level of the MtAHA4 gene in the NF10457 homozygous mutant; FIG. 2F shows the results of analysis of the expression level of the MtAHA9 gene in the NF3114 homozygous mutant;

FIG. 3 is a graph showing the results of analysis of proanthocyanidin content in the homozygous mutant Tnt1 insert provided in example 1 of the present application, wherein FIG. 3A shows the results of analysis of proanthocyanidin content in the homozygous mutants NF0707, NF12901, NF10457 and NF3114, and FIG. 3B shows the results of analysis of proanthocyanidin content in the homozygous mutants NF0707, NF19445 and NF7687 of the MtAHA5 gene;

FIG. 4 is a graph of the results of an assay providing that the MtAHA5 gene is capable of restoring the reduced proanthocyanidin phenotype in NF0707 mutants in example 2 of the present application;

FIG. 5 shows the localization of the MtAHA5 protein after co-injection of tobacco leaves with Agrobacterium harboring the MtAHA5-RFP gene and Agrobacterium of vac-ck-CFP in example 3 of the present application;

FIG. 6 is a graph showing the analysis result of gene expression patterns of key genes, mtANR and MtAHA5 genes in the biosynthesis pathway of Arabidopsis thaliana proanthocyanidin in test example 1 according to the present application during seed development;

FIG. 7 is a qualitative analysis result of the identification of the expression of the MtAHA5 gene and the content of proanthocyanidins in the transgenic line of the MtAHA5 gene genetic transformation aha mutant in test example 2 according to the present application, wherein FIG. 7A is a result of analysis of the expression of the MtAHA5 gene in the transgenic line of the MtAHA5 gene genetic transformation aha mutant, FIG. 7B is a seed coat color of the transgenic line seed of the MtAHA5 gene genetic transformation aha10 mutant, and FIG. 7C is a seed coat color of the transgenic line seed of the MtAHA5 gene genetic transformation aha10 mutant after DMACA staining;

FIG. 8 is a quantitative analysis result of proanthocyanidin content in transgenic line seeds of the MtAHA5 gene genetically transformed aha mutant in test example 2 of the present application;

FIG. 9 is a graph showing the result of anthocyanin content analysis of the insertion homozygous mutant NF0707 of Tnt provided in test example 3 of the present application;

FIG. 10 is a graph showing the results of anthocyanin content analysis of the insertion homozygous mutants NF0707, NF19445 and NF7687 into Tnt provided in test example 3 of the present application and plant photographs: wherein, fig. 10A is the anthocyanin observation results in the hypocotyl of seedlings three days in the homozygous mutants of NF0707, NF19445 and NF7687 of the MtAHA5 gene, fig. 10B is the anthocyanin observation in the seedling stage of the homozygous mutants of NF0707, NF19445 and NF7687 of the MtAHA5 gene, the dark color of the stem base is the anthocyanin accumulation, fig. 10C is the anthocyanin observation in the leaves of the homozygous mutants of NF0707, NF19445 and NF7687 of the MtAHA5 gene, and the anthocyanin spots are the anthocyanin accumulation, and fig. 10D is the analysis results in the leaves of the homozygous mutants of NF0707, NF19445 and NF7687 of the MtAHA5 gene.

Detailed Description

The application will now be described with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the application. Unless otherwise indicated, the technical means employed in the examples are conventional means well known to those skilled in the art, and the reagents and products employed are also commercially available. The various processes and methods not described in detail are conventional methods well known in the art, the sources of the reagents used, the trade names and those necessary to list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are the same as those indicated at the first occurrence.

Example 1 phenotypic analysis of the MtAHA5 Gene

In order to embody the role of the MtAHA5 gene in the accumulation process of the alfalfa proanthocyanidin in caltrops, the inventor purchased Tnt insertion mutant NF0707 of the gene, and simultaneously takes MtAHA3, mtAHA4 and MtAHA9 genes with the same gene expression pattern as that of key genes in the biosynthesis pathway of the alfalfa proanthocyanidin in the seed development process as controls, wherein the corresponding mutants are NF12901, NF10457 and NF3114 respectively.

Primers were designed based on the sequence of Tnt and Tnt1 insert, and PCR was used to identify Tnt insert homozygous mutants, and then RNA was extracted from the mutants by conventional methods. Then qRT-PCR is adopted to analyze the expression level of the genes in the mutant, and the identification result and the gene expression level result are shown in the figures 1 and 2, so that the test materials of the application are homozygous mutants, and NF0707 can be used as the test material for the MtAHA5 gene function analysis.

Meanwhile, the R108 and Tnt1 are planted to insert the homozygous mutant, and the planting method can be any method in the prior art, and the application is not limited. Mature R108 and Tnt1 insert homozygous mutant seeds were harvested and then qualitatively and quantitatively analyzed for proanthocyanidin content in the seeds, as shown in FIG. 3, and based on the analysis results of FIG. 3, it was found that only Tnt of MtAHA5 insert homozygous mutant NF0707 had significantly less proanthocyanidin content than the wild type.

In one embodiment of the present application, operations may be performed using Jun, J.H., liu, C., xiao, X. & Dixon, R.A et al, publication The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyanidin and anthocyanin pigmentation in Medicago trunk. Plant cell (2015) 27:2860-2879. Of course, those skilled in the art can also use other methods to perform qualitative and quantitative analysis, and the application is not limited.

From the above results, it can be seen that MtAHA5 plays a key role in the accumulation of alfalfa proanthocyanidins in caltrops.

The MtAHA5 gene provided by the application is obtained from a caltrop alfalfa genome (Medicago truncatula A r5.0 genome, website: https:// medium. Toulouse. Inra. Fr/Mtrunk A17r5.0-ANR /), and the nucleotide sequence of the MtAHA5 gene is shown as SEQ ID NO. 1. In one embodiment, the MtAHA5 gene may be obtained using the primer set shown as SEQ ID NO. 3-4. In one embodiment, the MtAHA5 gene level may also be detected using the detection primer set shown as SEQ ID NO. 5-6.

Example 2 complementation test

To verify that mutation of MtAHA5 gene is a direct factor of proanthocyanidin reduction in NF0707 mutant, the inventors genetically transformed with NF0707 mutant as material into MtAHA5 gene, and then quantitatively analyzed proanthocyanidin in the MtAHA5 gene-transferred plant, and the result is shown in fig. 4.

From the analysis of fig. 4 it can be seen that the soluble proanthocyanidin content was substantially restored to wild-type levels, and that the insoluble proanthocyanidin content was also significantly higher than the NF0707 mutant, but still lower than the wild-type levels. Overall, the MtAHA5 gene was able to restore the reduced proanthocyanidin phenotype in NF0707 mutants, further confirming that the MtAHA5 gene was able to alter the accumulation of proanthocyanidins in medicago truncatula.

Of course, the person skilled in the art can also construct a nucleotide sequence shown as SEQ ID NO.1 according to the MtAHA5 gene sequence provided by the present application.

EXAMPLE 3 analysis of MtAHA5 protein

In order to further explain the functional principle of the MtAHA5 gene, the application researches the MtAHA5 protein and performs subcellular localization on the MtAHA5 protein, specifically, fusion construction of the MtAHA5 gene and the RFP gene into a plant expression vector pCAMBIA1302, and transfer into agrobacterium GV3101, wherein the vector construction method and the transfer method are conventional methods in the field, and the application is not limited. The results of co-injection of Agrobacterium harboring the MtAHA5-RFP gene with Agrobacterium of vac-ck-CFP (Marker line of vacuole film) into tobacco leaves are shown in FIG. 5, which shows: the MtAHA5 protein is positioned in a vacuole membrane, and the amino acid sequence of the MtAHA5 protein is shown as SEQ ID NO. 2. The results of protein localization were consistent with the results of accumulation of proanthocyanidins in the vacuoles, further indicating that the MtAHA5 gene affects transport from the cytoplasm into the vacuoles during proanthocyanidins biosynthesis. It can be seen that the MtAHA5 gene alters the accumulation of proanthocyanidins in vacuoles by altering the transport of proanthocyanidin precursors from the cytoplasm into the vacuoles.

In order to further verify the effect of the MtAHA5 gene in proanthocyanidin accumulation, the inventor uses model plant Arabidopsis thaliana as an experimental plant for verification, and the specific experiment is as follows:

the following examples are only a part of the experiments conducted by the inventors in studying the MtAHA5 gene, and are merely illustrative of the functions of the MtAHA5 gene.

Test example 1 Gene expression Pattern analysis experiment of key genes in the proanthocyanidin biosynthetic pathway during seed development

In order to verify the effect of the MtAHA5 gene provided by the application on changing the content of plant proanthocyanidins, the inventor compares and analyzes the expression pattern of the MtAHA5 gene with the expression pattern of key genes in the biosynthesis pathway of arabidopsis proanthocyanidins in terms of gene expression pattern, and specifically comprises the following steps:

the proanthocyanidin is mainly accumulated in the seed coat, the biosynthesis of which starts in the bead hole region 1 to 2 days after double fertilization, and then gradually accumulates in the seed coat toward the junction until the biosynthesis in the junction region ends at about 5 to 6 days, so the present application first studied the expression patterns of key genes DFR, ANS, ANR, TT, TT19 and TT2 in the proanthocyanidin biosynthesis pathway of Arabidopsis and obtained from Arabidopsis eFP Browser the gene expression data of key genes (DFR, ANS, ANR, TT12, TT19 and TT 2) in the proanthocyanidin biosynthesis pathway at different stages of seed development. Gene expression data shows that arabidopsis thaliana maintains expression of these genes at relatively high levels early in seed development, i.e., before the cardiac phase, while their expression levels decrease rapidly after the cardiac phase, as shown in fig. 6. Of course, the above-described expression patterns of key genes in the proanthocyanidin biosynthetic pathway of Arabidopsis thaliana are also known from the prior art literature (e.g., jiang W, xia Y, su X, pang Y. ARF2 positively regulates flavonols and proanthocyanidins biosynthesis in Arabidopsis thiana. Planta 2022, 256: 44).

Meanwhile, the inventor also compares and analyzes the expression pattern of the MtAHA5 gene with the expression pattern of a typical gene MtANR in the biosynthesis pathway of the medicago sativa proanthocyanidin, and further verifies the function of the MtAHA5 gene in changing the content of the plant proanthocyanidin, which is specifically as follows:

the expression mode of the key gene MtANR in the biosynthesis pathway of the medicago truncatula proanthocyanidin is constructed, and the expression mode of the MtAHA5 gene is constructed, and the analysis result is shown in figure 6 through quantitative analysis of gene expression.

As can be seen from FIG. 6, the above-mentioned critical gene expression was maintained at a higher level at the early stage of the development of alfalfa seeds in which the MtANR gene was constructed, followed by rapid decline. Construction of the MtAHA5 gene of the application also showed that expression was maintained at a higher level at the early stages of development of alfalfa seeds, followed by rapid decline.

Thus, the MtAHA5 gene provided by the application may have similar functions to those of a key gene in the proanthocyanidin biosynthesis pathway of Arabidopsis thaliana and a typical gene in the proanthocyanidin biosynthesis pathway of alfalfa.

Experimental example 2 MtAHA5 gene mediated transport of proanthocyanidin precursors by generating a proton electrochemical potential gradient across the vacuole membrane

The method comprises the steps of (1) carrying out genetic transformation on a aha mutant of arabidopsis not capable of accumulating proanthocyanidins by using a biological conventional method to obtain an arabidopsis gene-transferred strain, wherein the identification result of the expression of the MtAHA5 gene is shown in fig. 7A, and then carrying out qualitative analysis on the proanthocyanidins content by using DMACA staining, wherein the result shows that: expression of the MtAHA5 gene in the aha mutant both before and after staining enabled the accumulation of proanthocyanidins similar to wild type, as shown in fig. 7B and 7C. The results of the quantitative analysis are shown in FIG. 8, and it can be seen that the MtAHA5 gene was able to restore the accumulation of proanthocyanidins in the Arabidopsis aha mutant.

It can be seen that the MtAHA5 gene is capable of restoring the accumulation of arabidopsis proanthocyanidins and enhancing the accumulation of proanthocyanidin precursors in the vacuoles by generating a proton electrochemical potential gradient across the vacuoles to drive the transport of proanthocyanidin precursors into the vacuoles.

Experimental example 3 Effect of MtAHA5 Gene on anthocyanin accumulation experiment

The application also purchases other 2 Tnt insertion mutants NF19445 and NF7687 of the MtAHA5 gene, and adopts the method in the embodiment 1 to carry out PCR identification result and gene expression level analysis, and the identification and analysis result shows that the mutants NF19445 and NF7687 are homozygous mutants of the MtAHA5 gene (as shown in figure 1) and can be used as experimental materials for the functional analysis of the MtAHA5 gene.

The application firstly plants harvested R108 and Tnt1 and inserts homozygous mutant NF0707 seeds, the anthocyanin content in the NF0707 mutant is observed, and the phenotype photo of the planted alfalfa seedlings is shown in figure 9: in fig. 9A, no anthocyanin accumulation was found in the stem base of NF0707 homozygous mutant, while anthocyanin accumulation was found in the stem base of wild-type R108, and dark color was shown, and in fig. 9B, no anthocyanin accumulation was found in the leaf of NF0707 homozygous mutant, while anthocyanin accumulation was found in the stem base of wild-type R108, and dark color was shown; the anthocyanin content of leaves in NF0707 homozygous mutant in fig. 9C was reduced compared to that in wild type leaves. It can be seen that the MtAHA5 gene may be involved in the regulation of anthocyanin.

To further verify whether the MtAHA5 gene was involved in anthocyanin regulation, the application re-planted seeds of the harvested R108 and Tnt1 insert homozygous mutants NF19445 and NF7687, observed anthocyanin phenotypes in three-day seedlings as shown in fig. 10A, and anthocyanin phenotypes in alfalfa seedling stage as shown in fig. 10B, 10C, while detecting anthocyanin content in leaves as shown in fig. 10D.

FIG. 10A shows that the three day seedling hypocotyls of NF0707 are colorless and have no anthocyanin accumulation, while NF19445 and NF7687 are both dark and have anthocyanin accumulation; fig. 10B shows that the stem base of NF0707 at seedling stage is colorless, no anthocyanin accumulation, while NF19445 and NF7687 are both dark in color, with anthocyanin accumulation; FIG. 10C shows that NF0707 leaves have no anthocyanin spots and no anthocyanin accumulation, while NF19445 and NF7687 have anthocyanin spots and anthocyanin accumulation; figure 10D shows that NF0707 has no anthocyanin accumulation, whereas NF19445 and NF7687 have anthocyanin accumulation.

Taken together, the anthocyanin of mutants NF19445 and NF7687 of the MtAHA5 gene was not affected by the mutation of the MtAHA5 gene. The anthocyanin content change in the NF0707 mutant is possibly regulated by other inserted genes in the mutant and is irrelevant to the MtAHA5 gene.

The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the application.

Claims

1. The application of the MtAHA5 gene in preparing alfalfa products for reducing the content of proanthocyanidins is characterized in that an exogenous nucleotide sequence is inserted into the MtAHA5 gene to inhibit the expression level of the MtAHA5 gene so as to reduce the accumulation of alfalfa proanthocyanidins, wherein the nucleotide sequence of the MtAHA5 gene is shown as SEQ ID NO. 1.

2. The application of the MtAHA5 gene in culturing transgenic alfalfa with reduced proanthocyanidin content is characterized in that an exogenous nucleotide sequence is inserted into the MtAHA5 gene to inhibit the expression level of the MtAHA5 gene of the alfalfa so as to reduce the accumulation of proanthocyanidin in the alfalfa, and the nucleotide sequence of the MtAHA5 gene is shown as SEQ ID NO. 1.

3. The application of the MtAHA5 gene in cultivation of alfalfa for preventing the occurrence of ruminant hooves is characterized in that an exogenous nucleotide sequence is inserted into the MtAHA5 gene to inhibit the expression level of the MtAHA5 gene of alfalfa so as to reduce the accumulation of alfalfa proanthocyanidins, wherein the nucleotide sequence of the MtAHA5 gene is shown as SEQ ID NO. 1; the proanthocyanidin content of the alfalfa is reduced to prevent the occurrence of ruminant hooves.

4. The use according to any one of claims 1 to 3, wherein the MtAHA5 gene is obtained by a primer pair consisting of the nucleotide sequences shown in SEQ ID No.3 to 4.

5. Use according to any one of claims 1 to 3 for detecting the expression level of the MtAHA5 gene by means of a primer set having the nucleotide sequence shown in SEQ ID No.5 to 6.