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

Abstract

The invention provides an application of MtAHA5 gene and MtAHA5 protein in alfalfa plants, relates to the fields of biotechnology and genetic engineering applications, and is specifically used in S1) regulating the proton electrochemical potential gradient across the tonoplast in alfalfa plants and promoting proanthocyanidin precursors. Transported into the vacuole of alfalfa plants; S2) Regulate the proanthocyanidins of alfalfa to increase the content of proanthocyanidins in alfalfa; S3) Regulate the proanthocyanidins of alfalfa to reduce the content of proanthocyanidins in alfalfa; S4) Preparation Application in alfalfa products with high content of proanthocyanidins; S5) Application of cultivating transgenic alfalfa with high content of proanthocyanidins; S6) Cultivating alfalfa that prevents the occurrence of ruminant bloat disease, which has a higher content of proanthocyanidins pigment. The present invention provides a practical way to regulate the content of proanthocyanidins in alfalfa plants. The method is simple and is of great significance to animal husbandry.

Description

Application of MtAHA5 gene and MtAHA5 protein in alfalfa plants

Technical field

The invention relates to the fields of biotechnology and genetic engineering applications, and in particular to a MtAHA5 gene, MtAHA5 protein and their applications.

Background technique

Proanthocyanidins (PA), also known as condensed tannins, their basic structural unit is flavan-3-ol, which is a polyphenolic compound produced by oxidation, dehydration and condensation under the action of acid, alkali or enzyme. Macromolecular compounds that are insoluble in water (see "Plant Tannin Chemistry" published by China Forestry Press and edited by Sun Dawang). Most proanthocyanidins in plants exist in the form of polymers. Proanthocyanidins are mainly found in plant leaves, stems, fruits, seeds, flowers and outer bark, and usually appear in the vacuoles of cells. Vegetables, fruits, forage grasses, trees, shrubs, legumes, cereals and seeds are rich in proanthocyanidins. It not only plays an important role in the regulation of seed dormancy, longevity and germination, but also participates in the regulation of biotic and abiotic stresses in plants (Debeaujon et al., 2003). In addition, it also has antioxidant, anti-inflammatory and anti-cancer effects that are beneficial to human health (Dixon et al., 2005).

Alfalfa is known as the "King of Forage" and has the characteristics of high yield, stable yield, easy cultivation, good feeding value and high protein content. However, ruminant animals such as cows will develop bloating disease after eating alfalfa, which seriously limits the utilization and nutritional potential of alfalfa in animal husbandry. Proanthocyanidins are a key factor in resisting bloating disease. When their content in alfalfa is higher than 2% of dry weight, they can effectively prevent the occurrence of bloating disease in ruminants (Verdier et al., 2012). This is due to the fact that proanthocyanidins are Anthocyanins combine with soluble proteins in the rumen of ruminants in a slightly acidic environment (pH 5.5-7.0) to form a more stable tannin-protein conjugate - rumen-passed protein. When it enters the abomasum (pH 2.5-3.5) and small intestine ( pH8.0-8.5), the pH value changes, and the protein is released and absorbed and utilized in the small intestine. This avoids the fermentation and decomposition of soluble protein in the rumen to produce gas, and reduces the degradation and dispersion of soluble protein in the rumen. The foam produced prevents the occurrence of bloating disease and protects rumen protein. Therefore, adding an appropriate amount of proanthocyanidins to the feed of ruminants has certain nutritional and physiological effects. Proanthocyanidins easily combine with proteins in the organism to form relatively stable complexes, which reduce the solubility and surface activity of proteins in the rumen and play a role in protecting proteins. At the same time, proanthocyanidins can inhibit ruminal protein-decomposing bacteria, ultimately improving protein utilization. In addition, the degradation products of proanthocyanidins will not have toxic effects on ruminants. However, excessive proanthocyanidin content (more than 3%) in forage can easily affect the feed intake of ruminants and the degradation rate of forage in the rumen, and reduce the overall digestibility of forage. Due to the astringent and astringent taste of proanthocyanidins, excessive consumption of proanthocyanidins will cause discomfort to the epithelium of the oral cavity and forestomach, thus affecting their palatability and further reducing the feed intake of ruminants. Ruminants that consume excessive amounts of grass rich in proanthocyanidins will suffer from poisoning symptoms and damage organs such as liver and kidney and their functions (Niu Julan et al., study on the protection of ruminal protein by tannins in red bean grass).

Some current research attempts to increase the proanthocyanidin content of alfalfa and other crops (Verdier et al., 2012; Escaray et al., 2014; Yuan and Grotewold, 2015; Li et al., 2016), but the results are not ideal and far from satisfactory. It cannot reach the level of 2% of dry weight, so it cannot effectively prevent the occurrence of bloat disease in ruminants such as cattle and sheep. Zhao et al. and Li et al. believe that the biggest obstacle to obtaining better improvement effects of proanthocyanidins is that proanthocyanidin biosynthesis and regulation are very complex, and there are still many problems that need to be solved (Zhao et al., 2010; Li et al. .,2016).

Contents of the invention

In order to regulate the content of proanthocyanidins in alfalfa plants at the genetic level, thereby obtaining alfalfa with plateau anthocyanin content and reducing the occurrence of bloat disease in ruminants, the present invention provides a method to change the proanthocyanidin content in alfalfa plants by applying the MtAHA5 gene or MtAHA5 protein. The biosynthesis of alfalfa proanthocyanidins makes the proanthocyanidin content in the plant adjustable and controllable, thereby obtaining alfalfa plants with higher proanthocyanidin content.

In order to achieve the technical purpose of the present invention, the first aspect of the present invention provides an application of the MtAHA5 gene, which is S1) or S2) or S3) or S4) or S5) or S6):

S1) Regulate the electrochemical potential gradient of protons across the tonoplast in alfalfa plants and promote the transport of proanthocyanidin precursors into the vacuoles of alfalfa plants;

S2) Regulate the proanthocyanidins of alfalfa to increase the proanthocyanidin content in alfalfa;

S3) Regulate the proanthocyanidins of alfalfa to reduce the content of proanthocyanidins in alfalfa;

S4) Application in preparing alfalfa products with high content of proanthocyanidins;

S5) Application of cultivating transgenic alfalfa with high content of proanthocyanidins;

S6) Cultivation of alfalfa that prevents the occurrence of ruminant bloat disease and contains high proanthocyanidin content.

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

In particular, the MtAHA5 gene is obtained through a primer pair whose nucleotide sequence is shown in SEQ ID NO. 3-4.

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

The second aspect of the invention provides an application of MtAHA5 protein, which is K1) or K2) or K3) or K4) or K5) or K6):

K1) Regulate the electrochemical potential gradient of protons across the tonoplast in alfalfa plants and promote the transport of proanthocyanidin precursors into the vacuoles of alfalfa plants;

K2) Regulate the proanthocyanidins of alfalfa to increase the proanthocyanidin content in alfalfa;

K3) Regulate the proanthocyanidins of alfalfa to reduce the content of proanthocyanidins in alfalfa;

K4) Application in preparing alfalfa products with high content of proanthocyanidins;

K5) Application of cultivating transgenic alfalfa with high content of proanthocyanidins;

K6) Cultivate alfalfa that prevents the occurrence of ruminant bloat disease. This alfalfa contains high proanthocyanidin content.

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

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

B1) Nucleic acid molecules encoding MtAHA5 protein;

B2) An expression cassette containing the nucleic acid molecule described in B1);

B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

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

The third aspect of the present invention provides a regulator of alfalfa proanthocyanidin content, which has the above-mentioned MtAHA5 gene, the above-mentioned MtAHA5 protein, or the above-mentioned biological material described in B1) to B3).

A fourth aspect of the present invention provides a method for cultivating transgenic plants. By increasing the expression of MtAHA5 protein in recipient plants, transgenic plants are obtained; compared with recipient plants, the content of proanthocyanidins in transgenic alfalfa is increased;

Wherein, increasing the expression level of the MtAHA5 protein in the recipient plant is achieved by introducing a nucleic acid molecule encoding the MtAHA5 protein into alfalfa.

Description of the drawings

Figure 1 is a diagram showing the identification and analysis results of the Tnt1 insertion mutant of the MtAHA5 gene provided in Example 1 and Experimental Example 3 of the present invention. Figure 1A shows the Tnt1 insertion position of NF0707, NF19445, and NF7687 mutants, and Figure 1B shows the Tnt1 insertion position of NF0707. The PCR identification results of Tnt1 insertion homozygous mutants. Figure 1C shows the PCR identification results of the Tnt1 insertion homozygous mutants of NF7687. Figure 1D shows the PCR identification results of the Tnt1 insertion homozygous mutants of NF19445. Figure 1E shows the NF0707 homozygous mutations. The results of the expression level analysis of the MtAHA5 gene in the body. Figure 1F shows the results of the expression level analysis of the MtAHA5 gene in the NF7687 homozygous mutant. Figure 1G shows the results of the expression level analysis of the MtAHA5 gene in the NF19445 homozygous mutant.

Figure 2 is a diagram showing the identification and analysis results of the Tnt1 insertion mutants of the MtAHA3, MtAHA4 and MtAHA9 genes provided in Example 1 of the present invention. Figure 2A is the PCR identification result of the Tnt1 insertion homozygous mutant of NF12901; Figure 2B is the PCR identification result of the Tnt1 insertion mutant of NF10457. PCR identification results of Tnt1 insertion homozygous mutants; Figure 2C is the PCR identification results of Tnt1 insertion homozygous mutants of NF3114; Figure 2D is the expression level analysis results of MtAHA3 gene in NF12901 homozygous mutants; Figure 2E is NF10457 homozygous The expression level analysis results of the MtAHA4 gene in the mutant; Figure 2F shows the expression level analysis results of the MtAHA9 gene in the NF3114 homozygous mutant;

Figure 3 is a diagram showing the proanthocyanidin content analysis results of the Tnt1 insertion homozygous mutant provided in Example 1 of the present invention. Figure 3A is the analysis result of the proanthocyanidin content in the NF0707, NF12901, NF10457 and NF3114 homozygous mutants. Figure 3B is the analysis result of proanthocyanidin content in the NF0707, NF19445 and NF7687 homozygous mutants of the MtAHA5 gene;

Figure 4 is an analysis result diagram showing that the MtAHA5 gene provided in Example 2 of the present invention can restore the phenotype of reduced proanthocyanidins in the NF0707 mutant;

Figure 5 is the localization result of MtAHA5 protein after co-injection of Agrobacterium carrying the MtAHA5-RFP gene and vac-ck-CFP into tobacco leaves in Example 3 of the present invention;

Figure 6 is the gene expression pattern analysis results of the key genes in the proanthocyanidin biosynthetic pathway of Arabidopsis thaliana, MtANR and MtAHA5 genes during seed development in Test Example 1 of the present invention;

Figure 7 is the identification result of MtAHA5 gene expression and the qualitative analysis result of proanthocyanidin content in the transgenic line of the aha10 mutant genetically transformed with the MtAHA5 gene in Experimental Example 2 of the present invention. Figure 7A shows the results of the aha10 mutant genetically transformed with the MtAHA5 gene. Results of MtAHA5 gene expression analysis in transgenic lines. Figure 7B shows the seed coat color of the seeds of the transgenic line genetically transformed into the aha10 mutant with the MtAHA5 gene. Figure 7C shows the seed coat color of the seeds of the transgenic line genetically transformed into the aha10 mutant with the MtAHA5 gene after DMACA staining. Seed coat color;

Figure 8 is the quantitative analysis result of proanthocyanidin content in the seeds of the transgenic line of the aha10 mutant genetically transformed with the MtAHA5 gene in Experiment 2 of the present invention;

Figure 9 is an anthocyanin content analysis result diagram and plant photos of the Tnt1 insertion homozygous mutant NF0707 provided in Experimental Example 3 of the present invention;

Figure 10 is an anthocyanin content analysis result diagram and a plant photo of the Tnt1 insertion homozygous mutants NF0707, NF19445, and NF7687 provided in Experimental Example 3 of the present invention: Figure 10A shows the NF0707, NF19445, and NF7687 homozygous mutants of the MtAHA5 gene. The observation results of anthocyanins in the hypocotyl of three-day seedlings. Figure 10B shows the observation of anthocyanins in the seedling stage of the NF0707, NF19445 and NF7687 homozygous mutants of the MtAHA5 gene. The dark color at the base of the stem indicates the accumulation of anthocyanins. Figure 10C is the observation of anthocyanin in the leaves of the NF0707, NF19445 and NF7687 homozygous mutants of the MtAHA5 gene. The anthocyanin spots represent anthocyanin accumulation. D in Figure 10 is the observation of the anthocyanin in the leaves of the NF0707, NF19445 and NF7687 homozygous mutants of the MtAHA5 gene. Analysis results of anthocyanin content in leaves.

Detailed ways

The present invention will be described below with reference to specific examples. It should be noted that these examples are only illustrative and should not be construed as limitations of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and products used are also commercially available. Various processes and methods that are not described in detail are well-known conventional methods in the art. The source and trade name of the reagents used, as well as if it is necessary to list their components, are indicated when they first appear. The same reagents used thereafter will be used unless otherwise specified. Descriptions are the same as those first indicated.

Example 1 Phenotypic analysis of MtAHA5 gene

In order to reflect the role of the MtAHA5 gene in the accumulation of proanthocyanidins in Medicago truncatula, the inventor purchased the Tnt1 insertion mutant NF0707 of this gene, and also used it with the key genes in the proanthocyanidin biosynthetic pathway of Medicago truncatula during seed development. MtAHA3, MtAHA4 and MtAHA9 genes with the same gene expression pattern were used as controls, and the corresponding mutants were NF12901, NF10457 and NF3114 respectively.

First, primers were designed based on the sequence of Tnt1 and the flanking sequences of Tnt1 insertion, and PCR was used to identify Tnt1 insertion homozygous mutants. Then, RNA from the mutants was extracted using conventional methods. Then qRT-PCR was used to analyze the expression levels of genes in the mutants. The identification results and gene expression level results are shown in Figures 1 and 2. It can be seen that the test materials of the present invention are all homozygous mutants, and NF0707 can function as the MtAHA5 gene. Analyzed experimental materials.

R108 and Tnt1 insertion homozygous mutants are planted at the same time. The planting method can be any method in the current technical field, and the present invention is not limited. Harvest mature R108 and Tnt1 insertion homozygous mutant seeds, and then qualitatively and quantitatively analyze the proanthocyanidin content in the seeds. The results are shown in Figure 3. According to the analysis results in Figure 3, it can be found that only the Tnt1 insertion homozygous mutant NF0707 of MtAHA5 The content of proanthocyanidins was significantly less than that of the wild type.

Among them, in one embodiment of the present invention, the transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyanidin and anthocyaninpigmentation in Medicago truncatula published by Jun, J.H., Liu, C., Xiao, Plant Cell. (2015) 27: 2860-2879. Operate. Of course, those skilled in the art can also use other methods to conduct qualitative and quantitative analysis, which is not limited by the present invention.

It can be seen from the above results that MtAHA5 plays a key role in the accumulation of proanthocyanidins in Medicago truncatula.

The MtAHA5 gene provided by the present invention is obtained from the Medicago truncatula A17r5.0 genome, website: https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/. The nucleotide sequence of the MtAHA5 gene As shown in SEQ ID NO.1. In one embodiment, the MtAHA5 gene can be obtained using the primer set shown in SEQ ID NO. 3-4. In one embodiment, the MtAHA5 gene level can also be detected using the detection primer set shown in SEQ ID NO. 5-6.

Example 2 Complementary test

In order to verify that the mutation of the MtAHA5 gene is a direct factor in the reduction of proanthocyanidins in the NF0707 mutant, the inventor used the NF0707 mutant as a material for genetic transformation and introduced the MtAHA5 gene, and then quantified the proanthocyanidins in the plants transformed with the MtAHA5 gene. Analysis, the results are shown in Figure 4.

From the analysis results in Figure 4, it can be seen that the soluble proanthocyanidin content has basically returned to the wild type level, and the insoluble proanthocyanidin content is also significantly higher than that of the NF0707 mutant, but still lower than the wild type level. Overall, the MtAHA5 gene can restore the proanthocyanidin reduction phenotype in the NF0707 mutant, further confirming that the MtAHA5 gene can change the accumulation of proanthocyanidins in Medicago truncatula.

Of course, those skilled in the art can also construct based on the MtAHA5 gene sequence provided by the present invention, such as the nucleotide sequence shown in SEQ ID NO. 1.

Example 3 Analysis of MtAHA5 protein

In order to further elucidate the functional principle of the MtAHA5 gene, this application studies the MtAHA5 protein and conducts subcellular localization of the MtAHA5 protein. Specifically, the MtAHA5 gene and the RFP gene are fused into the plant expression vector pCAMBIA1302 and transferred into Agrobacterium GV3101. The vector construction method and transfer methods are common methods in this field, and are not limited by the present invention. Agrobacterium carrying the MtAHA5-RFP gene and vac-ck-CFP (Marker line of tonoplast) were co-injected into tobacco leaves. The results are shown in Figure 5. The figure shows: MtAHA5 protein is localized in the tonoplast, MtAHA5 The amino acid sequence of the protein is shown in SEQ ID NO.2. The protein localization results are consistent with the accumulation of proanthocyanidins in the vacuole, further indicating that the MtAHA5 gene affects the transport from the cytoplasm to the vacuole during the biosynthesis of proanthocyanidins. It can be seen that the MtAHA5 gene changes the accumulation of proanthocyanidins in the vacuole by changing the transport of proanthocyanidin precursors from the cytoplasm to the vacuole.

In order to further verify the role of the MtAHA5 gene provided by the present invention in the accumulation of proanthocyanidins, the inventor used the model plant Arabidopsis thaliana as an experimental plant for verification. The specific experiments are as follows:

It should be noted that the following experimental examples are only part of the inventor's experimental process of studying the MtAHA5 gene, and are only exemplary to illustrate the function 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 present invention in changing the content of proanthocyanidins in plants, the inventors compared and analyzed the expression pattern of the MtAHA5 gene with the expression pattern of key genes in the proanthocyanidin biosynthetic pathway of Arabidopsis thaliana in terms of gene expression patterns. ,details as follows:

In Arabidopsis, proanthocyanidins are mainly accumulated in the testa. Their biosynthesis starts in the micropyle area 1 to 2 days after double fertilization, and then gradually accumulates in the inner testa toward the chalaza until about 5 to 6 days. The biosynthesis of Tianshi ends in the chalazal region, so the present invention first studies the expression patterns of key genes DFR, ANS, ANR, TT12, TT19 and TT2 in the proanthocyanidin biosynthetic pathway of Arabidopsis thaliana, and obtains them from Arabidopsis eFP Browser Gene expression data of key genes (DFR, ANS, ANR, TT12, TT19 and TT2) in the proanthocyanidin biosynthetic pathway at different stages of seed development were obtained. Gene expression data show that in Arabidopsis, the expression of these genes is maintained at a relatively high level in the early stage of seed development, before the heart-shaped stage, and their expression levels decrease rapidly after the heart-shaped stage, as shown in Figure 6. Of course, the above-mentioned expression patterns of key genes in the proanthocyanidin biosynthesis pathway of Arabidopsis can also be found from existing technical literature (for example, Jiang W, Xia Y, Su X, Pang Y. ARF2 positively regulates flavonols and proanthocyanidins biosynthesis in Arabidopsis thaliana. Planta 2022, 256:44).

At the same time, the inventor also compared and analyzed the expression pattern of the MtAHA5 gene with the expression pattern of the typical gene MtANR in the proanthocyanidin biosynthetic pathway of Medicago truncatula to further verify the function of the MtAHA5 gene provided by the present invention in changing the content of plant proanthocyanidins. details as follows:

The expression pattern of the key gene MtANR in the proanthocyanidin biosynthesis pathway of Medicago truncatula was constructed, and the expression pattern of the MtAHA5 gene of the present invention was constructed at the same time. Through quantitative analysis of gene expression, the analysis results are shown in Figure 6.

As can be seen from Figure 6, the expression of the above-mentioned key genes is maintained at a high level in the early stages of seed development of Medicago truncatula constructed with the MtANR gene, and then decreases rapidly. The MtAHA5 gene constructed in the present invention also shows that the expression is maintained at a high level in the early stages of Medicago truncatula seed development, and then decreases rapidly.

It can be seen that the MtAHA5 gene provided by the present invention may have similar functions to the key genes in the proanthocyanidin biosynthetic pathway of Arabidopsis thaliana and the typical genes in the proanthocyanidin biosynthetic pathway in Medicago truncatula.

Experimental Example 2 MtAHA5 gene mediates the transport of proanthocyanidin precursors by generating a proton electrochemical potential gradient across the tonoplast

Apply conventional biological methods to genetically transform the MtAHA5 gene into the aha10 mutant of Arabidopsis thaliana that cannot accumulate proanthocyanidins, and obtain a transgenic Arabidopsis thaliana gene line. The identification results of MtAHA5 gene expression are shown in Figure 7A, and then stained with DMACA Qualitative analysis of the content of proanthocyanidins showed that the expression of the MtAHA5 gene in the aha10 mutant could make the accumulation of proanthocyanidins similar to that of the wild type before and after staining, as shown in Figures 7B and 7C. The results of quantitative analysis are shown in Figure 8. It can be seen that the MtAHA5 gene can restore the accumulation of proanthocyanidins in the Arabidopsis aha10 mutant.

It can be seen that the MtAHA5 gene can restore the accumulation of proanthocyanidins in Arabidopsis and drive the transport of proanthocyanidin precursors into the vacuole by generating a proton electrochemical potential gradient across the tonoplast membrane, thereby increasing the accumulation of anthocyanin precursors in the vacuole.

Test Example 3 Experiment on the influence of MtAHA5 gene on anthocyanin accumulation

This application also purchased two other Tnt1 insertion mutants of the MtAHA5 gene, NF19445 and NF7687, and used the method in Example 1 to conduct PCR identification results and gene expression level analysis. The identification and analysis results showed that both mutants NF19445 and NF7687 were The homozygous mutant of the MtAHA5 gene (shown in Figure 1) can be used as experimental material for functional analysis of the MtAHA5 gene.

This application first planted the harvested R108 and Tnt1 homozygous mutant NF0707 seeds, and observed the anthocyanin content in the NF0707 mutant. The phenotypic photos of the planted alfalfa seedlings are shown in Figure 9: In the NF0707 homozygous mutant in Figure 9A There is no anthocyanin accumulation at the base of the stem, while there is anthocyanin accumulation at the base of the wild-type R108 stem, showing a dark color. Figure 9B shows that there is no anthocyanin accumulation in the leaves of the NF0707 homozygous mutant, while there is anthocyanin accumulation at the base of the wild-type R108 stem. The anthocyanin content in the leaves of the NF0707 homozygous mutant in Figure 9C is lower than that in the wild-type leaves. It can be seen that the MtAHA5 gene may be involved in the regulation of anthocyanins.

In order to further verify whether the MtAHA5 gene is involved in the regulation of anthocyanins, the harvested seeds of R108 and Tnt1 insertion homozygous mutants NF19445 and NF7687 were planted again and the anthocyanin phenotypes in three-day seedlings were observed, as shown in Figure 10A , as well as the anthocyanin phenotype of alfalfa at the seedling stage, as shown in Figures 10B and 10C, and the anthocyanin content in the leaves was detected at the same time, as shown in Figure 10D.

Figure 10A shows that the hypocotyls of three-day seedlings of NF0707 are colorless and have no anthocyanin accumulation, while NF19445 and NF7687 are both dark and have anthocyanin accumulation; Figure 10B shows that the stem base of NF0707 seedlings is colorless. There is no anthocyanin accumulation, while NF19445 and NF7687 are both dark and have anthocyanin accumulation. Figure 10C shows that NF0707 leaves have no anthocyanin spots and no anthocyanin accumulation, while NF19445 and NF7687 both have anthocyanin spots and have anthocyanin accumulation. Anthocyanin accumulation; Figure 10D shows that NF0707 does not accumulate anthocyanin, while NF19445 and NF7687 have anthocyanin accumulation.

In summary, it can be seen that the anthocyanins of the MtAHA5 gene mutants NF19445 and NF7687 are not affected by the MtAHA5 gene mutation. The changes in anthocyanin content in the NF0707 mutant may be regulated by other inserted genes in the mutant and have nothing to do with the MtAHA5 gene.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.

Claims (5)

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.