Application of gene MtUMAMIT8 in regulating growth and quality of forage grass

Abstract

The present application relates to the field of biological agriculture, and in particular to the application of the gene MtUMAMIT8 in regulating the growth and development and quality of forage grass. The biological function of the gene MtUMAMIT8 is identified for the first time, the MtUMAMIT8 gene is screened and locked through multi-omics analysis, and the phenotype of the homozygous mutant NF8465 / NF12630 is analyzed, confirming that the down-regulation of the gene MtUMAMIT8 leads to the growth of Medicago truncatula being hindered but the lignin content being reduced, and that the gene MtUMAMIT8 positively regulates the growth and development of the plant height, branch number and leaf size of Medicago truncatula and negatively regulates the synthesis of lignin, thereby providing a new gene resource for regulating the growth and development and quality of forage grass and breeding forage grass varieties with low lignin content.

Description

Technical Field

[0001] This invention relates to the field of bio-agriculture, specifically to genes. MtUMAMIT8 Application in regulating the growth, development, and quality of forage. Background Technology

[0002] Transport proteins, a class of important functional proteins embedded in cell membranes or organelle membranes, precisely mediate the transmembrane transport of ions, small organic molecules (such as carbohydrates and amino acids), and signaling molecules. They play an irreplaceable core role in fundamental life activities such as nutrient uptake, metabolic waste excretion, cell signal transduction, and plant organ morphogenesis. However, due to the large size of the transport protein family, its highly complex gene composition, and significant functional redundancy among members, there are still considerable gaps in the elucidation of the specific physiological functions of most transport proteins, and further research is urgently needed.

[0003] UMAMIT transporters, as important members of the protein transport superfamily, contain 44 and 53 family members respectively in model plants such as Arabidopsis and rice. Although some... UMAMIT The biological functions of genes in Arabidopsis thaliana have been preliminarily revealed, but overall research is still in its early stages, and the functions of many genes remain unclear. This is especially true in alfalfa, a legume. UMAMIT Gene family members MtUMAMIT There are no literature reports on this gene, nor have we seen any reports on its function. Even those skilled in the art cannot predict its function. The discovery and utilization of this gene resource has significant research and application value.

[0004] Tribulus terrestris ( Medicago truncatula As an annual herbaceous plant belonging to the genus *Alfalfa* in the legume family, *Alfalfa* has become an ideal model plant for genetic research in legumes due to its rich natural population genetic diversity, extensive mutant and ecotype resource pool, short growth cycle, compact genome structure, and efficient and stable genetic transformation system. Meanwhile, *Alfalfa truncatum* and *Alfalfa purpurea*, known as the "King of Forage," are... Medicago sativa The high degree of lignin similarity makes it a promising candidate for genetic improvement of forage crops. However, the high lignin content in forage has been a long-standing technical challenge in forage breeding and livestock production, affecting the absorption of nutrients by animals. There is an urgent need to develop forage with low lignin content. However, traditional breeding methods are difficult to precisely control lignin content and have long breeding cycles. There is an urgent need to find new technical solutions to address the problem of high lignin content in forage. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention screens candidate genes from the *Alfalfa tribulus* genome through multi-omics analysis and obtains the genes via gene recombination technology. MtUMAMIT8 Further screening yielded homozygous mutants of the gene, and in-depth research on the gene's function revealed that the gene plays a regulatory role in the growth and development of alfalfa and its lignin content, providing new gene resources for improving the yield and quality of alfalfa and other forage crops.

[0006] On the one hand, this invention provides genes MtUMAMIT8 The gene's application in regulating forage growth and development. MtUMAMIT8 The coding region nucleotide sequence is shown in SEQ ID NO:1; the gene MtUMAMIT8 The sequence encoding the protein is shown in SEQ ID NO:2.

[0007] Furthermore, in the aforementioned application, the forage is alfalfa.

[0008] Furthermore, in the aforementioned application, reducing genes MtUMAMIT8 Expression levels of the gene in alfalfa were observed to inhibit forage growth. MtUMAMIT8 It plays a positive regulatory role in the growth and development of alfalfa.

[0009] Secondly, this invention provides genes. MtUMAMIT8 The gene's application in regulating forage quality. MtUMAMIT8 The coding region nucleotide sequence is shown in SEQ ID NO:1; the gene MtUMAMIT8 The sequence encoding the protein is shown in SEQ ID NO:2; the regulation of forage quality is to reduce the lignin content in the forage.

[0010] Furthermore, in the aforementioned application, the forage is alfalfa.

[0011] Furthermore, in the aforementioned application, reducing genes MtUMAMIT8 The expression level in alfalfa improves the quality of alfalfa, wherein the improvement in quality of alfalfa is achieved by reducing the lignin content in alfalfa.

[0012] Thirdly, the present invention also provides a method for regulating the growth, development, and / or quality of forage, the method comprising regulating genes in forage. MtUMAMIT8 The expression of the gene MtUMAMIT8 The nucleotide sequence is shown in SEQ ID NO:1.

[0013] Fourthly, the present invention also provides a method for reducing the lignin content of forage grass, the method comprising downregulating genes in the forage grass. MtUMAMIT8 The expression of the gene MtUMAMIT8 The nucleotide sequence is shown in SEQ ID NO:1.

[0014] Fifthly, the present invention also provides a method for detecting genes. MtUMAMIT8 The primer pair consists of the upstream primer shown in SEQ ID NO:3 and the downstream primer shown in SEQ ID NO:4.

[0015] Finally, this invention also provides genes. MtUMAMIT8 The application of this gene in the breeding of forage varieties with low lignin content MtUMAMIT8 The coding region nucleotide sequence is shown in SEQ ID NO:1.

[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0017] This invention screens and obtains genes through multi-omics analysis. MtUMAMIT8 Further research on genes MtUMAMIT8 The function, through homozygous mutants MtUMAMIT8 -1 and MtUMAMIT8 Phenotypic analysis of -2 confirmed the downregulated gene. MtUMAMIT8 This results in stunted growth of alfalfa but also a decrease in lignin content. Reducing lignin content can significantly improve the palatability and nutritional value of alfalfa and other forages, solving the technical problem of low digestibility of roughage in animal husbandry.

[0018] In addition, genes MtUMAMIT8 It can be used as a molecular marker to select low-lignin forage varieties, accelerate the breeding process through CRISPR knockout or RNAi technology; and be used to regulate the growth and development of forage and to improve forage quality.

[0019] This invention provides new genetic resources for regulating the growth, development, and quality of forage grasses and for breeding forage varieties with low lignin content. Attached Figure Description

[0020] Figure 1 The gene of alfalfa tribulus MtUMAMIT8 CDS and Arabidopsis homologous genes AT1G75500 CDS sequence alignment results.

[0021] Figure 2 The amino acid sequence of the MtUMAMIT8 protein from alfalfa is homologous to that of Arabidopsis thaliana. AT1G75500 The corresponding amino acid sequence alignment results.

[0022] Figure 3 The gene of alfalfa tribulus MtUMAMIT Identification results of homozygous Tnt1 mutant in gene 8. A represents the alfalfa truncatum gene. MtUMAMIT8 Results of homozygous screening for Tnt1 insertion mutants; B represents the alfalfa gene. MtUMAMIT8 Expression level; C represents the gene expression level in the homozygous mutant. MtUMAMIT8 The relative expression level.

[0023] Figure 4 This is a schematic diagram of the flanking sequences of the NF8465 mutant.

[0024] Figure 5 The image shows the sequence alignment results for the NF8465 mutant flanking segments.

[0025] Figure 6 This is a schematic diagram of the flanking sequences of the NF12360 mutant.

[0026] Figure 7 The image shows the sequence alignment results for the NF12360 mutant flanking segments.

[0027] Figure 8 alfalfa MtUMAMIT8 Mutant phenotypic identification results. A is alfalfa tribulus. MtUMAMIT8 Root phenotype of mutant; B is alfalfa tribulus. MtUMAMIT8 Root length statistics of mutants; C represents alfalfa. MtUMAMIT8 Statistical graph of lateral root count in mutants.

[0028] Figure 9 alfalfa MtUMAMIT8 Mutant plant height and branching phenotype. A is alfalfa tribulus. MtUMAMIT8 Phenotypic diagrams of mutant plant height and branch number; B represents alfalfa truncatum. MtUMAMIT8 Statistical graph of mutant plant height; C represents alfalfa truncatum. MtUMAMIT8 Statistical chart of the number of branches in mutants.

[0029] Figure 10 alfalfa MtUMAMIT8 Leaf area phenotype of mutants. A represents alfalfa burjorn. MtUMAMIT8 Leaf area phenotypic diagram of mutants; B represents alfalfa. MtUMAMIT8 Statistical chart of leaf area of ​​mutant.

[0030] Figure 11 alfalfa MtUMAMIT8 Secondary branching phenotype of the mutant. A represents alfalfa burdock. MtUMAMIT8 Secondary branching phenotypic diagram of mutants; B represents alfalfa tribulus. MtUMAMIT8 Statistical diagram of the number of secondary branches in mutants; C represents alfalfa truncatum. MtUMAMIT8 A statistical chart showing the number of nodes on each of the four secondary branches of the mutant, with the number indicating the growth rate.

[0031] Figure 12 alfalfa MtUMAMIT8 Results of lignin content determination in mutants. Detailed Implementation

[0032] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0034] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0035] Example 1

[0036] This embodiment is for obtaining the Medicago truncatum gene. MtUMAMIT8 .

[0037] Genes were screened from the genome of Alfalfa tribulus through multi-omics analysis. MtUMAMIT8 (GeneID: LOC25490244 , MtrunA17_Chr3g0142131 Design upstream primers MtUMAMIT8 -F and downstream primers MtUMAMIT8 -R, use MtUMAMIT8 -F and MtUMAMIT8 The -R primer pair was used to amplify the gene via PCR using cDNA from alfalfa as a template. MtUMAMIT8 The full-length CDS was obtained. The PCR reaction enzyme used was the high-fidelity enzyme 2xPhanta FlashMaster Mix (catalog number: P520) from Novizan Biosciences to ensure the fidelity of the amplified products. After PCR electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results of the PCR products are shown in SEQ ID NO:1, which is the gene. MtUMAMIT8 The CDS sequence of the MtUMAMIT8 protein is shown in SEQ ID NO:2.

[0038] Genes obtained through sequencing MtUMAMIT8 The CDS sequence is homologous to that in Arabidopsis thaliana. AT1G75500 Gene multiple sequence alignment was performed, and the alignment results are as follows: Figure 1 As shown, the two have a 68.18% similarity. The amino acid sequence of protein MtUMAMIT8 was compared with... AT1G75500 Multiple sequence alignment was performed on the corresponding protein, and the alignment results are as follows: Figure 2 As shown, the similarity between the two is 74.56%. The results indicate that the gene... MtUMAMIT8 with Arabidopsis AT1G75500 Significant differences in gene CDS and amino acid sequence; genes MtUMAMIT8 This is a novel gene that has not been previously reported.

[0039] Example 2

[0040] This embodiment is MtUMAMIT8 Screening of Tnt1 insertion homozygous mutant materials.

[0041] Purchase from the Tribulus terrestris mutant library (https: / / medicago-mutant.dasnr.okstate.edu) MtUMAMIT8 Mutants NF8465 and NF12630 were developed. Upstream primers NF8465-F and NF12630-F were designed 200 bp before the Tnt1 insertion site; downstream primers NF8465-R and NF12630-R were designed 200 bp after the Tnt1 insertion site. The Tnt1 flanking primers LTR6 and LTR31 were then used to connect to the gene... MtUMAMIT8 The mutant upstream and downstream primers (NF8465-F, NF12630-F, NF8465-R, and NF12630-R) were used to identify homozygotes. The identification results are as follows: Figure 3 As shown in Figure A.

[0042] PCR amplification using NF8465-F as the upstream primer and NF8465-R as the downstream primer showed no bands in the electrophoresis image. PCR amplification using NF8465-F and Tnt1 flanking primers LTR6 and LTR31 showed one band in the electrophoresis image. PCR amplification using NF8465-R and Tnt1 flanking primers LTR6 and LTR31 also showed one band in the electrophoresis image. This indicates that NF8465 is a gene. MtUMAMIT8 Homozygous Tnt1 insertion mutants were labeled as MtUMAMIT8 -1. PCR amplification using NF12630-F as the upstream primer and NF12630-R as the downstream primer showed no bands in the electrophoresis image. PCR amplification using NF12630-F and Tnt1 flanking primers LTR6 and LTR31 showed one band in the electrophoresis image. PCR amplification using NF12360-R and Tnt1 flanking primers LTR6 and LTR31 also showed one band in the electrophoresis image. This indicates that NF12360 is a gene. MtUMAMIT8 Homozygous Tnt1 insertion mutants were labeled as MtUMAMIT8 -2.

[0043] The homozygous mutant materials obtained by screening MtUMAMIT8 -1 and MtUMAMIT8 Propagation was carried out at -2°C, and the harvested seeds were stored at 4°C. Wild-type alfalfa (WT) and alfalfa... MtUMAMIT8After sterilization, mutant seeds were plated on 1 / 2 MS plates and vernalized at 4°C for 48 h. They were then transferred to an incubator and cultured at 24°C under 16 h light / 8 h dark conditions for 7 days. Three healthy and uniformly growing plants were selected and RNA extracted from them. The RNAprep Pure plant total RNA extraction kit (catalog number: DP432) from Tiangen Biotech Co., Ltd. was used as instructed. The integrity of the extracted RNA was assessed by gel electrophoresis. Specific primers were designed. MtUMAMIT8 -CF and MtUMAMIT8 -CR, internal reference gene primers MtActin7 -F and MtActin7 -R Detection of Genes MtUMAMIT8 The expression level was determined. First-strand cDNA was synthesized via reverse transcription using the HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper) (catalog number: R412-01) from Novizan Biosciences. RT-PCR experiments were performed using the cDNA as a template with 2 × Rapid Taq Master Mix. The semi-quantitative PCR results are shown below. Figure 3 As shown in B, the internal reference gene is displayed. MtActin7 In wild-type alfalfa (WT), mutant MtUMAMIT8- 1 and mutant MtUMAMIT8 The uniform brightness of the bands in sample -2 indicates that the amount of cDNA used was consistent. (Target gene) MtUMAMIT8 In mutants MtUMAMIT8 -1 and mutants MtUMAMIT8 The band brightness in the cDNA of -2 is lower than that of wild-type alfalfa (WT), indicating that the alfalfa mutant gene... MtUMAMIT8 The expression level of the gene was significantly lower than that of the wild-type gene. MtUMAMIT8 The expression level. qPCR experiments were performed using TaqPro Universal SYBR Qpcr Master Mix with cDNA as a template. The qPCR results are as follows: Figure 3 As shown in C. Wild-type genes MtUMAMIT8 The expression level was 1, while the mutant... MtUMAMIT8 -1 and mutants MtUMAMIT8 In -2, the gene expression levels were 0.02 and 0.01, respectively, indicating that the gene... MtUMAMIT8 The expression level was significantly reduced in the mutant.

[0044] The alfalfa genome website has identified NF8465 and NF12630 as genes. MtUMAMIT8 The mutant NF8465 and NF12630 also exhibit Tnt1 sequence insertions in other genes. To verify that NF8465 and NF12630 are, and only are, gene mutations... MtUMAMIT8 Based on the above analysis, the following supplementary explanations were provided regarding the Tnt1 insertion mutant.

[0045] (1) Enter the following into the website for the Medicago-mutant mutant (medicago-mutant.dasnr.okstate.edu): MtUMAMIT8 The -1 mutant's ID, NF8465, can be used to obtain or display all flanking sequences of this mutant, as shown in the following example. Figure 4 As shown.

[0046] (2) The above flanking sequences were BLAST aligned on the Alfalfa genome website (https: / / medicago.toulouse.inra.fr / MtrunA17r5.0-ANR / ), and the results are as follows. Figure 5 As shown in the figure and Table 1.

[0047] From the above alignment results, sequences with an alignment rate of 95% or higher were selected and sorted from highest to lowest. A total of 5 sequences were obtained (these 5 sequences belong to 4 genes). MtrunA17_Chr4g0039341 No flanking label indicates that no Tnt1 flanking sequence was inserted; MtrunA17_Chr3g0136981 The Tnt1 flanking sequence is inserted at an intron location, but not at an exon location; therefore, the inserted sequence cannot cause gene mutation. Thus, in this invention, NF8465 is and only is a gene. MtUMAMIT8 Tnt1 insertion mutant.

[0048] Table 1. Gene alignment results of NF8465 mutant

[0049]

[0050] Similarly, enter the following into the website for the Medicago-mutant mutant (medicago-mutant.dasnr.okstate.edu): MtUMAMIT8 By using the mutant number -2, i.e., NF12360, all flanking sequences of that mutant can be obtained or displayed. Figure 6 As shown.

[0051] (2) The above flanking sequences were BLAST aligned on the Alfalfa genome website (https: / / medicago.toulouse.inra.fr / MtrunA17r5.0-ANR / ), and the results are as follows. Figure 7 As shown in the figure and Table 2.

[0052] From the above alignment results, sequences were sorted from highest to lowest alignment rate, and those with an alignment rate of over 95% were selected, resulting in two sequences (belonging to two different genes). MtrunA17_Chr6g0454371 The absence of flanking tags indicates the absence of Tnt1 flanking sequence insertion. Therefore, NF12360 in this invention is and only is a gene. MtUMAMIT8 Tnt1 insertion mutant.

[0053] Table 2. Gene alignment results of NF12360 mutant

[0054]

[0055] Example 3

[0056] This embodiment is MtUMAMIT8 Mutant phenotype identification.

[0057] Observe wild-type alfalfa (WT) and clover cultured vertically for 7 days on 1 / 2 MS plates. MtUMAMIT8 -1 mutant alfalfa and MtUMAMIT8 -2 mutant alfalfa seedling root phenotype. Results are as follows. Figure 8 As shown, it indicates MtUMAMIT8 -1 mutant alfalfa and MtUMAMIT8 The -2 mutant alfalfa had significantly shorter root length and fewer lateral roots than the wild type.

[0058] The above-mentioned wild-type alfalfa and MtUMAMIT8 -1 and MtUMAMIT8 -2 mutant alfalfa seedlings were transplanted into soil and cultured under the same conditions for one month. The phenotype of one-month-old plants was then observed. Results Figure 9 and Figure 10 As shown, the results indicate that compared to the wild type, MtUMAMIT8 -1 and MtUMAMIT8 The -2 mutant alfalfa had significantly reduced leaf area, plant height, and number of lateral branches.

[0059] Statistical analysis of wild type and mutants separately MtUMAMIT8 -1 and MtUMAMIT8 -2 The number of secondary branches on each primary branch of *Alfalfa tribulus*, as shown in the results. Figure 11 As shown, this indicates the mutants on each first-order branch. MtUMAMIT8 -1 and MtUMAMIT8 -2 The number of secondary branches in Medicago sativa 'Berberis' was significantly lower than that in the wild type.

[0060] This embodiment demonstrates the gene MtUMAMIT8 Genes play a crucial role in root development, aboveground growth (plant height, leaf area), and branching pattern (primary and secondary branching) of alfalfa. MtUMAMIT8 It has a positive regulatory effect on the growth of alfalfa.

[0061] Example 4

[0062] This embodiment is MtUMAMIT8 Test for determining the lignin content of mutants.

[0063] Take 2-month-old wild-type alfalfa (WT) and mutants MtUMAMIT8 -1 and MtUMAMIT8 -2. The stem base, middle, and top of *Alfalfa tribulus* were dried at 80℃ to constant weight, ground, and 3 mg was weighed and placed in a centrifuge tube. The lignin content of wild-type *Alfalfa tribulus* and... (The sentence is incomplete and requires further context to translate accurately.) MtUMAMIT8 Lignin content in the mutant alfalfa. Results are as follows: Figure 12 As shown, compared to wild-type alfalfa, MtUMAMIT8 -1 and MtUMAMIT8 The -2 mutant alfalfa showed significantly reduced lignin content in the base, middle, and top of the stem. This indicates that the gene... MtUMAMIT8 It plays a negative regulatory role in lignin synthesis in alfalfa.

[0064] The primer information involved in the above embodiments is shown in Table 3:

[0065] Table 3 Primer sequence list

[0066]

[0067] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Reduce genetic risk MtUMAMIT8 The application of expression levels in forage grasses in regulating forage grass growth and development is characterized by... The gene MtUMAMIT8 The coding region nucleotide sequence is shown in SEQ ID NO:1; the gene MtUMAMIT8 The sequence encoding the protein is shown in SEQ ID NO:2; the regulation of forage growth and development is to reduce the root length, number of lateral roots, leaf area, plant height, number of lateral branches or number of branches of the forage, and the forage is alfalfa.

2. Reduce gene levels MtUMAMIT8 The application of expression levels in forage in regulating forage quality is characterized by, The gene MtUMAMIT8 The coding region nucleotide sequence is shown in SEQ ID NO:1; the gene MtUMAMIT8 The sequence encoding the protein is shown in SEQ ID NO:2; the regulation of forage quality is to reduce the lignin content of the forage, and the forage is alfalfa.

3. A method for regulating the growth and development of forage grasses, characterized in that, Including reducing the genetic content of forage MtUMAMIT8 The expression of the gene MtUMAMIT8 The nucleotide sequence is shown in SEQ ID NO:

1. The regulation of forage growth and development is to reduce the root length, number of lateral roots, leaf area, plant height, number of lateral branches or number of branches of the forage. The forage is alfalfa.

4. A method for reducing the lignin content of forage grass, characterized in that, Including downregulating genes in forage MtUMAMIT8 The expression of the gene MtUMAMIT8 The nucleotide sequence is shown in SEQ ID NO:1, and the forage is alfalfa.

5. Reduce gene density MtUMAMIT8 The application of expression levels in forage for the selection of forage varieties with low lignin content is characterized by, The gene MtUMAMIT8 The coding region nucleotide sequence is shown in SEQ ID NO:1, and the forage is alfalfa.

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