Application of MtPDF1 gene in regulation and control of plant growth and development and feed quality

Abstract

The invention discloses application of an MtPDF1 gene in regulation and control of plant growth and development and feed quality, and belongs to the field of genetic engineering, the MtPDF1 gene is derived from medicago truncatula, the nucleotide sequence of the MtPDF1 gene is shown as SEQ ID NO: 1, and the MtPDF1 gene is used for regulating and controlling plant growth and development and feed quality; tnt1 is obtained through screening and inserted into homozygous mutants MtPDF1-1 and MtPDF1-2, and expression analysis shows that the expression quantity of the gene in the mutants is remarkably reduced; phenotype identification shows that the MtPDF1 gene participates in regulating and controlling the plant height, the branch thickness, the root neck thickness and the floret number of each inflorescence; component determination shows that the MtPDF1 gene participates in regulating and controlling the content of lignin, cellulose and pectin; the invention provides important gene resources and technical schemes for plant type improvement, seed yield increase and feed quality genetic improvement of forage grass such as alfalfa.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically the application of the MtPDF1 gene in regulating plant growth and development and feed quality. Background Technology

[0002] In the growth and development of terrestrial plants, their epidermis plays a crucial role in protection and the exchange of gases, water, and nutrients. Research has found that the differentiation of plant epidermis begins in the embryogenesis stage and accompanies it throughout its life cycle. The development of plant epidermal cells originates from the shoot apical meristem. The apical meristem is composed of different cell layers. The outermost layer (L1) forms the epidermal cells of the primary branches through oblique cell division, while the L2 and L3 cell layers produce the internal parts of the plant.

[0003] Although several genes expressed only in L1 have been identified, the molecular mechanisms underlying L1 establishment and maintenance remain unclear. The Arabidopsis meristem layer 1 (ATML1) gene encodes a transcription factor with the homologous domain GLABRA2 (HD-GL2), specifically expressed in the L1 of the developing embryo's protodermis and shoot apex. Its homologs show similar expression patterns in Phalaenopsis, maize, and rice. In Arabidopsis, the PROTODERMAL FACTOR 1 (PDF1) gene was cloned using cDNA abundance differences between wild-type and terminal flower (tfl1) shoot apex meristems, suggesting it encodes a cell wall protein. Inhibition of AtPDF1 expression leads to male sterility in transgenic Arabidopsis. Researchers at the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences reported at an academic conference that PDF1 is highly expressed in the stamens of fertile rapeseed plants, and subcellular localization revealed that the protein is located on the cytoplasmic membrane. Further research suggests that PDF1 may be a suppressor of programmed cell death (PCD). Although the biological functions of some epidermal factor (PDF) genes in plants have been preliminarily revealed, overall research is still in its early stages, and the functions of many genes remain unclear. Particularly in alfalfa, a legume, there are no literature reports on members of the PDF gene family, let alone functional reports. Even those skilled in the art cannot predict their functions. Therefore, the exploration and utilization of this gene resource has significant research and application value.

[0004] Medicago truncatula is an annual herbaceous plant belonging to the genus *Medicago* in the legume family. Due to its small genome, self-pollination, short growth cycle, highly efficient and stable genetic transformation system, and rich natural genetic diversity, mutant resources, and ecotype library, it is internationally recognized as an ideal model plant for legume research. It plays a crucial role in basic research such as symbiotic nitrogen fixation, compound leaf development, and the discovery of stress-resistance genes. Furthermore, its close genetic relationship with high-quality forage alfalfa (*Medicago sativa*) gives it enormous application potential in the field of genetic improvement of forage crops.

[0005] However, in alfalfa breeding, the thickness of the alfalfa stem is a major factor affecting forage quality. The lignin content in the stem has always been a key technical challenge in alfalfa quality breeding, as it affects animal nutrient absorption. Therefore, there is an urgent need to develop forage with low lignin content to improve alfalfa nutrient conversion rate. my country heavily relies on imports for high-quality forage and ecological restoration seeds, and the supply of high-yield, high-quality alfalfa seeds falls short of demand. The number of florets per inflorescence is a factor affecting seed yield; therefore, research on genes related to alfalfa stem and floret development has significant value for alfalfa quality and high-yield breeding. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention screened the MtPDF1 gene from the alfalfa genome using multi-omics analysis and obtained it through gene recombination technology. Homozygous alfalfa mutant materials MtPDF1-1 and MtPDF1-2 were obtained through genomic PCR identification. RT-PCR and RT-qPCR tests showed that the expression level of this gene was significantly reduced in the homozygous mutant materials. Phenotypic observation and index detection revealed that mutants MtPDF1-1 and MtPDF1-2 significantly increased the number of florets per inflorescence compared to the wild type; the lignin and cellulose content of the mutant plants were significantly higher than the control. This indicates that the MtPDF1 gene regulates alfalfa growth and development, significantly controls the number of florets, and positively regulates the lignin and cellulose content in the plant. This will provide excellent genetic resources for improving the yield and quality of alfalfa and other forage crops, as detailed below: An application of the MtPDF1 gene for regulating plant growth and / or regulating the feed quality of plants; the MtPDF1 gene is derived from alfalfa, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0007] Preferably, the coding region nucleotide sequence of the MtPDF1 gene is shown in SEQ ID NO: 2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 3.

[0008] Preferably, the regulation of plant growth and development includes: regulating plant height, branch thickness, root collar thickness and / or the number of florets per inflorescence.

[0009] Preferably, the specific method for regulating plant growth and development is as follows: inhibiting or reducing the expression of the MtPDF1 gene, thereby increasing the number of florets per inflorescence, reducing plant height, increasing branch thickness, and increasing root collar thickness.

[0010] Preferably, the regulation of the feed quality of the plant includes regulating the lignin content, cellulose content and / or pectin content in the plant stem.

[0011] Preferably, the specific method for regulating the feed quality of plants is as follows: enhancing or overexpressing the MtPDF1 gene to increase the pectin content in the plant stem; or inhibiting or reducing the expression of the MtPDF1 gene to reduce the pectin content in the plant stem.

[0012] Preferably, the specific method for regulating the feed quality of plants is as follows: inhibiting or reducing the expression of the MtPDF1 gene to increase the lignin and / or cellulose content in the plant stem; or enhancing or overexpressing the MtPDF1 gene to reduce the lignin and / or cellulose content in the plant stem.

[0013] Preferably, the plant is alfalfa.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to clone and identify the MtPDF1 gene from the legume model plant *Alfalfa tribulus*. Sequence alignment showed that the CDS sequence of this gene is only 52.38% identical to that of *Arabidopsis thaliana* AtPDF1, and the amino acid sequence identity is 45.31%, confirming that MtPDF1 is a previously unreported new gene and providing a novel gene resource for the study of epidermal factor function in legumes.

[0015] 2. This invention reveals for the first time the regulatory function of the MtPDF1 gene in plant growth and development. Phenotypic identification shows that the MtPDF1 gene is involved in regulating multiple agronomic traits of alfalfa: after mutation of this gene, the plant height decreases, while the thickness of primary branches, root collar thickness and the number of florets per inflorescence all increase significantly, providing a new direction for plant type improvement.

[0016] 3. This invention clarifies for the first time the association between the MtPDF1 gene and key traits of forage quality. Component analysis results show that the MtPDF1 gene is involved in regulating the synthesis and accumulation of cell wall components. After mutation of this gene, the lignin and cellulose content in the stem base increases significantly, while the pectin content decreases, providing a theoretical basis and genetic resources for cultivating high-quality forage with moderate lignin content. Attached Figure Description

[0017] Figure 1 The CDS sequence alignment results of the MtPDF1 gene and the Arabidopsis thaliana AT2G42840 gene are shown. Figure 2 The amino acid sequence alignment results between the MtPDF1 gene and the Arabidopsis AT2G42840 gene; Figure 3 Schematic diagram of the insertion sites for MtPDF1-1 and MtPDF1-2 mutants; Figure 4 A represents homozygous material of the Tnt1 mutant of the MtPDF1 gene in alfalfa; B represents the homozygous screening of the Tnt1 insertion mutant of the MtPDF1 gene in alfalfa; C represents the RT-PCR results of the MtPDF1 gene in alfalfa; and C represents the relative gene expression level in the MtPDF1 homozygous material. Figure 5 The following are the 60-day phenotypes of the alfalfa MtPDF1 mutant: A is the plant height phenotype of the alfalfa MtPDF1 mutant; B is the branch thickness phenotype of the alfalfa MtPDF1 mutant; C is the plant height statistics of the alfalfa MtPDF1 mutant; D is the branch thickness statistics of the alfalfa MtPDF1 mutant material; and E is the root collar thickness statistics of the alfalfa MtPDF1 mutant material. Figure 6 Phenotypic and statistical analysis of floret number in *Alfalfa* MtPDF1 mutant materials; Figure 7 The following parameters were determined for the MtPDF1 mutant material of alfalfa: A represents the lignin content of the MtPDF1 mutant material, B represents the cellulose content of the MtPDF1 mutant material, and C represents the total pectin content of the MtPDF1 mutant material. Detailed Implementation

[0018] Example 1: Cloning of the MtPDF1 gene from Alfalfa tribulus The gene MtPDF1 (ID: LOC11420158, MtrunA17_Chr3g0096661) was obtained from the *Alfalfa truncatum* genome through multi-omics analysis. PCR amplification was performed using upstream primer MtPDF1-F (SEQ ID NO: 4) and downstream primer MtPDF1-R (SEQ ID NO: 5). The high-fidelity enzyme 2xPhanta FlashMaster Mix (catalog number: P520) from Novizan Biosciences was selected for the PCR reaction to ensure the fidelity of the amplified products and reduce sequence mutations caused by human manipulation. After electrophoresis, the products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correct CDS sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 3) were obtained by comparing the sequencing results with the electronic sequences. The CDS sequence of the gene MtPDF1 obtained by sequencing showed the highest homology with the AT2G42840 gene in Arabidopsis thaliana, and this Arabidopsis gene is the previously reported AtPDF1. Multiple sequence alignment was performed on the CDS sequences of MtPDF1 and the Arabidopsis thaliana AT2G42840 gene; the alignment results are as follows. Figure 1 As shown, the similarity between the two is 52.38%. Multiple sequence alignment was performed on the amino acid sequences of the MtPDF1 gene and the Arabidopsis AT2G42840 gene; the alignment results are shown below. Figure 2 As shown, the similarity between the two was 45.31%. The comparison results showed that the CDS and amino acid sequences of gene MtPDF1 were significantly different from those of Arabidopsis thaliana AT2G42840, and gene MtPDF1 is a novel gene that has not been reported before.

[0019] Example 2 Screening of MtPDF1 Tnt1 insertion homozygous mutant materials MtPDF1 mutants NF12356 (MtPDF1-1) and NF4268 (MtPDF1-2) were purchased from the alfalfa mutant library. The insertion sites of the two mutants are as follows: Figure 3 As shown. Upstream primer NF12356-F (SEQ ID NO: 6) and downstream primer NF12356-R (SEQ ID NO: 7) for mutant NF12356 (MtPDF1-1) were designed 200 bp before and after the insertion site. Similarly, upstream primer NF4268-F (SEQ ID NO: 8) and downstream primer NF4268-R (SEQ ID NO: 9) for mutant NF4268 (MtPDF1-2) were designed using the same method. Homozygous materials were identified using Tnt1 flanking primers LTR6 (SEQ ID NO: 10) and LTR31 (SEQ ID NO: 11) and gene-specific primers. Figure 4A). PCR amplification using NF12356-F as the upstream primer and NF12356-R as the downstream primer showed no bands in the electrophoresis image. PCR amplification using NF12356-F and Tnt1 flanking primers LTR6 and LTR31 showed one band in the electrophoresis image. PCR amplification using NF12356-R and Tnt1 flanking primers LTR6 and LTR31 also showed one band in the electrophoresis image. This indicates that NF12356 is a homozygous Tnt1 insertion mutant of the MtPDF1 gene, labeled as MtPDF1-1. PCR amplification was performed using NF12630-F as the upstream primer and NF4268-R as the downstream primer, and no bands were observed in the electrophoresis image. PCR amplification was performed using NF4268-F and Tnt1 flanking primers LTR6 and LTR31, and one band was observed in the electrophoresis image. PCR amplification was performed using NF12360-R and Tnt1 flanking primers LTR6 and LTR31, and one band was observed in the electrophoresis image. This indicates that NF12360 is a homozygous Tnt1 insertion mutant of the MtPDF1 gene, and is labeled as MtPDF1-2.

[0020] The obtained homozygous mutant material was propagated, and the harvested seeds were stored at 4℃. Wild-type alfalfa and alfalfa... MtPRP4 After sterilization, mutant seeds were plated on 1 / 2 MS plates and vernalized at 4°C for 48 hours. They were then transferred to an incubator and cultured at 24°C under 16 hours of light / 8 hours of darkness for 7 days. Three healthy and uniformly growing plants were selected and RNA was 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 MtPDF1-qF (SEQ ID NO: 12) and MtPDF1-qR (SEQ ID NO: 13), and internal control gene primers MtActin7-F (SEQ ID NO: 14) and MtActin7-R (SEQ ID NO: 15) were designed to detect gene expression levels. Using the AccurSTART One Step RT-PCR Kit (catalog number: P613-01) from Novizam Biotechnology, specific primers MtPDF1-qF and MtPDF1-qR, and internal control primers MtActin7-F and MtActin7-rRNA, with RNA as a template, RT-PCR experiments were performed to verify the results. Figure 4B. In the wild-type (WT) group, the MtPDF1 band is clearly visible; however, in the two mutants, MtPDF1-1 and MtPDF1-2, the MtPDF1 band is very weak or almost invisible. This clearly demonstrates that, at the transcriptional level, MtPDF1 gene expression in the mutants has been significantly suppressed. (Refer to Novozymes' HiScript IV 1) st The Strand cDNA Synthesis Kit (+gDNAwiper) (Catalog No.: R412-01) is used to synthesize the first strand of cDNA via reverse PCR. RT-qPCR experiments are then performed using the cDNA as a template with the Taq Pro Universal SYBR Qpcr Master Mix. Figure 4 (C) The results showed that the expression level of MtPDF1 in mutants MtPDF1-1 and MtPDF1-2 was significantly reduced compared with wild type (WT).

[0021] Example 3: Phenotypic Identification of MtPDF1 Mutants Gently scratch the outer skin of alfalfa seeds with sandpaper to allow them to absorb water and swell. Then, place the seeds in a petri dish lined with moist filter paper and vernalize them at 4°C for 3 days. After vernalization, transfer them to a photocatalytic incubator for normal cultivation. When the seed roots are about 1-2 cm long, transplant the seedlings into 10 cm diameter square pots filled with nutrient soil and place them in a cultivation chamber. Greenhouse growing conditions are: 16 hours of light, 8 hours of darkness, temperature 22-24°C, humidity 60%-70%, and light intensity 150 μmol / m². -2 s -1 Plants were cultured under the same conditions, and the phenotypes of 2-month-old plants were observed. The results, as shown in the figure, indicate that compared to the wild type, the MtPDF1-1 and MtPDF1-2 mutant plants had significantly lower plant height, but significantly higher thickness of primary branches, root collar thickness, and the number of florets per inflorescence than the control. Figure 5 , Figure 6 ). Figure 5 The figures show the 60-day phenotypes of the alfalfa MtPDF1 mutant. 5A shows the plant height phenotype of the alfalfa MtPDF1 mutant; 5B shows the branch thickness of the alfalfa MtPDF1 mutant; 5C shows the plant height statistics of the alfalfa MtPDF1 mutant; 5D shows the branch thickness statistics of the alfalfa MtPDF1 mutant material; and 5E shows the root collar thickness statistics of the alfalfa MtPDF1 mutant material. Figure 5 A and Figure 5 C shows that compared with the wild type (WT), the plant height of the two mutants MtPDF1-1 and MtPDF1-2 was significantly reduced, indicating that the MtPDF1 gene plays a positive regulatory role in promoting plant height growth. Figure 5 B and Figure 5The D-scan showed that, compared to the wild-type (WT), the primary branches of both mutants were thicker, and the difference was significant. This indicates that the MtPDF1 gene plays a negative regulatory role in branch thickness. Figure 5 E showed that compared with the wild type, the root necks of the two mutants were thicker and the difference was significant, indicating that the MtPDF1 gene also plays a negative regulatory role in the thickening growth of the root neck. Figure 6 Phenotypic and statistical analysis of floret number in MtPDF1 mutant materials of alfalfa showed that, compared with the wild type (WT), the number of florets per inflorescence of the two mutants MtPDF1-1 and MtPDF1-2 was significantly increased. The number of florets per inflorescence of mutants MtPDF1-1 and MtPDF1-2 was significantly higher than that of the wild type, and the difference was statistically significant, proving that the MtPDF1 gene plays a negative regulatory role in floret number of alfalfa.

[0022] In summary, MtPDF1 plays a key role in the aboveground growth and development of alfalfa, positively regulating plant height, while negatively regulating branch thickness, root neck thickness, and the number of florets per inflorescence.

[0023] Example 4: Determination of lignin, cellulose, and pectin content in MtPDF1 mutants Take 2-month-old wild-type alfalfa and mutants MtPDF1- 1 and MtPDF1-2 The stem base of *Alfalfa tribulus* was dried at 80℃ to constant weight, ground, passed through a 40-mesh sieve, and 5 mg was weighed and placed in a centrifuge tube. The lignin content of wild-type *Alfalfa tribulus* and its derivatives was determined using a lignin content assay kit (catalog number: BC4200) from Beijing Solarbio Science & Technology Co., Ltd. MtPDF1 Lignin content in the mutant alfalfa. Results are as follows: Figure 7 As shown in A, it indicates MtPDF1-1 and MtPDF1-2 The lignin content at the base of the stem of the mutant material was significantly higher than that of the wild-type material.

[0024] Take 2-month-old wild-type alfalfa and mutants MtPDF1-1 and MtPDF1-2 0.3g of the stem base of Alfalfa tribulus was used to extract cell wall material using a cellulose content detection kit (catalog number: BC4280) from Beijing Solarbio Science & Technology Co., Ltd., and 5mg of the dried cell wall material was used to determine the cellulose content. Figure 7 B), the results show MtPDF1-1 and MtPDF1-2 The cellulose content at the base of the stem of the mutant material is higher than that of the wild type material.

[0025] Take 2-month-old wild-type alfalfa and mutants MtPDF1-1 and MtPDF1-20.05g of the stem base of *Alfalfa truncatum* was analyzed using a lignin content assay kit (catalog number: BC1400) from Beijing Solarbio Science & Technology Co., Ltd. to determine the lignin content of wild-type *Alfalfa truncatum* and... MtPDF1-1 and MtPDF1-2 Total pectin content in the mutant alfalfa burdock. Results are as follows: Figure 7 C indicates a mutant. MtPDF1 The total pectin content of alfalfa tribulus material is lower than that of wild-type material.

Claims

1. An application of the MtPDF1 gene, characterized in that, The MtPDF1 gene is used to regulate plant growth and / or to regulate the feed quality of plants; the MtPDF1 gene is derived from alfalfa, and its nucleotide sequence is shown in SEQ ID NO: 1; the plant is alfalfa.

2. The application as described in claim 1, characterized in that, The coding region nucleotide sequence of the MtPDF1 gene is shown in SEQ ID NO: 2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:

3.

3. The application according to claim 1, characterized in that, The regulation of plant growth and development includes: regulating plant height, branch thickness, root collar thickness, and / or the number of florets per inflorescence.

4. The application according to claim 3, characterized in that, The specific method for regulating plant growth and development is as follows: inhibiting or reducing the expression of the MtPDF1 gene, thereby increasing the number of florets per inflorescence, reducing plant height, increasing branch thickness, and increasing root collar thickness.

5. The application according to claim 1, characterized in that, The regulation of the feed quality of plants includes regulating the lignin content, cellulose content, and / or pectin content in the plant stems.

6. The application according to claim 5, characterized in that, The specific method for regulating the feed quality of plants is as follows: enhancing or overexpressing the MtPDF1 gene to increase the pectin content in the plant stem; or inhibiting or reducing the expression of the MtPDF1 gene to reduce the pectin content in the plant stem.

7. The application according to claim 5, characterized in that, The specific method for regulating the feed quality of plants is as follows: inhibiting or reducing the expression of the MtPDF1 gene to increase the lignin and / or cellulose content in the plant stem; or enhancing or overexpressing the MtPDF1 gene to reduce the lignin and / or cellulose content in the plant stem.

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