MsGRF1c regulates root nodule senescence and biomass in alfalfa

Abstract

This application provides the use of the MsGRF1c gene in regulating alfalfa nodule senescence, nitrogen fixation efficiency, and biomass. This application demonstrates that the MsGRF1c gene can delay alfalfa nodule senescence, improve nodule nitrogen fixation efficiency, and thereby increase plant biomass under low-nitrogen conditions. This new discovery complements the regulatory mechanism for nodule senescence in legumes and has important utility in reducing nitrogen fertilizer application.

Description

Technical Field

[0001] The present application belongs to the field of molecular biology. Specifically, the present application provides an application of the MsGRF1c gene in regulating alfalfa nodule senescence and biomass. Background Art

[0002] The symbiotic nitrogen fixation system formed by legumes and rhizobia is the most effective and economical way for plants to obtain nitrogen, and is also an important nitrogen input pathway in agricultural ecosystems. According to statistics, legumes and rhizobia in the soil symbiotically fix about 1.75×10 8 The amount of nitrogen available to plants per ton is approximately 4.73 times the global output of industrial nitrogen fertilizer. Symbiotic nitrogen fixation accounts for 30%-50% of the nitrogen in agricultural land. Therefore, efficient utilization of symbiotic nitrogen fixation is crucial for sustainable agricultural development.

[0003] Alfalfa is an important perennial legume forage grass with high protein content and good palatability. It is known as the "king of forage grasses" and is a vital forage grass for maintaining the sustainable development of my country's animal husbandry. In recent years, with the increasing demand for meat, eggs, and milk in the residents' diet, my country's demand for high-quality alfalfa has also increased year by year. Currently, increasing the use of nitrogen fertilizers remains one of the important ways to ensure a significant increase in the yield of alfalfa and other crops. However, the large-scale use of nitrogen fertilizers not only reduces the efficiency of alfalfa's biological nitrogen fixation system, but also causes a series of ecological problems such as eutrophication of water bodies and nitrate pollution. In the face of the ecological and economic pressures caused by fertilization, fully leveraging and utilizing the biological nitrogen fixation capacity of legumes such as alfalfa is an important way to achieve sustainable agricultural development and protect the ecological environment.

[0004] Growth-Regulating Factors (GRFs) belong to a family of plant-specific transcription factors and are highly conserved in terrestrial plants. Different plant species contain varying numbers of GRFs, such as 9 in Arabidopsis thaliana, 12 in rice (Oryza sativa), 8 in grape (Vitis vinifera), 19 in poplar (Populus trichocarpa), and 10 in Chinese pear (Pyrus bretschneideri). These members exhibit either functional redundancy or specificity. Most GRFs contain miR396 binding sites, resulting in post-transcriptional cleavage by miR396, leading to their repression.

[0005] GRFs regulate the growth and development of plant organs such as roots, stems, leaves, flowers, fruits, and seeds, but their role in nodule growth in legumes is less well-researched. GRFs also participate in regulating several important agronomic traits in plants, such as nitrogen utilization, biomass, and grain yield. OsGRF4 has been reported to promote and integrate nitrogen uptake, carbon fixation, and growth, making it a rare growth and metabolism integrator. AtGRF5 has great potential for improving plant productivity. Overexpressing its gene results in higher photosynthetic rates and improved tolerance to growth on nitrogen-deficient media. OsGRF1 / 4 / 6 / 8 regulate rice grain yield. PvGRF9 promotes biomass accumulation in switchgrass stems. Currently, no studies have examined the role of GRFs in nitrogen utilization and biomass in the legume alfalfa. GRFs also play a crucial role in regulating leaf lifespan in Arabidopsis thaliana. Studies have reported that AtGRF1, AtGRF3, AtGRF4, and AtGRF5 delay leaf senescence. However, studies examining the role of GRFs in regulating nodule senescence have yet to be reported. Summary of the Invention

[0006] In one aspect, the present application provides the use of the MsGRF1c gene in regulating root nodule senescence in alfalfa plants.

[0007] Furthermore, in the application, the MsGRF1c gene is overexpressed to inhibit nodule senescence.

[0008] On the other hand, the present application provides the use of the MsGRF1c gene in regulating the nitrogen fixation efficiency of alfalfa plants.

[0009] Furthermore, in the application, the MsGRF1c gene is overexpressed to improve nitrogen fixation efficiency.

[0010] On the other hand, the present application provides the use of the MsGRF1c gene in regulating the biomass of alfalfa plants.

[0011] Furthermore, in the application, the MsGRF1c gene is overexpressed to increase biomass.

[0012] Furthermore, the regulating the biomass of alfalfa plants is regulating the biomass of alfalfa plants under low nitrogen conditions.

[0013] Furthermore, in the application, an overexpression vector is constructed to overexpress the MsGRF1c gene.

[0014] Furthermore, the MsGRF1c gene sequence is SEQ ID NO.1 or SEQ ID NO.3.

[0015] Furthermore, the alfalfa plant is alfalfa.

[0016] The overexpression means in this application can adopt overexpression means known in the field of molecular biology, and are not limited to the construction of overexpression vectors. Available overexpression vectors are also not limited to the types used in the examples. Those skilled in the art can routinely select and construct any type known to be applicable to alfalfa plants.

[0017] The present invention discovered that the MsGRF1c gene can delay alfalfa nodule senescence, improve nodule nitrogen fixation efficiency, and thus increase plant biomass under low-nitrogen conditions. This invention supplements the regulatory mechanism of leguminous plant nodule senescence and has important application value in reducing nitrogen fertilizer application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In situ PCR analysis of MsGRF1c in nodules (15 dpi); Note: The scale bar represents 0.25 mm. M: meristem and infection zone; NF: nitrogen-fixing zone; VB: vascular bundle;

[0019] Figure 2 Alignment of the nucleotide (a) and amino acid sequences (b) of cloned MsGRFc with the search sequence (MSAD-280575); Note: The boxes indicate the target sequence sites of miR396

[0020] Figure 3 Schematic diagram of the T-DNA region of MsGRF1c-related vectors; (a) Schematic diagram of the synonymous mutant bases (red bases) of rMsGRF1c; the yellow sequence represents the DNA sequence of the miR396 recognition site; (b) T-DNA region of the pZh01-rMsGRF1c plant expression vector; (c) T-DNA region of the pZh01-MsGRF1c-SRDX plant expression vector. Note: HygR: hygromycin resistance gene; NOS: terminator; CaMV: tobacco mosaic virus; LB and RB: left and right ends of the T-DNA.

[0021] Figure 4 PCR identification of transgenic plants overexpressing and suppressing MsGRF1c; (a) PCR identification of transgenic plants overexpressing rMsGRF1c (rMsGRF1c-OX, rOX); (b) PCR identification of transgenic plants overexpressing MsGRF1c-SRDX (MsGRF1c-SRDX, SR). Note: M: marker (DL2000); WT: wild-type plant, serving as a negative control; +: pZh01-rMsGRF1c plasmid (a) and pZh01-MsGRF1c-SRDX plasmid (b), serving as positive controls; -: blank control.

[0022] Figure 5The expression levels of rMsGRF1c-overexpressing transgenic plants are shown in Figure 2. Note: rOX: rMsGRF1c-overexpressing transgenic plants; WT: wild-type plants. L: low expression; M: moderate expression; H: high expression. Values represent mean ± SD (n = 3).

[0023] Figure 6 The expression levels of transgenic plants overexpressing MsGRF1c-SRDX are shown in Figure 2. SR: transgenic plants overexpressing MsGRF1c-SRDX; WT: wild-type plants. L: low expression; M: moderate expression; H: high expression. Values represent mean ± SD (n = 3).

[0024] Figure 7 Statistical analysis of plant morphology and phenotypes of MsGRF1c-related transgenic plants; (a) Photograph of a one-month-old rMsGRF1c-overexpressing transgenic plant; (b) Photograph of a one-month-old MsGRF1c-SRDX-overexpressing transgenic plant; (c) Morphology of the fourth leaf from the top of a one-month-old rMsGRF1c-overexpressing transgenic plant; (d) Morphology of the fourth leaf from the top of a one-month-old MsGRF1c-SRDX-overexpressing transgenic plant; (e) Plant height of one-month-old plants; (f) Stem diameter of the fourth node from the top of a one-month-old M plant; (g) and (h) Length (g) and width (h) of the fourth leaf from the top of a one-month-old plant. Note: rOX: rMsGRF1c-overexpressing transgenic plant; SR: MsGRF1c-SRDX-overexpressing transgenic plant; WT: wild-type plant. L: low expression; M: moderate expression; H: high expression. The scale bar represents 1 cm. The values are mean ± SD (n = 3). Different lowercase letters in the values indicate significant differences.

[0025] Figure 8 Figure 3. Aboveground phenotypes of MsGRF1c-overexpressing and -repressed transgenic plants under low-nitrogen rhizobium inoculation conditions. (a) and (b) Morphology of transgenic plants overexpressing rMsGRF1c (a) and MsGRF1c-SRDX (b) 60 days after rhizobium inoculation. (c) Plant height 60 days after rhizobium inoculation. (d) Plant dry weight 60 days after rhizobium inoculation. Note: WT: wild-type plant; rOX: rMsGRF1c-overexpressing transgenic plant; SR: MsGRF1c-SRDX-overexpressing transgenic plant. Values are mean ± SD (n = 7). "*" indicates a significant difference between transgenic and wild-type plants ("*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001).

[0026] Figure 9Phenotypic analysis of roots and nodules (60 dpi) in MsGRF1c-overexpressing and -repressed transgenic plants; (a), (b), and (c) Photographs of roots of WT (a), rMsGRF1c-overexpressing (b), and MsGRF1c-repressed (c) transgenic plants (bars represent 2 cm); (d), (e), and (f) Photographs of nodules of WT (d), rMsGRF1c-overexpressing (e), and MsGRF1c-repressed (f) transgenic plants (bars represent 5 mm); (g) Nodule size (n = 90); (h) Percentage of effective nodules (n = 3). Note: WT: wild-type plant; SR: MsGRF1c-SRDX-overexpressing transgenic plant; rOX: rMsGRF1c-overexpressing transgenic plant. Values represent mean ± SD. “*” indicates significant differences between transgenic and wild-type plants (“*” indicates P < 0.05, “**” indicates P < 0.01, and “***” indicates P < 0.001).

[0027] Figure 10 Staining analysis of nodules (21 dpi) from MsGRF1c-overexpressing and -repressed transgenic plants; (a) (b) (c) Toluidine blue staining of WT (a), MsGRF1c-repressed (b), and rMsGRF1c-overexpressing (c) transgenic nodules (top: intact nodule section; bottom: close-up of a nodule section near the root tip); (d) (e) (f) DAB staining of WT (d), MsGRF1c-repressed (e), and rMsGRF1c-overexpressing (f) transgenic nodules. Note: WT: wild type; SR: MsGRF1c-SRDX-overexpressing transgenic plant; rOX: rMsGRF1c-overexpressing transgenic plant. Scale bars for intact nodules represent 0.5 mm; scale bars for close-up nodules represent 0.2 mm.

[0028] Figure 11 Figure 2: DCFH-DA reactive oxygen species assay in nodules (21 dpi) of MsGRF1c-overexpressing and -repressed transgenic plants. Note: WT: wild-type plant; SR: MsGRF1c-SRDX-overexpressing plant; rOX: rMsGRF1c-overexpressing plant. M: meristem and infection zone; UNF: upper nitrogen-fixing zone; LNF: lower nitrogen-fixing zone. Scale bar represents 0.25 mm. Values represent mean ± SD (n = 8). "*" indicates a significant difference between transgenic and wild-type nodules ("*" indicates P < 0.05, "***" indicates P < 0.001). Different lowercase letters indicate significant differences between different nodule compartments of the same line (P < 0.05). DETAILED DESCRIPTION

[0029] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and in no way limit the scope of protection of the present invention.

[0030] Unless otherwise specified, the equipment and reagents used in each example are commercially available.

[0031] Example 1 Analysis of the expression pattern of MsGRF1c in nodules

[0032] In situ PCR experiments of MsGRF1c in nodules revealed that MsGRF1c was mainly expressed in the meristem, infection zone and vascular bundle of nodules at 15 dpi (e.g. Figure 1 ).

[0033] Example 2: Cloning of the alfalfa MsGRF1c gene

[0034] Based on the obtained MsGRF1c gene sequence, primers covering the full-length sequence were designed and the corresponding sequence was cloned from the cDNA of 'Zhongmu No. 1'. The cloned sequence of MsGRF1c differed from the search sequence by one base ( Figure 2 a), the corresponding amino acid sequence differs by one amino acid but has conserved QLQ and WRC domains ( Figure 2 b). The actual amplified sequence (MsGRF1c) has a CG difference at nucleotide position 636 with the query sequence (MSAD_280575.t1). >MsGRF1c nucleotide sequence (SEQ ID NO.1)

[0035] ATGATGAATGGAAGAAACAGGTATCCTTTACTCCAACTCAGTGGCAAGAGCTTGAACATCAAGCTCTTATCTACAAATA

[0036] CATGGCTTCTGGTATTTCCATTCCTCCTGATCTTCTACTCACTATCAAAAGAAGCTACTTAGACTCTTCAAGACTCTCAC

[0037] CTCATCAGTCTCAACACTTTGGATGGAACTATTTACCAATGGGATTGGGAAGAAAAATAGACCCAGAGCCAGGGAGGTGC

[0038] AGAAGAACAGATGGAAAGAAATGGAGGTGTTCAAAAGAAGCATATCCAGATTCAAAGTACTGTGAAAGGCACATGCATAG

[0039] AGGGAAAAACCGTTCAAGAAAGCCTGTGGAAGTTTTGAAAACAACAACAAACACAGATGCTCCTGCTGCTACTTCAACAA

[0040] TCTTATCAATCACTAAAAATAGTAGTCTTGATTCTGATCATAACACTGCAGCACTAACATCAACAACTCATGACACTAAT

[0041] TACCATTACCCTCAACACTCTTCTTATGCTTCTTCCCATCTTAACCACCCTTTCCTCTACCATAACCCTCCATCCTCAAG

[0042] GCCTAACTTCTCATTTCAAGACAATAGTGCTCCCTTGTTTCTTGACAATACTGGTTCTTCCTCTCACAATAACAACAACA

[0043] CTGATTGCAGGTATGTTTATGGACTGAAAGAGGAGGTGGATGAACATGCTTTCTTCACTGAACCTTCTGGAACTATAAGA

[0044] AGTTTATCAGCTTCTTCAATGGATGATTCATGGCAACTCACACCACTTACCATAAGCTCCTCTTCCTCTTTTAAACAGAG

[0045] AAATTGTTCTGGTTTATCCAATGACAATGAGTACTCTTACTTGCAACTTGACAACAACTCAAAACAACCACAGCAAGATC

[0046] AAGGTTGTTACATCTCAGGTAGTGAAGAAACATTCATGAAACATGGGAAAGAAGAACCTCAGAAGATTTTTCATCGTTTC

[0047] TTTGATGAATGGCCACCCAAAAGCAGCAGAAACTCATGGCTTGATTTGGATGATAAATCATCCACCACTCAGCTTTCAAT

[0048] CTCCATTCCTACATCTTCTCATGATTTCACAACTTTTAGTTCCACAAACCAACGAGATGGTTGA

[0049] >Amino acid sequence of MsGRF1c (SEQ ID NO.2)

[0050] MMNGRNRYPFTPTQWQELEHQALIYKYMASGISIPPDLLLTIKRSYLDSSRLSPHQSQHFGWNYLPMGLGRKIDPEPGRC

[0051] RRTDGKKWRCSKEAYPDSKYCERHMHRGKNRSRKPVEVLKTTTNTDAPAATSTILSITKNSSLDSDHNTAALTSTTHDTN

[0052] YHYPQHSSYASSHLNHPFLYHNPPSSRPNFSFQDNSAPLFLDNTGSSSHNNNNTDCRYVYGLKEEVDEHAFFTEPSGTIR

[0053] SLSASSMDDSWQLTPLTISSSSSFKQRNCSGLSNDNEYSYLQLDNNSKQPQQDQGCYISGSEETFMKHGKEEPQKIFHRF

[0054] FDEWPPKSSRNSWLDLDDKSSTTQLSISIPTSSHDFTTFSSTNQRDG

[0055] >Nucleotide sequence of Growth-regulating factor-like protein MAKER:8.1:0.14:Medtr4g125490 (SEQ ID NO.3)

[0056] ATGATGAATGGAAGAAACAGGTATCCTTTTACTCCAACTCAGTGGCAAGAGCTTGAACATCAAGCTCTTATCTACAAATA

[0057] CATGGCTTCTGGTATTTCCATTCCTCCTGATCTTCTACTCACTATCAAAAGAAGCTACTTAGACTCTTCAAGACTCTCAC

[0058] CTCATCAGTCTCAACACTTTGGATGGAACTATTTACCAATGGGATTGGGAAGAAAAATAGACCCAGAGCCAGGGAGGTGC

[0059] AGAAGAACAGATGGAAAGAAATGGAGGTGTTCAAAAGAAGCATATCCAGATTCAAAGTACTGTGAAAGGCACATGCATAG

[0060] AGGGAAAAACCGTTCAAGAAAGCCTGTGGAAGTTTTGAAAACAACAACAAACACAGATGCTCCTGCTGCTACTTCAACAA

[0061] TCTTATCAATCACTAAAAATAGTAGTCTTGATTCTGATCATAACACTGCAGCACTAACATCAACAACTCATGACACTAAT

[0062] TACCATTACCCTCAACACTCTTCTTATGCTTCTTCCCATCTTAACCACCCTTTCCTCTACCATAACCCTCCATCCTCAAG

[0063] GCCTAACTTCTCATTTCAAGACAATAGTGCTCCCTTGTTTCTTGACAATACTGGTTCTTCCTCTCACAATAACAAGAACA

[0064] CTGATTGCAGGTATGTTTATGGACTGAAAGAGGAGGTGGATGAACATGCTTTCTTCACTGAACCTTCTGGAACTATAAGA

[0065] AGTTTATCAGCTTCTTCAATGGATGATTCATGGCAACTCACACCACTTACCATAAGCTCCTCTTCCTCTTTTAAACAGAG

[0066] AAATTGTTCTGGTTTATCCAATGACAATGAGTACTCTTACTTGCAACTTGACAACAACTCAAAACAACCACAGCAAGATC

[0067] AAGGTTGTTACATCTCAGGTAGTGAAGAAACATTCATGAAACATGGGAAAGAAGAACCTCAGAAGATTTTTCATCGTTTC

[0068] TTTGATGAATGGCCACCCAAAAGCAGCAGAAACTCATGGCTTGATTTGGATGATAAATCATCCACCACTCAGCTTTCAAT

[0069] CTCCATCCTACATCTTCTCATGATTTCACAACTTTTAGTTCCACAAACCAACGAGATGGTTGA

[0070] >MSAD_280575.t1 amino acid sequence (SEQ ID NO.3)

[0071] MMNGRNRYPFTPTQWQELEHQALIYKYMASGISIPPDLLLTIKRSYLDSSRLSPHQSQHFGWNYLPMGLGRKIDPEPGRC

[0072] RRTDGKKWRCSKEAYPDSKYCERHMHRGKNRSRKPVEVLKTTTNTDAPAAATSTILSITKNSSLDSDHNTAALTSTTHDTN

[0073] YHYPQHSSYASSHLNHPFLYHNPPSSRPNFSFQDNSAPLFLDNTGSSSHNNKNTDCRYVYGLKEEVDEHAFFTEPSGTIR

[0074] SLSASSMDDSWQLTPLTISSSSSFKQRNCSGLSNDNEYSYLQLDNNSKQPQQDQGCYISGSEETFMKHGKEEPQKIFHRF

[0075] FDEWPPKSSRNSWLDLDDKSSTTQLSISIPTSHDFTTFSSTNQRDG

[0076] Example 3 Construction of MsGRF1c Overexpression and Suppression Expression Vectors

[0077] According to the principle of codon degeneracy, the seven bases of the miR396 target site on the cloned MsGRF1c were subjected to synonymous mutation using the Overlap-PCR method to obtain the rMsGRF1c gene sequence ( Figure 3a). Using the double enzyme digestion method, rMsGRF1c was recombined into the pZh01 structural backbone between the BamHI and KpnI restriction sites to obtain the mutant rMsGRF1c overexpression vector pZh01-rMsGRF1c ( Figure 3 b). The stop codon of MsGRF1c was removed and the sequence was ligated into the existing 35s-NAC60-SRDX vector between the XbaI and BamHI restriction sites by double enzyme digestion. The MsGRF1c-SRDX fusion gene was then recombined into the pZh01 structural backbone between the XbaI and SalI restriction sites by double enzyme digestion to obtain the plant expression vector pZh01-MsGRF1c-SRDX that inhibits MsGRF1c activity ( Figure 3 c).

[0078] Example 4 PCR Identification of MsGRF1c Overexpression and Suppression Transgenic Plants

[0079] Through Agrobacterium-mediated genetic transformation and the laboratory's existing alfalfa tissue culture technology, 52 overexpressing rMsGRF1c and 47 overexpressing MsGRF1c-SRDX resistant regenerated plants were obtained after 10 mg / L hygromycin screening. The plants were identified by PCR using the corresponding vector upstream and gene downstream primers. The results showed that the transgenic positive plants and the plasmid positive control could amplify the target fragment, while the non-transgenic, wild type and blank controls could not amplify the target fragment. Therefore, this experiment obtained a total of 44 rMsGRF1c overexpressing transgenic positive plants (rOX), 30 MsGRF1c-SRDX overexpressing transgenic positive plants (SR) ( Figure 4 ).

[0080] Example 5 Identification of expression levels in MsGRF1c overexpressing and suppressed transgenic plants

[0081] In order to detect the expression of target genes in each transgenic plant, this experiment used qRT-PCR to detect the expression of target genes in MsGRF1c overexpression and suppression transgenics. The results showed that compared with the wild type, except for rOX27 and rOX24, where the expression of MsGRF1c was only about 1 times, the expression of other rOX transgenic plants ranged from 3 times to 2900 times ( Figure 5 The rOX plants with MsGRF1c expression levels lower than 15 times were designated as the low expression group (rOX-L), the rOX plants with expression levels higher than 15 times but lower than 250 times were designated as the medium expression group (rOX-M), and the rOX plants with expression levels higher than 250 times were designated as the high expression group (rOX-H) ( Figure 5Similarly, SR transgenic plants were divided into three groups based on the expression level of MsGRF1c. SR plants with an expression level of 1.5 to 3.5 times that of the wild type were classified as the low expression group (SR-L), SR plants with an expression level between 8 and 13 times that of the wild type were classified as the medium expression group (SR-M), and SR plants with an expression level between 43 and 208 times that of the wild type were classified as the high expression group (SR-H) ( Figure 6 ).

[0082] Example 6: MsGRF1c regulates alfalfa plant shape and leaf size

[0083] By observing the plant type and leaf morphology of rOX and SR transgenic plants, it was found that MsGRF1c is involved in regulating the growth of the aboveground part of alfalfa plants. That is, high overexpression of MsGRF1c promotes leaf size, while moderate and high overexpression of MsGRF1c-SRDX inhibits plant growth, reduces leaf size, and causes leaf developmental deformities ( Figure 7 a, b, c and d). After statistical analysis, there was no significant difference in plant height between rOX plants and wild type ( Figure 7 e). The stem thickness of the rOX-H plant at the fourth node from top to bottom was significantly larger than that of the wild type by about 0.2 mm ( Figure 7 f). The length and width of the fourth leaf from the top to the bottom of the rOX-H plant were significantly larger than those of the wild type by approximately 0.3 cm and 0.2 cm, respectively. Figure 7 g and h). In contrast, the height of SR-M and SR-H plants was significantly lower than that of wild-type plants, about 10cm-13cm. The stem diameter of SR plants from the top to the fourth node was smaller than that of wild-type plants, and the stem diameter of SR-H (0.96mm) was significantly different from that of wild-type plants (1.37mm). Figure 7 f). The length of the fourth leaf from top to bottom in SR-M and SR-H plants was significantly smaller than that in wild type by about 0.6 cm ( Figure 7 g). The width of the fourth leaf from top to bottom of the SR plant is between 0.7-0.9 mm, which is significantly smaller than that of the wild-type plant (1.1 mm) ( Figure 7 h).

[0084] Example 7: MsGRF1c regulates the morphology, size, and nitrogen fixation of alfalfa nodules

[0085] The MsGRF1c overexpression and suppression transgenic plants were inoculated with rhizobia under low nitrogen conditions, and the plant growth and nodulation were observed and analyzed. Figure 8 As shown, 60 days after low nitrogen inoculation with rhizobia, the height of rMsGRF1c overexpressing transgenic plants (except rOX50 line) was significantly higher than that of wild type, while inhibition of MsGRF1c expression significantly inhibited the growth of plant height ( Figure 8a, b and c). The dry weight of rMsGRF1c overexpressing transgenic plants was significantly lower than that of wild type (approximately 205mg-332mg), while the dry weight of MsGRF1c knockdown plants (28-133mg) was significantly lower than that of wild type (313mg). Figure 8 d). We further found that the root size of the rMsGRF1c overexpressing transgenic plant (rOX50) with the highest MsGRF1c expression was no different from that of the wild type, while the root size of the MsGRF1c suppressed transgenic plant (SR8) was significantly smaller than that of the wild type ( Figure 9 a, b, and c). Compared with the wild type, rOX50 plants had more pink nodules on their roots and the nodules were larger overall, while SR8 plants had fewer pink nodules on their roots and the nodules were smaller overall ( Figure 9 d, e and f). Quantitative analysis revealed that the nodules of MsGRF1c-inhibited transgenic plants were significantly smaller than those of the wild type; the nodules of rOX50 plants were significantly larger than those of the wild type, while the nodules of other rMsGRF1c-overexpressing transgenic plants were no different in size from those of the wild type ( Figure 9 g). The percentage of effective nodules in rMsGRF1c overexpressing transgenic roots was significantly higher than that in wild type; while the percentage of effective nodules in MsGRF1c suppressed transgenic roots was significantly lower than that in wild type ( Figure 9 h). Compared with wild-type nodules, the nitrogenase activity of rMsGRF1c-overexpressing transgenic nodules was significantly enhanced, while the nitrogenase activity of MsGRF1c-inhibited transgenic nodules was significantly reduced ( Figure 9 i).

[0086] Example 8 MsGRF1c regulates alfalfa nodule senescence

[0087] To further investigate the function of MsGRF1c in regulating nodule senescence, this study used toluidine blue staining, DAB staining, and ROS fluorescence quantitative detection on the nodules of wild-type and transgenic plants inoculated with rhizobia for 21 days. Figure 10 As shown in a, b, and c, toluidine blue staining showed that WT nodules had senescent zones, SR nodules had more severe senescence than WT nodules, and rOX50 nodules had no senescent zones. DAB staining showed that the brown deposits near the roots of SR nodules were darker than those of the wild type, while the staining near the roots of rOX nodules was lighter than that of the wild type; the infected area of SR nodules was lighter than that of other parts, and the brown color of the upper part (distal part of the root system) of rOX nodules was darker than that of the lower part (proximal part of the root system) ( Figure 10 d, e and f). Similar results were obtained by DCFH-DA reactive oxygen species detection ( Figure 11 a and b). The DCF fluorescence intensity in the meristem and infection zones of SR nodules was lower than that of the wild type (the difference was not significant), and was significantly lower than that of the nitrogen-fixing zone itself; similarly, the DCF fluorescence intensity in the lower half of the nitrogen-fixing zone was significantly higher than that of the wild type ( Figure 11a and b). In contrast, the DCF fluorescence intensity in the meristem and infection zones of rOX nodules was the strongest, the upper nitrogen-fixing zone was in the middle, and the lower nitrogen-fixing zone was the weakest. Among them, the DCF fluorescence intensity in the meristem and infection zones was significantly stronger than that of the wild type, while that in the lower nitrogen-fixing zone was significantly lower than that of the wild type ( Figure 11 a and b). These results indicate that MsGRF1c regulates nodule senescence by regulating the accumulation and distribution of ROS in nodules.

Claims

1. Use of the MsGRF1c gene in regulating nodule senescence in alfalfa plants, wherein the MsGRF1c gene is overexpressed to inhibit nodule senescence. The nucleotide sequence of the MsGRF1c gene is SEQ ID NO.

1.

2. The use according to claim 1, wherein an overexpression vector is constructed to overexpress the MsGRF1c gene.

3. The use according to claim 1, wherein the alfalfa plant is alfalfa.

4. Use of the MsGRF1c gene in regulating nitrogen fixation efficiency in alfalfa plants, wherein the MsGRF1c gene is overexpressed to improve nitrogen fixation efficiency. The nucleotide sequence of the MsGRF1c gene is SEQ ID NO.

1. The use according to claim 4 , wherein an overexpression vector is constructed to overexpress the MsGRF1c gene.

6. The use according to claim 4, wherein the alfalfa plant is alfalfa.

Images