Application of Medicago polymorpha MtNOOT1 and MtNOOT2 genes in regulating plant yield and leaf protein content
By regulating the MtNOOT1 and MtNOOT2 genes in alfalfa through genetic engineering, the problems of growth period, plant height, and leaf protein content were solved, resulting in an extended growth period, increased plant height, and improved protein content, thereby enhancing yield and quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG UNIV
- Filing Date
- 2026-02-26
- Publication Date
- 2026-06-19
AI Technical Summary
Existing technologies are insufficient to effectively control the growth period, plant height, above-ground fresh weight, and leaf protein content of alfalfa, resulting in poor yield and quality.
By using genetic engineering techniques, the simultaneous knockout, knockdown, or silencing of the MtNOOT1 and MtNOOT2 genes in alfalfa can regulate the plant's growth period, plant height, and leaf protein content, thereby improving plant yield and quality.
Extending the growing season of alfalfa increases plant height and above-ground fresh weight, while also increasing leaf protein content, thereby improving alfalfa yield and quality.
Smart Images
Figure CN122235199A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the MtNOOT1 and MtNOOT2 genes of alfalfa in regulating plant yield and leaf protein content. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Alfalfa (Medicago sativa L.) is rich in carbohydrates, crude protein, vitamins, and other trace elements, making it an important forage crop for animal husbandry. With the development of animal husbandry, the demand for alfalfa products is increasing, thus, cultivating high-quality forage grasses has significant agricultural and strategic importance.
[0004] Alfalfa's yield primarily comes from its leaves and stems, with leaves contributing up to 60% of the total yield. Leaves are not only crucial indicators of alfalfa growth, yield composition, and varietal characteristics, but also primary research subjects for cultivation management and pest and disease monitoring. Furthermore, leaves store 70% of alfalfa's protein; therefore, the greater the proportion of leaves, the higher the nutritional value, palatability, and overall utilization value of the alfalfa. In addition, the length of the growing season is closely related to forage yield indicators such as leaf yield and quality. A longer growing season results in higher forage yield, but because forage stems are coarse and hard, leaves are few and yellowed, palatability and digestibility decrease, and quality is poor in the later stages of growth. Therefore, in practical applications, it is necessary to balance the length of the growing season and forage quality. Summary of the Invention
[0005] To overcome the above problems, this invention provides the application of the MtNOOT1 and MtNOOT2 genes of alfalfa in regulating plant yield and leaf protein content.
[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides the use of the MtNOOT1 and MtNOOT2 genes of alfalfa in any of the following: a1) Regulating plant growth period; a2) Regulating plant height; a3) Regulate the fresh weight of the above-ground parts of plants; a4) Regulates the protein content in plant leaves; The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO: 2.
[0007] In one or more embodiments, the regulation is suppression.
[0008] In one or more embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
[0009] A second aspect of the present invention provides a method for extending the growth period of plants, comprising: using genetic engineering to simultaneously inhibit the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa. The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO: 2.
[0010] In one or more embodiments, the method for simultaneously inhibiting the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa is to simultaneously knock out the MtNOOT1 and MtNOOT2 genes in alfalfa, simultaneously knock down the MtNOOT1 and MtNOOT2 genes in alfalfa, or simultaneously silence the MtNOOT1 and MtNOOT2 genes in alfalfa.
[0011] In one or more embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
[0012] A third aspect of the present invention provides a method for increasing plant height, comprising: using genetic engineering to simultaneously inhibit the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa. The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO: 2.
[0013] In one or more embodiments, the method for simultaneously inhibiting the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa is to simultaneously knock out the MtNOOT1 and MtNOOT2 genes in alfalfa, simultaneously knock down the MtNOOT1 and MtNOOT2 genes in alfalfa, or simultaneously silence the MtNOOT1 and MtNOOT2 genes in alfalfa.
[0014] In one or more embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
[0015] A fourth aspect of the present invention provides a method for increasing the fresh weight of aboveground parts of plants, comprising: using genetic engineering to simultaneously inhibit the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa. The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO: 2.
[0016] In one or more embodiments, the method for simultaneously inhibiting the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa is to simultaneously knock out the MtNOOT1 and MtNOOT2 genes in alfalfa, simultaneously knock down the MtNOOT1 and MtNOOT2 genes in alfalfa, or simultaneously silence the MtNOOT1 and MtNOOT2 genes in alfalfa.
[0017] In one or more embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
[0018] A fifth aspect of the present invention provides a method for increasing the protein content in plant leaves, comprising: using genetic engineering to simultaneously inhibit the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa. The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO: 2.
[0019] In one or more embodiments, the method for simultaneously inhibiting the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa is to simultaneously knock out the MtNOOT1 and MtNOOT2 genes in alfalfa, simultaneously knock down the MtNOOT1 and MtNOOT2 genes in alfalfa, or simultaneously silence the MtNOOT1 and MtNOOT2 genes in alfalfa.
[0020] In one or more embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
[0021] A sixth aspect of the present invention provides the application of transgenic plants obtained by the methods described in the second, third, fourth, or fifth aspects in plant breeding.
[0022] In one or more embodiments, the breeding method includes transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.
[0023] The beneficial effects of this invention are as follows: The mutant lines of alfalfa with simultaneous knockout of the MtNOOT1 and MtNOOT2 genes constructed in this invention exhibit increased plant height, increased above-ground fresh weight, and increased leaf protein content compared to the wild type at the end of the growth period. Therefore, simultaneous knockout of the MtNOOT1 and MtNOOT2 genes in alfalfa can prolong the growth period of alfalfa and increase leaf protein content, thereby improving alfalfa yield and quality. Attached Figure Description
[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0025] Figure 1 The phenotypes of mutants and double mutants of the MtNOOT1 and MtNOOT2 genes in alfalfa are shown; where A represents Tnt1 insertion into the exons of the MtNOOT1 and MtNOOT2 genes, respectively, and B represents alfalfa. mtnoot1-mtnoot2 Phenotypes of double mutants and wild-type (WT) plants; Figure 2 alfalfa mtnoot1-mtnoot2 Biomass measurements of double mutant and wild-type plants, where A is plant height and B is fresh weight of aboveground parts; Figure 3 alfalfa mtnoot1-mtnoot2 Results of leaf protein content determination in double mutant and wild-type plants. Detailed Implementation
[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.
[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0029] Example 1 Obtaining mutants of the MtNOOT1 and MtNOOT2 genes from alfalfa and analyzing the phenotypes of the double mutants: Using thermal asymmetric interlaced-PCR (TAIL-PCR) technology, a mutant library of alfalfa marked with Tnt1 was screened. From 22,000 mutant lines, mutant lines in which Tnt1 was inserted into the MtNOOT1 and MtNOOT2 genes were selected. mtnoot1 and mtnoot2 Molecular biological identification results showed that Tnt1 was inserted into the exons of the MtNOOT1 and MtNOOT2 genes, respectively. Figure 1 (A). Constructed through hybridization. mtnoot1-mtnoot2 Double mutant, alfalfa mtnoot1-mtnoot2 The double mutant plants were significantly taller than the wild type ( Figure 1 (B)
[0030] Example 2 Tribulus terrestris alfalfa mtnoot1-mtnoot2 Biomass determination of double mutant plants: alfalfa mtnoot1-mtnoot2 Phenotypic analysis and biomass determination were performed on the aboveground parts of the double mutant plants, and the results are as follows: Figure 2 As shown, during the same growth period, compared with the wild type, the mutant plants were about 64% taller and about 117% heavier above-ground parts (n=9).
[0031] Example 3 Tribulus terrestris alfalfa mtnoot1-mtnoot2Determination of leaf protein content in double mutant plants: alfalfa mtnoot1-mtnoot2 The protein content of leaves of the double mutant plants was measured, and the results showed that, at the same growth stage, the protein content of leaves of the mutant plants was about 30% higher than that of the wild type.
[0032] The mutant lines of alfalfa with simultaneous knockout of the MtNOOT1 and MtNOOT2 genes constructed in this invention exhibit increased plant height, increased above-ground fresh weight, and increased leaf protein content compared to the wild type at the end of the growth period. Therefore, simultaneous knockout of the MtNOOT1 and MtNOOT2 genes in alfalfa can prolong the growth period of alfalfa and increase leaf protein content, thereby improving alfalfa yield and quality.
[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. Application of the MtNOOT1 and MtNOOT2 genes of alfalfa in any of the following: a1) Regulating plant growth period; a2) Regulating plant height; a3) Regulate the fresh weight of the above-ground parts of plants; a4) Regulates the protein content in plant leaves; The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO:
2.
2. The application as described in claim 1, characterized in that, The regulation referred to is inhibition.
3. The application as described in claim 1, characterized in that, The plant is a monocotyledonous or dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, common bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
4. A method for extending the growth period of plants, characterized in that, This includes: using genetic engineering to simultaneously suppress the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa; The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO:
2.
5. A method for increasing plant height, characterized in that, This includes: using genetic engineering to simultaneously suppress the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa; The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO:
2.
6. A method for increasing the fresh weight of the above-ground parts of a plant, characterized in that, This includes: using genetic engineering to simultaneously suppress the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa; The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO:
2.
7. A method for increasing the protein content in plant leaves, characterized in that, This includes: using genetic engineering to simultaneously suppress the expression of the MtNOOT1 and MtNOOT2 genes in alfalfa; The nucleotide sequence of the alfalfa MtNOOT1 is shown in SEQ ID NO: 1; The nucleotide sequence of the alfalfa MtNOOT2 is shown in SEQ ID NO:
2.
8. The method according to any one of claims 4 to 7, characterized in that, The methods to simultaneously suppress the expression of MtNOOT1 and MtNOOT2 genes in alfalfa are to simultaneously knock out MtNOOT1 and MtNOOT2 genes, simultaneously knock down MtNOOT1 and MtNOOT2 genes, or simultaneously silence MtNOOT1 and MtNOOT2 genes in alfalfa.
9. The method according to any one of claims 4 to 7, characterized in that, The plant is a monocotyledonous or dicotyledonous plant; preferably a legume; more preferably, the legume includes alfalfa, birdsfoot root, soybean, pea, peanut, common bean, mung bean, red bean, broad bean, cowpea, milkvetch, licorice and astragalus, preferably alfalfa.
10. The use of the transgenic plant obtained by the method according to any one of claims 4 to 7 in plant breeding.