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CN-121991985-A - MsCCoAOMTc3 gene, recombinant vector, engineering bacterium and application thereof in improving drought tolerance of alfalfa

CN121991985ACN 121991985 ACN121991985 ACN 121991985ACN-121991985-A

Abstract

The invention belongs to the technical fields of genetic engineering and plant biology, and discloses an alfalfa drought-tolerant gene MsCCoAOMTc, the nucleotide sequence of which is shown as SEQ ID NO. 1. Meanwhile, the invention relates to a recombinant vector, an expression cassette and engineering bacteria containing the gene, and a preparation method thereof. The invention also relates to a method for preparing transgenic alfalfa plants with improved drought tolerance by using the gene or the vector and application of the gene or the vector in improving the drought tolerance of alfalfa or cultivating drought-tolerant varieties. Experiments show that the over-expression MsCCoAOMTc < 3 > can obviously enhance drought tolerance of alfalfa.

Inventors

  • YANG PEIZHI
  • MA FANG
  • HUANG SHUDI
  • LIANG YUNFEI
  • Zhan Xiangqiang
  • SHEN XIAOXIA

Assignees

  • 西北农林科技大学

Dates

Publication Date
20260508
Application Date
20260128

Claims (10)

  1. 1. An isolated nucleic acid molecule, wherein said nucleic acid molecule has the nucleotide sequence set forth in SEQ ID No. 1 under the designation MsCCoAOMTc gene.
  2. 2. A recombinant vector comprising the nucleic acid molecule of claim 1 and a plant expression regulatory element that drives expression thereof, said plant expression regulatory element comprising a 5 'CaMV 35S promoter and a 3' NOS terminator.
  3. 3. The recombinant vector according to claim 2, wherein the recombinant vector is obtained by ligating the nucleic acid molecule according to claim 1 with an over-expression vector PHELLSGATE.
  4. 4. A plant expression cassette comprising the nucleic acid molecule of claim 1, and linked to its 5 'end a CaMV 35S promoter and linked to its 3' end a NOS terminator.
  5. 5. An engineering bacterium which is characterized by comprising the recombinant vector of claim 2 or 3 or the plant expression cassette of claim 4, wherein the engineering bacterium is agrobacterium GV3101.
  6. 6. A method for preparing the recombinant vector of claim 3, which is characterized by comprising the following steps of (1) taking cDNA of Xinjiang leaves of alfalfa as a template, carrying out PCR amplification by using a forward primer with a sequence shown as SEQ ID NO.2 and a reverse primer with a sequence shown as SEQ ID NO.3 to obtain the nucleic acid molecule of claim 1, (2) carrying out double enzyme digestion on an over-expression vector PHELLSGATE by using restriction enzymes XbaI and XhoI to obtain a linearization vector, (3) connecting the PCR amplification product obtained in the step (1) with the linearization vector obtained in the step (2) to obtain a recombinant product, and (4) converting the recombinant product into escherichia coli competent cells, and screening to obtain the recombinant vector.
  7. 7. The method according to claim 6, wherein the PCR amplification in the step (1) is performed by using 1. Mu.L of the primer shown in SEQ ID NO.2, 2. Mu.L of the primer 1. Mu. L, ddH 2 O7.5. Mu. L, cDNA template shown in SEQ ID NO.3, 0.5. Mu.L of the 2X Phanta Max Buffer 12.5.5. Mu. L, dNTP mixed solution, and 0.5. Mu.L of Phanta Max Super-FIDELITY DNA polymerase, wherein the reaction procedure is 95℃for 5 minutes, 95℃for 30 seconds, 57℃for 30 seconds, 72℃for 80 seconds, 35 cycles total, and 72℃for 5 minutes.
  8. 8. A method for preparing a alfalfa transformant with improved drought tolerance, which is characterized by comprising the steps of (a) providing a recombinant vector containing the nucleic acid molecule of claim 1, (b) infecting a mechanically scratched leaf explant of 'Xinjiang big leaf' of alfalfa with an agrobacterium GV3101 invasion solution containing the recombinant vector, and (c) screening and cultivating transformed cells to obtain a transgenic plant.
  9. 9. A transgenic alfalfa plant obtained by the method of claim 8 having the nucleic acid molecule of claim 1 integrated into its genome.
  10. 10. Use of the nucleic acid molecule of claim 1, or the recombinant vector of claim 2 or 3, or the plant expression cassette of claim 4, for improving drought tolerance of alfalfa or for breeding drought tolerant alfalfa varieties.

Description

MsCCoAOMTc3 gene, recombinant vector, engineering bacterium and application thereof in improving drought tolerance of alfalfa Technical Field The invention belongs to the technical fields of genetic engineering and plant biology, and particularly relates to MsCCoAOMTc genes, recombinant vectors and engineering bacteria thereof and application of the MsCCoAOMTc genes in improving drought tolerance of alfalfa. Background Alfalfa (Medicago satival) is regarded as the most important high-quality leguminous forage grass worldwide, and is known as the 'forage grass king' because of high crude protein content, stable yield and strong adaptability. It not only provides key feed for animal husbandry, but also has ecological functions of water and soil conservation and soil improvement, and is an important component of sustainable agricultural system. With the increase of the demand of China for high-quality livestock products, the strategic position of the alfalfa industry is increasingly prominent. However, alfalfa production in China faces serious challenges. On one hand, the main planting areas (northwest, north China and northeast China) are subjected to drought stress throughout the year, the global climate change further causes continuous increase of drought frequency and intensity, and directly restricts the growth and yield of alfalfa, and on the other hand, in order to ensure the grain safety, the alfalfa is produced and mostly utilizes medium-low yield fields or marginal lands, and the water conditions of the lands are poor. In addition, the alfalfa seeds in China have high self-raising yield and insufficient stress-resistant varieties. Therefore, the cultivation of new alfalfa varieties with high drought tolerance is not only a key for breaking through the industrial bottleneck, but also an urgent need for responding to the national strategy of seed source attack and guaranteeing the autonomy of the grass industry. From the technical approach, improving plant stress resistance by using genetic engineering means is the core direction of modern breeding. Lignin is used as a key component of cell walls, not only provides mechanical support, but also plays an important role in resisting abiotic stress such as drought and the like of plants. caffeoyl-coa-O-methyltransferase (CCoAOMT) is a key enzyme in monolignol synthesis, whose function is closely related to drought resistance in plants. Studies have shown that PoCCoAOMT gene is induced to express by drought in peony (Paeonia ostii), and drought resistance is enhanced by promoting lignin accumulation and Reactive Oxygen Species (ROS) removal, and that the drought resistance performance is improved by over-expressing the elephant grass (Pennisetum purpureum) PpCCoAOMT gene in tobacco, and also by increasing lignin and flavonoid content and enhancing antioxidant capacity. In contrast, the Arabidopsis ccoaomt mutant exhibited hypersensitivity to drought. These studies agree that the CCoAOMT gene has a conserved and important forward regulatory function in drought resistance in plants. Although the drought-resistant function of the CCoAOMT gene in a variety of plants has been recognized, whether a specific homologous gene in this important forage grass, alfalfa, has a similar function has not been reported yet. The unique CCoAOMT gene of the alfalfa is discovered and verified, so that not only can a new clue be provided for analyzing drought-resistant molecular mechanisms of the alfalfa, but also key gene resources needed by cultivating new varieties of drought-resistant alfalfa with independent intellectual property rights are provided. Disclosure of Invention The invention aims at providing MsCCoAOMTc gene, recombinant vector, engineering bacteria and application thereof in improving drought resistance of alfalfa, aiming at the problems that the specific sequence of CCoAOMT gene family members in alfalfa and the function thereof in drought resistance are not disclosed in the prior art. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: In a first aspect the invention provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO. 1 under the name MsCCoAOMTc gene. The MsCCoAOMTc gene and the application thereof provided by the invention can effectively improve drought tolerance of alfalfa. The transgenic alfalfa plant ('Xinjiang big leaf') obtained by the gene shows a survival rate which is obviously higher than that of a wild plant after drought stress and rehydration, the relative conductivity of the leaf and the content of Malondialdehyde (MDA) are obviously reduced, the activities of superoxide dismutase (SOD), peroxidase (POD) and Catalase (CAT) are obviously enhanced, meanwhile, the accumulation of active oxygen (O 2 & gtand H 2O2) is reduced, and in addition, the lignin content of roots, stems, leaves and the whole plant of the plant is obviously improved. The experimental res