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CN-121991995-A - Recombinant rhizobium OEuspA with stress resistance, plant salt-resistant bacterial agent and application thereof

CN121991995ACN 121991995 ACN121991995 ACN 121991995ACN-121991995-A

Abstract

The invention discloses a recombinant rhizobium OEuspA with stress resistance, a plant salt-resistant microbial inoculum and application thereof, and belongs to the technical field of genetic engineering. The invention identifies a new uspA gene from the stem nodule nitrogen fixing rhizobia ORS571 for the first time. Then carrying the uspA gene into the rhizobium azotembotryoides by using a plasmid vector, and constructing the recombinant rhizobium with the uspA gene being over-expressed. Compared with wild rhizobia, the stress resistance of the recombinant rhizobia of the over-expressed uspA gene constructed by the invention is obviously improved. After the recombinant rhizobia which over-expresses the uspA gene is symbiotic with sesbania glaucescens, wheat and corn, the growth of plants under the condition of salt stress can be promoted. The uspA gene can be used for constructing a recombinant rhizobium material with stronger stress resistance and growth promotion capability, and has potential and wide application prospects in the field of genetic engineering reconstruction of rhizobium or other bacteria, rhizobium preparation industry, bean planting industry in agricultural production and saline-alkali soil development.

Inventors

  • XIE ZHIHONG
  • ZHOU YANAN
  • WANG DANDAN
  • GENG NA
  • WANG SHUAIBING
  • Pu Zitian
  • BI WENJIE

Assignees

  • 山东农业大学

Dates

Publication Date
20260508
Application Date
20260116

Claims (10)

  1. Use of the uspa gene in (1) or (2) as follows: (1) The stress resistance of rhizobia is improved; (2) Constructing rhizobium engineering strains with stress resistance; The uspA gene is a nucleic acid molecule as shown in (i) or (ii): (i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 1; (ii) A nucleic acid molecule other than (i) encoding the amino acid sequence shown in SEQ ID NO. 2.
  2. 2. The use according to claim 1, wherein the stress resistance is resistance to salt stress, heavy metal stress and/or oxidative stress.
  3. The application of UspA protein in improving the stress resistance of rhizobia, wherein the UspA protein is shown in the following (A1) or (A2): (A1) A protein with an amino acid sequence shown as SEQ ID NO. 2; (A2) A protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.
  4. 4. Use of a substance that promotes the expression of the uspA gene or a substance that increases the activity and/or content of the uspA gene in (1) or (2) as follows: (1) The stress resistance of rhizobia is improved; (2) Constructing rhizobium engineering strain with stress resistance.
  5. 5. The use according to claim 4, wherein the substance that promotes the expression of the uspA gene is any one of the following: C1 An expression cassette comprising a uspA gene; C2 A recombinant vector containing the uspA gene, or a recombinant vector containing the expression cassette of C1); C3 A recombinant microorganism containing the uspA gene, or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2).
  6. 6. Use of a recombinant rhizobia that overexpresses the uspA gene in at least one of the following (1) - (4): (1) Promoting plant growth under salt stress; (2) Increasing the root nodule number of the plant under salt stress; (3) Increasing the number of stem tumors of the plant under salt stress; (4) Preparing the plant salt-resistant microbial agent.
  7. 7. The use according to claim 6, wherein the recombinant rhizobia that overexpresses the uspA gene is constructed by the following method: cloning the nucleotide sequence shown in SEQ ID NO.1 onto a plasmid to obtain a recombinant plasmid, and introducing the recombinant plasmid into a stem nodule nitrogen fixing rhizobium host cell to construct the recombinant rhizobium with the over-expressed uspA gene.
  8. 8. The use according to claim 7, wherein the plasmid is pBBR1MCS-2.
  9. 9. The use according to claim 6, wherein the plant is a leguminous plant or a gramineous plant, preferably sesbania glauca (Sesbania rostrata), maize or wheat.
  10. 10. The use according to claim 6, wherein the salt stress condition is 0.3% NaCl concentration.

Description

Recombinant rhizobium OEuspA with stress resistance, plant salt-resistant bacterial agent and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to a recombinant rhizobium OEuspA with stress resistance, a plant salt-resistant microbial inoculum and application thereof. Background Rhizobia is a class of gram-negative bacteria that establishes a symbiotic relationship with leguminous plants. Through symbiotic action, the leguminous plants can convert nitrogen in the atmosphere into nitrogen compounds available to plants, so that the nitrogen utilization capacity of the plants is remarkably expanded. The rhizobium azotemlobum ORS571 is a special rhizobium which has symbiotic and autogenous nitrogen fixation capability, and can nodulate at the root of sesbania glaucescens which is a host plant and also nodulate at the stem of sesbania glaucescens. The special nitrogen fixation capability can effectively promote the growth of the sesbania glauca and promote the adaptability of the sesbania glauca to the saline-alkali environment. Soil salinization is an important problem facing current agricultural production, and salt stress causes ion toxicity, osmotic stress, nutrient deficiency and oxidative stress, severely affecting crop yield. By planting the salt-tolerant plants, the long-term improvement of the saline-alkali soil can be realized, and the salt resistance of the plants can be improved by methods of improving the microbial inoculum and the like. In the saline-alkali soil environment, rhizobia is often subjected to various environmental stresses such as saline-alkali stress, oxidative stress, heavy metal stress, acid-base stress and the like in a free state or in a symbiotic process with plants. These environmental stresses can reduce the viability of rhizobia, thereby affecting the colonization of the host plant by the rhizobia and promoting the ability of the host plant to grow under stress. Therefore, how to improve the stress resistance of rhizobia is a problem to be solved at present. Disclosure of Invention Aiming at the prior art, the invention aims to provide a recombinant rhizobium OEuspA with stress resistance, a plant salt-resistant microbial agent and application thereof. In order to achieve the above purpose, the invention adopts the following technical scheme: In a first aspect of the invention there is provided the use of the uspA gene in (1) or (2) as follows: (1) The stress resistance of rhizobia is improved; (2) Constructing rhizobium engineering strains with stress resistance; The uspA gene is a nucleic acid molecule as shown in (i) or (ii): (i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 1; (ii) A nucleic acid molecule other than (i) encoding the amino acid sequence shown in SEQ ID NO. 2. In the above application, the stress resistance is resistance to salt stress, heavy metal stress and/or oxidative stress. According to the invention, a new uspA gene is identified from the rhizobium azotemozolomide ORS571 for the first time, and the uspA gene is overexpressed in the rhizobium azotemozolomide, so that the stress resistance of the rhizobium can be remarkably improved. In a second aspect, the invention provides an application of UspA protein in improving stress resistance of rhizobia, wherein the UspA protein is shown in the following (A1) or (A2): (A1) A protein with an amino acid sequence shown as SEQ ID NO. 2; (A2) A protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag. Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc. In a third aspect of the present invention there is provided the use of a substance that promotes the expression of the uspA gene or a substance that increases the activity and/or content of the uspA gene in (1) or (2) as follows: (1) The stress resistance of rhizobia is improved; (2) Constructing rhizobium engineering strain with stress resistance. In some preferred embodiments of the invention, the substance that promotes the expression of the uspA gene may be any of the following: C1 An expression cassette comprising a uspA gene; C2 A recombinant vector containing the uspA gene, or a recombinant vector containing the expression cassette of C1); C3 A recombinant microorganism containing the uspA gene, or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2). In a fourth aspect of the present invention, there is provided the use of a recombinant rhizobia that overexpresses the uspA gene in at l