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CN-122012581-A - Application of soybean gene GmACR to regulation of salt and alkali tolerance of plants

CN122012581ACN 122012581 ACN122012581 ACN 122012581ACN-122012581-A

Abstract

The invention relates to the field of plant genetic engineering, in particular to a novel application of a soybean ACR gene family member GmACR in improving salt and alkali tolerance of plants. The invention provides application of an isolated gene in preparing a product for improving salt and alkali tolerance of plants, wherein the gene codes a nucleotide sequence shown as SEQ ID NO. 1 or codes a protein which has at least 80% of identity with SEQ ID NO. 2 and has the function of improving the salt and alkali tolerance of the plants. The gene is up-regulated under the stress of salt and alkali, and the salt and alkali tolerance of transgenic plants can be obviously improved by over-expressing the gene. The invention also provides an expression vector containing the gene, an sgRNA molecule targeting a promoter thereof, a CRISPR activation system, related reagents, a kit, a method for cultivating saline-alkali tolerant plants and a detection means. The invention provides a new gene resource and molecular breeding tool for cultivating new varieties of saline-alkali tolerant crops.

Inventors

  • WANG LIANXIA
  • MENG FANLI
  • YUAN MING
  • CAO YINGXUE

Assignees

  • 黑龙江省农业科学院齐齐哈尔分院

Dates

Publication Date
20260512
Application Date
20260203

Claims (10)

  1. 1. Use of soybean gene GmACR24 or a protein encoded thereby for modulating salt tolerance in a plant, said soybean gene GmACR or a protein encoded thereby selected from the group consisting of: (1) A nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO. 2, or a nucleotide sequence encoding a protein having at least 80% identity with SEQ ID NO. 2 and having a function of improving the saline-alkali tolerance of a plant; (2) A nucleotide sequence shown as SEQ ID NO. 1; (3) A protein with an amino acid sequence shown as SEQ ID NO.2, or a protein which has at least 80% of identity with SEQ ID NO.2 and has the function of improving the saline-alkali tolerance of plants; optionally, the expression level of the soybean gene GmACR is increased for increasing the salt tolerance of the plant; Optionally, the increased expression of the soybean gene GmACR, 24 is achieved by over-expression of the gene; optionally, the over-expression is achieved by introducing an expression vector containing the GmACR gene into the plant genome.
  2. 2. An sgRNA molecule, wherein the sgRNA molecule targets a promoter or enhancer region of soybean gene GmACR; Optionally, the sgRNA molecule is suitable for increasing the transcriptional activity of soybean gene GmACR 24; optionally, the soybean gene GmACR has a nucleotide sequence as set forth in SEQ ID NO. 1; Optionally, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO. 5.
  3. 3. An expression vector comprising the soybean gene GmACR of claim 1, or an expression cassette expressing the sgRNA molecule of claim 2; Optionally, the expression vector further comprises a nucleic acid sequence encoding a fusion protein of a nuclease-inactivated Cas protein and a transcriptional activator; Optionally, the expression vector is an agrobacterium transformation vector or a plant viral vector.
  4. 4. A CRISPR activation system, comprising: The sgRNA molecule of claim 2, and a fusion protein of an inactivated Cas9 protein and a transcriptional activator.
  5. 5. A reagent, comprising: the sgRNA molecule of claim 2, the expression vector of claim 3, or the CRISPR activation system of claim 4.
  6. 6. A kit of parts for the manufacture of a kit, characterized by comprising the following steps: the sgRNA molecule of claim 2, the expression vector of claim 3, or the CRISPR activation system of claim 4.
  7. 7. A method for improving the saline-alkali tolerance of a plant, comprising: increasing the expression level of soybean gene GmACR in a plant, said GmACR gene having the nucleotide sequence defined in claim 1; optionally, the increasing the expression level is achieved by: transforming the sgRNA molecule of claim 2 or the expression vector of claim 3 into a plant cell or tissue, or Transcriptional activation of a GmACR gene endogenous to a plant using the CRISPR activation system of claim 4; Optionally, the plant is soybean.
  8. 8. A host cell carrying the expression vector of claim 3, or having integrated and expressed in the genome the GmACR gene of claim 1; optionally, the host cell is a bacterial, fungal or plant cell; Optionally, the host cell is an agrobacterium, agrobacterium tumefaciens or yeast cell.
  9. 9. A transgenic plant cell comprising the exogenously introduced soybean gene GmACR of claim 1 or comprising the sgRNA molecule of claim 2, wherein the soybean gene GmACR is expressed in the plant cell in a higher amount than in an untransformed wild type control cell.
  10. 10. Use of the soybean gene GmACR of claim 1 or a protein encoded thereby, the sgRNA molecule of claim 2 or a primer pair for specifically amplifying the soybean gene GmACR of claim 1 in the preparation of a reagent or kit for screening saline-alkali tolerant plants.

Description

Application of soybean gene GmACR to regulation of salt and alkali tolerance of plants Technical Field The invention belongs to the technical field of plant genetic engineering, in particular to application of soybean gene GmACR to regulation and control of salt and alkali tolerance of plants, and more particularly relates to an expression vector, sgRNA molecules, a CRISPR activation system, a reagent, a kit, a method for improving the salt and alkali tolerance of plants and related application thereof. Background Saline-alkali stress is one of the major abiotic stress that limits crop growth and productivity. Large-area salinized lands exist in China and the world, and the grain safety is seriously threatened. Soybean (Glycine max) is an important food and oil crop, and improvement of salt and alkali tolerance thereof is an important goal of breeding. The molecular biological means is utilized to discover key salt and alkali tolerant genes and clarify the functions of the genes, and the gene is an effective way for cultivating new salt and alkali tolerant varieties. Amino acid metabolism plays a key role in plant stress response. Proteins containing the ACT domain (Aromatic amino acid, chorismate mutase, and TyrA) (ACR proteins) are a family of proteins that are widely involved in amino acid metabolism and regulation, members of which typically comprise 2-4 repeated ACT domains, which can ACT as amino acid sensors or metabolic regulators. Studies have shown that the ACR gene family has important functions in plant growth and development and stress response. In the model plant Arabidopsis thaliana (Arabidopsis thaliana), 12 ACR gene family members have been identified. Of these, atACR4 was demonstrated to be a receptor kinase involved in regulating embryonic development, atACR was localized to chloroplasts, demonstrated to be involved in glutamine metabolism and nitrogen assimilation processes, and possibly involved in defensive reactions by regulating active oxygen and salicylic acid accumulation. In rice (Oryza sativa), a total of 9 ACR genes were identified by the study. For example OsACR and OsACR9 were demonstrated to have glutamine sensing function and correlate with the spatial and temporal expression of nitrogen assimilation genes. These studies indicate that the ACR gene family has a conserved and diverse function in plant basal metabolism and stress adaptation. However, the current research on ACR gene families is mainly focused on pattern plants such as arabidopsis and rice. In soybean, an important cash crop, the systematic identification, molecular characteristics and specific functions of the gene family in abiotic stress (especially saline-alkali stress) response are not known, and the potential application value of the gene family is not developed. Therefore, the system identifies the soybean ACR gene family, screens and verifies the key salt and alkali tolerance functional genes, has important significance for analyzing the salt and alkali tolerance molecular mechanism of the soybean and pushing molecular breeding practice, and is also a problem to be solved in the technical field at present. Disclosure of Invention The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the application provides the application of the soybean gene GmACR to the regulation and control of the salt and alkali tolerance of plants. The present application has been completed based on the following findings by the inventors: The inventor finds that the expression quantity of the member GmACR24 is obviously up-regulated under the condition of saline-alkali stress when systematically researching the soybean ACR gene family. Further, through the verification experiment of functions, the GmACR gene is over-expressed in soybean, so that the survival rate, biomass (plant height, fresh weight and dry weight) and the activity of key antioxidant enzyme (SOD, POD, CAT) of transgenic plants under the stress of salt and alkali can be obviously improved, and the salt and alkali tolerance of the plants can be effectively enhanced. This indicates that GmACR gene plays a positive regulatory role in soybean response to saline-alkali stress. Based on the above findings, the present invention provides a use of soybean gene GmACR in regulating salt and alkali tolerance of a plant, wherein soybean gene GmACR comprises any one of the nucleotide sequences shown in (1) a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, or a nucleotide sequence encoding a protein having at least 80% identity with SEQ ID NO: 2 and having a function of improving salt and alkali tolerance of a plant, and (2) a nucleotide sequence shown in SEQ ID NO: 1. In a second aspect of the invention, the invention provides an sgRNA molecule. According to an embodiment of the invention, the sgRNA molecule targets the promoter or enhancer region of