阅读说明：本技术 基因在提高植物耐盐中的应用 (Application of gene in improving salt tolerance of plant ) 是由 宝力格 刘敏轩 陆平 于 2020-07-17 设计创作，主要内容包括：本发明公开了基因在提高植物耐盐中的应用,具体的公开了LOC110433180和LOC8070651在耐盐高粱中高表达,且在盐胁迫试验中,LOC110433180和LOC8070651对盐胁迫产生响应,呈现与耐盐株相同的表达趋势。可以将LOC110433180和LOC8070651应用于植物耐盐的改良。(The invention discloses application of genes in improving salt tolerance of plants, and particularly discloses high expression of LOC110433180 and LOC8070651 in salt tolerant sorghum, wherein in a salt stress test, LOC110433180 and LOC8070651 respond to salt stress and show the same expression trend as salt tolerant plants. LOC110433180 and LOC8070651 can be applied to the improvement of salt tolerance of plants.)

1. A sorghum salt tolerance related gene and a protein coded by the same are characterized in that the gene is selected from the following genes: LOC110433180 and/or LOC 8070651.

Application of LOC110433180 and/or LOC8070651 in screening salt-tolerant sorghum.

Application of LOC110433180 and/or LOC8070651 in sorghum breeding.

4. A method for identifying salt tolerance of sorghum is characterized in that the salt tolerance of sorghum is predicted by detecting the expression level of LOC110433180 and/or LOC8070651 in a sample.

5. The method according to claim 4, characterized in that the expression level of LOC110433180 and/or LOC8070651 in the sample is detected by nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification techniques, protein immunization techniques.

6. The method according to claim 5, characterized in that the expression level of LOC110433180 and/or LOC8070651 in a sample is detected by a nucleic acid amplification technique.

7. The method according to any one of claims 4-6, characterized in that LOC110433180 and LOC8070651 are up-regulated in salt tolerant sorghum.

8. A product for predicting sorghum salt tolerance, comprising an agent for detecting the expression level of LOC110433180 and/or LOC 8070651.

9. A method for improving salt tolerance of sorghum, comprising increasing the expression level of LOC110433180 and/or LOC 8070651.

10. The method according to claim 9, wherein LOC110433180 and/or LOC8070651 nucleic acids are transferred into sorghum.

Technical Field

The invention belongs to the technical field of biology, and relates to application of a gene in improving salt tolerance of plants.

Background

Salt damage is one of important adverse hazards in current agricultural production, and the continuous development of grain production and agriculture is severely restricted. Currently, about 10 hundred million hm is around the world²The land of (1) has different degrees of salinization (Zhu J K. plant salt)The pesticide is mainly distributed in arid areas, semi-arid areas and coastal areas with large soil evaporation capacity and low precipitation quantity, wherein the pesticide is prepared from the materials of trees in Plant Science,6(2) and 66-71. The saline soil in China has large area, various types and wide distribution. According to the recent research report, the salinized soil area is about 3693.3 ten thousand hm²The residual salinized soil area is about 4486.7 kilohm²The potential salinized soil area is 1733.3 kilohm²The total area of various saline-alkali soil reaches 9913.3 ten thousand hm²(Li Bin, Wang Shi Chun, Sun Shi Gao, Chen Yuan, Yang Fu. 2005. Chinese saline-alkali soil resource and sustainable utilization research. agricultural research in arid area, 23(2):154 and 158.). There is also an increase in the rate of millions of hectares per year due to irrigation expansion, unreasonable exploitation and management, and climate change. Approximately half of the irrigated land in the world is affected by salinization (Rhoads J D, Loveday J.1990.Salinity in irrigated aggregations. in American society of social networks, irrigation of agrichural crops, 1089-. Soil salinization has become a worldwide problem, and is closely related to human population, resources, environment, food and other problems.

Plants are very closely related to the problem of soil salinization. The plants participate in the occurrence and evolution process of specific types of salinization, and meanwhile, the salinization causes the change of the habitat conditions of the plants, and the growth process and the dry matter accumulation of the plants are seriously influenced. On the other hand, the pressure of population, resources and grains facing the world requires people to fully excavate the production potential of the salinized farmland and develop and utilize different types of salinized soil resources so as to increase the effective farmland area and improve the utilization efficiency of the salinized soil resources and the land productivity. In the process of development, utilization and management of saline soil, the role of plants is highly valued by people. Since salinization is the major abiotic stress form of plants worldwide, efforts have been made to study plant salt tolerance to improve crop quality. The salt-tolerant characteristics of plants, the development and utilization of salt-tolerant and halophytes, the cultivation of salt-tolerant crop varieties, the improvement of the salt-resistant capability of crops and the like become important problems which need to be solved urgently in the development, utilization and improvement of saline soil resources.

Disclosure of Invention

The invention aims to provide a gene for enhancing the salt stress resistance of plants and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, a sorghum salt tolerance related gene and a protein coded by the same are provided, wherein the gene is selected from the group consisting of: LOC110433180 and/or LOC 8070651.

In a second aspect of the present invention, there is provided the use of LOC110433180 and/or LOC8070651 in screening salt tolerant sorghum.

In a third aspect of the present invention, there is provided the use of LOC110433180 and/or LOC8070651 in sorghum breeding.

In a fourth aspect of the present invention, a method for identifying salt tolerance of sorghum is provided, wherein the salt tolerance of sorghum is predicted by detecting the expression level of LOC110433180 and/or LOC8070651 in a sample.

In a preferred embodiment, the expression level of LOC110433180 and/or LOC8070651 in a sample is detected by nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification techniques, protein immunization techniques.

More preferably, the expression level of LOC110433180 and/or LOC8070651 in the sample is detected by a nucleic acid amplification technique.

More preferably, LOC110433180 and LOC8070651 are up-regulated in salt tolerant sorghum.

In a fifth aspect of the present invention, there is provided a product for predicting sorghum salt tolerance, said product comprising an agent for detecting the expression level of LOC110433180 and/or LOC 8070651.

In a sixth aspect of the present invention, there is provided a method for improving salt tolerance of sorghum, said method comprising increasing the expression level of LOC110433180 and/or LOC 8070651.

In a preferred example, LOC110433180 and/or LOC8070651 nucleic acids are transferred into sorghum.

The invention has the advantages and beneficial effects that:

the invention discovers that LOC110433180 or LOC8070651 is related to plant salt tolerance for the first time, and has very wide application prospect in the identification and screening of salt-tolerant plants and the field of salt-tolerant genetic improvement of crops.

Drawings

FIG. 1 is a diagram of the expression of molecular markers LOC110433180 and LOC8070651 in salt-sensitive strains and salt-tolerant strains; wherein, A is LOC 110433180; fig. B is LOC 8070651;

FIG. 2 is a graph of the expression of LOC110433180 and LOC8070651 in salt stress; wherein, the A is LOC110433180, and the B is LOC 8070651.

Detailed Description

The method provided by the invention carries out transcriptome sequencing on the salt-tolerant sorghum and the salt-tolerant sorghum, utilizes a genetic means to search the difference of salt-tolerant and salt-tolerant gene expression profiles, simultaneously detects a salt response gene through a salt stress test, excavates a salt-tolerant functional gene, analyzes a complex molecular mechanism of the salt-tolerant functional gene, lays a theoretical foundation, and provides an important means for identification and breeding of the salt-tolerant sorghum.

As used herein, the criteria for dividing an environmental term, salt stress environment or normal environment, in the present invention are consistent with the prior art. For example, for most plants, generally "salt stress tolerance" refers to the ability to tolerate salt concentrations of 0.1% to 0.2% or higher.

As used herein, the expression of LOC110433180 and LOC8070651 is up-regulated in salt-tolerant sorghum, which means that LOC110433180 and LOC8070651 are up-regulated compared to the conventional values of LOC110433180 and LOC8070651 expression in salt-tolerant sorghum, and LOC110433180 and LOC8070651 are used as a known gene, which can be easily known by those skilled in the art, and methods for comparing the expression of genes and their encoded proteins are well known, such as nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification techniques, and protein immunization techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., mRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

The present invention can amplify nucleic acids (e.g., mRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Protein immunization techniques include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

The invention relates to LOC110433180 and/or LOC8070651 nucleic acids, which can be polynucleotides encoding proteins, and can also be polynucleotides comprising additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides, which may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

In the present invention, LOC110433180 and/or LOC8070651 polynucleotide sequences may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

When expressed in higher eukaryotic cells, the polynucleotides will provide enhanced transcription when enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. The transformed plant may be transformed by Agrobacterium transformation or particle gun transformation.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.