CN116333077B - Application of corn ZmLAC9 gene in plant adaptation to salt stress - Google Patents
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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Abstract
The invention discloses cornZmLAC9The application of the gene in adapting to salt stress of plants. The gene sequence is shown in a sequence table SEQ ID NO: 1. The invention discovers that the expression level of the corn ZmLAC9 gene is increased under the condition of corn salt stress, and the condition that the corn suffers from salt stress can be relieved by over-expressing the corn ZmLAC9 gene in the corn, and the discovery lays a good theoretical foundation for breeding high-salt-tolerance corn varieties.
Description
Technical Field
The invention belongs to the technical field of biology, and in particular relates to cornZmLAC9The application of the gene in adapting to salt stress of plants.
Background
Excessive Na in salt stress soil + The osmotic potential of the soil solution is reduced, the plants are prevented from absorbing moisture and nutrients from the soil, and the soil solution is toxic to cytoplasm and organelles. Salt stress has become a major environmental factor worldwide limiting plant growth and crop yield. Although the total yield of corn in the primary grain ranks first, the global corn yield is largely limited by salt stress. Therefore, the development of related genes of corn for tolerance to salt stress and the cultivation of salt-tolerant varieties are one of key targets of modern corn breeding.
To accommodate salt stress, plants evolved a series of strategies that balance growth and stress responses, including regulation of osmotic pressure, exclusion and sequestration of ions, activation of ROS scavenging mechanisms, and metabolic reprogramming. Plant cell walls are a dynamic structure that helps determine plant morphology and protects plants from abiotic or biotic stresses. The plant cell wall is mainly composed of cellulose, hemicellulose, pectin, lignin and structural proteins. Numerous studies in plants currently show salt stressAffecting plant cell wall biosynthesis. Lignin is synthesized from monolignol (p-coumarol, coniferyl alcohol, and Xin Nachun) by phenylpropanol route oxidative polymerization. Oxidative polymerization of monolignols depends on peroxidases and Laccases (LACs). Lignin, as a second only biopolymer to cellulose, is also subject to salt stress. Hu et al found that BpNAC012 directly activated lignin-related gene expression and that overexpression of BpNAC012 increased the salt tolerance of Betula alba (Hu P, zhang K, yang C).BpNAC012positively regulates abiotic stress Responses and secondary wall biosynthesis, plant Physiology, 2019, 179, 700-717); over-expression of BpMYB46 by Guo et al has been found to increase birch lignin deposition and secondary cell wall thickness, thereby increasing birch salt tolerance and osmotic stress capacity (Guo H, wang Y, wang L, hu P, wang Y, jia Y, zhang C, zhang Y, zhang Y, wang C, et al Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula playphyllla. Plant Biotechnology Journal, 2017, 15, 107-121). The lignin content in plants is related to plant salt tolerance stress, however, the application of laccase genes in participating in corn salt tolerance has not been reported yet.
Disclosure of Invention
The invention aims to provide cornZmLAC9The application of the gene in adapting to salt stress of plants.
CornZmLAC9The gene has a gene sequence shown in a sequence table SEQ ID NO: 1.
CornZmLAC9The amino acid sequence of the gene expressed protein is shown in a sequence table SEQ ID NO: 2.
Comprising said cornZmLAC9Vector of gene.
Comprising said cornZmLAC9Recombinant bacteria or cell lines of the gene vector.
Amplifying the corn of claimZmLAC9Primers for any fragment of the gene.
The cornZmLAC9The application of the gene in improving the adaptation of plants to salt stress.
The plant is corn, wheat, soybean, sorghum, rape or cotton.
The invention has the beneficial effects that: the invention discovers that the expression level of the corn ZmLAC9 gene is increased under the condition of corn salt stress, and the condition that the corn suffers from salt stress can be relieved by over-expressing the corn ZmLAC9 gene in the corn, and the discovery lays a good theoretical foundation for breeding high-salt-tolerance corn varieties.
Drawings
FIG. 1 shows the salt treatment before and afterZmLAC9The relative expression amount of mRNA of the gene varies.
FIG. 2 is a turnZmLAC9mRNA relative expression amount in the gene corn plants is changed.
FIG. 3 is a schematic view ofZmLAC9Lignin staining map of the genetic maize plants.
FIG. 4 is a wild type and trans-formZmLAC9Phenotype comparison of the genetic maize plants.
FIG. 5 wild type and transZmLAC9Leaf phenotype comparison of the genetic maize plants.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental materials used in the following examples: maize inbred material: b73 and KN5585; strains: coli strain DH 5. Alpha. And Agrobacterium tumefaciens strain GV3101; the over-expression vector is CUB.
Example 1 real-time fluorescent quantitative PCR analysisZmLAC9Response to salt stress
And (3) experimental material treatment: the maize B73 inbred line is firstly subjected to water planting treatment, and the formula of the water planting total nutrient solution is as follows: 0.75 mM K 2 SO 4 、0.65 mM MgSO 4 •7H 2 O、0.1 mM KCl、2 mM Ca(NO 3 ) 2 •4H 2 O、0.2 mM Fe-EDTA、0.001 mM H 3 BO 3 、1 μM MnSO 4 •H 2 O、1 μM ZnSO4•7H 2 O、0.1 μM CuSO 4 、0.005 μM (NH 4 ) 6 Mo 7 O 24 •4H 2 O、250 μM KH 2 PO 4 The pH of the nutrient solution is 5.8-6.0. The nutrient solution is changed every 2 days during the water planting period. The culture conditions of the plants were 14 h light/10 h dark, 28 ℃/22 ℃. After 11 days of water culture, the whole nutrient solution containing 0 mM NaCl, 100 mM NaCl, 125 mM NaCl and 150 mM NaCl was used for treatment, and the two-leaf was sampled at the time points of treatment 24h and 48h, respectively.
Extracting total RNA from B73 leaves subjected to water culture for 24 hours and 48 hours by using 150 mM NaCl, carrying out reverse transcription to generate a first-chain cDNA, carrying out real-time fluorescence quantitative PCR amplification by using the first-chain cDNA as a template and adopting primer pairs consisting of 5'-ACCACCACGAGTTCGTTATC-3' and 5'-CAGTCCTTATCTGGCGGATG-3', and identifyingZmLAC9Relative expression levels of genes. Selecting and usingUbiThe gene is a reference gene (the primer pair used for identifying the reference gene consists of 5'-GCTGCCGATGTGCCTGCGTCG-3' and 5'-CTGAAAGACAGAACATAATGAGCACAG-3'). Real-time fluorescence quantification was performed using Applied Biosystems 7500 Real Time PCR system (ABI, USA), with 3 replicates per parallel assay setup. By 2 -ΔΔCT The relative expression level was calculated by the method.
The results are shown in FIG. 1, and compared with the control, when 150 mM NaCl is subjected to water culture for 24 hours and 48 hours,ZmLAC9the relative expression level of the gene is significantly increased.
Example 2 cornZmLAC9Preparation of overexpressing transgenic plants
CUB-LAC9Construction of an over-expression vector: PCR amplification was performed using cDNA of the aerial part of maize inbred line B73 as a template under hydroponic conditions, using the upstream primer 5'-TAGAGGATCGGTCACCATGGCGTCGTCGTCCTCGTCCCGGCT-3' and the downstream primer 5'-CCCGCGGTACGGTGACGCACATGGGAAGATCAACTG-3', and purified to recover about 1500 bpZmLAC9Gene DNA fragment, use ofBamH I endonuclease (NEB) cleaves the CUB vector and the cleaved product is recovered. By In-Fusion (Clontech)ZmLAC9Recovering the fragment and ligating to the linearized CUB vector to obtain a recombinant vectorCUB-LAC9。
ZmLAC9Preparation of overexpressing transgenic plants: by maize inbred lineKN5585 as background material, use of a material containingCUB- LAC9Infecting young maize embryo with agrobacterium GV3101 strain of the vector to obtain T 0 Generating transgenic positive plants; will T 0 The generation positive transgenic plant is selfed for 2 generations to obtain homozygosityZmLAC9The over-expression transgenic corn material is selected from two over-expression homozygous lines #1 and #2, and a primer pair consisting of 5'-ACCACCACGAGTTCGTTATC-3' and 5'-CAGTCCTTATCTGGCGGATG-3' is adopted for real-time fluorescence quantitative PCR detection.
The results are shown in FIG. 2, in which the relative expression level of the ZmLAC9 gene was significantly increased in the ZmLAC9-OE #1 and #2 strain plants as compared to the wild-type plants.
Example 3 lignin staining observations of corn under salt treatment conditionsZmLAC9Lignin content in leaves of over-expressed transgenic plants
The test plants were:ZmLAC9over-expressed transgenic materials [ ]#1And#2) Wild type material (KN 5585).
Leaf lignin staining: taking wild type and transgenic plant penultimate leaves (6 to cm from leaf tips) which are subjected to water culture treatment for 4 days with 150 mM NaCl, and embedding the materials with 3% agarose; fixing the embedding material on a base of a slicing machine, slicing by using a Leica VT 1000s oscillation slicing machine, wherein the slicing thickness is 50 mu m; adding a drop of water in the middle of a glass slide, picking up a complete slice material, placing the slice material on the glass slide, absorbing water after the material is unfolded, dripping 5% phloroglucinol for dyeing for 2 min, dripping an equal volume of concentrated hydrochloric acid, standing at room temperature for 2 min, dripping 40% glycerol, and observing and photographing by a Leica CTR6 microscope.
The results are shown in FIG. 3, after 150 mM NaCl treatment, compared to wild type plantsZmLAC9-OEThe lignin staining color was darkened in plants of lines #1 and #2, indicating an increased lignin content.
Example 4 plant phenotype observations under salt treatment of maize ZmLAC9 overexpressing transgenic plants
Experimental material treatment and phenotypic observation: wild-type material (KN 5585) andZmLAC9over-expressed transgenic materials [ ]# 1And#2) 150 mM NaCl water culture treatment is carried out, and the phenotype of the transgenic material under salt treatment is observed.
As shown in FIGS. 4 and 5, leaves of wild-type corn wilt after 150 mM NaCl treatmentZmLAC9Leaves that overexpress plant material remain green and expand.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (2)
1. CornZmLAC9Use of a gene for increasing plant adaptation to salt stress, characterized in that the maizeZmLAC9The nucleotide sequence of the gene is shown in a sequence table SEQ ID NO: 1.
2. The corn of claim 1ZmLAC9The application of the gene in improving plant adaptation to salt stress is characterized in that the plant is corn, wheat, soybean, sorghum, rape or cotton.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5652799B1 (en) * | 2014-02-17 | 2015-01-14 | 独立行政法人国際農林水産業研究センター | The gene qNaCl3 controlling the salt tolerance located on the chromosome 3 of soybean and its utilization |
| CN106416773A (en) * | 2016-10-09 | 2017-02-22 | 山东省农业科学院玉米研究所 | Screening and identification method of salt tolerance of corns at seedling stage |
| CN112063597A (en) * | 2019-06-10 | 2020-12-11 | 山东大学 | Maize multi-copper oxidase coding gene ZmDEK559-2 and application thereof |
| CN114807172A (en) * | 2022-05-23 | 2022-07-29 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Salt-responsive corn functional gene |
| CN115094070A (en) * | 2022-06-22 | 2022-09-23 | 南通大学 | Discovery method and application of corn salt-tolerant gene Zm00001d033878 |
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