CN111334514A

CN111334514A - Method for improving plant aluminum resistance by applying peanut AhGSNOR gene

Info

Publication number: CN111334514A
Application number: CN202010121986.3A
Authority: CN
Inventors: 何龙飞; 潘春柳; 詹洁; 肖冬; 王爱勤
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-02-26
Filing date: 2020-02-26
Publication date: 2020-06-26
Anticipated expiration: 2040-02-26
Also published as: CN111334514B

Abstract

The invention discloses a method for improving plant aluminum resistance by applying peanut AhGSNOR gene, wherein the AhGSNOR gene has a polynucleotide sequence comprising: (a) has the sequence shown in SEQ ID NO: 1; (b) encoding the amino acid sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. The AhGSNOR gene is transformed into a tobacco plant through a transgenic technology, and the tolerance of the tobacco plant to aluminum stress is found to be remarkably improved, which shows that the gene plays a positive role in resisting the aluminum stress of the plant. The invention has important significance for improving the aluminum resistance of crops and creating plant aluminum-resistant germplasm by utilizing a genetic engineering technology.

Description

Method for improving plant aluminum resistance by applying peanut AhGSNOR gene

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a method for improving plant aluminum resistance by applying peanut AhGSNOR genes.

Background

Aluminum is the most abundant metal in the crust, its chemical morphology depends on pH, in acid soils (pH < 4.5), Al³⁺Is the main soluble and toxic form of aluminum. Thus, the adverse effect of acid soils on plant growth and Al³⁺The toxicity of (2) is closely related. Soluble Al³⁺Can inhibit root growth, influence plant water and nutrient absorption ability, and limit plant growth and productivity. In recent years, a great deal of research has been carried out around the mechanism of aluminum toxicity removal of plants and the mechanism of internal and external adaptation of plants to aluminum toxicity, however, limited gene function research limits the understanding of the mechanism of aluminum resistance of plants and the application of aluminum-resistant germplasm. The genetic engineering transformation of plants by using breeding means and the creation and cultivation of aluminum-resistant germplasm have become important targets of agricultural scientific research.

Related studies indicate that the toxicity of aluminum to plants is related to Nitric Oxide (NO) homeostasis, and GSNOR (S-nitrosoglutathione reductase) plays an important role in regulating NO homeostasis and participates in various physiological processes of plants. It has been found that overexpression of GSNOR can increase the saline tolerance of tomato and increase the ROS (Reactive oxygen species) scavenging efficiency in saline-alkali stress (Gong B, Wen D, Wang X, et al, S-nitro glutamatergic reaction-modulated redox signaling control and sodium alginate L [ J ] plant and Cell Physiology 2015, 56 (4): 790) 802.). Conversely, inhibition of GSNOR expression causes a reduction in the antioxidant capacity of tomato plants and leads to ROS outbreaks and PCD (programmed cell death) occurrences. Another study found that Arabidopsis thaliana GSNOR mutant Plant (atgsnor1-3) exhibited early flowering, suppressed root elongation, and reduced cotyledon phenotypes (Lee U, Wie C, Fernandez BO, et al.Modulation of sexual stress by S-nisotoglutathione production for clinical research and Plant growth in Arabidopsis [ J ]. The Plant Cell, 2008, 20 (3): 786-. However, the application of the gene in improving the aluminum resistance of economic crops such as peanuts, tobaccos and the like is not found at present.

Disclosure of Invention

It is an object of the present invention to address at least the above-mentioned deficiencies and to provide at least the advantages which will be described hereinafter.

The invention also aims to provide application of the peanut AhGSNOR gene in improving the aluminum resistance of plants.

Another object of the present invention is to provide a method for improving aluminum tolerance of plants by using the peanut AhGSNOR gene.

To achieve these objects and other advantages in accordance with the purpose of the invention, the present invention provides a use of an AhGSNOR gene of peanut as a gene for improving aluminum resistance of a plant, the AhGSNOR gene having a sequence as set forth in SEQ ID NO: 1.

Further, SEQ ID NO:1 is the coding sequence of the AhGSNOR gene.

Further, the AhGSNOR gene codes an amino acid sequence shown in SEQ ID NO:2, respectively.

Further, the plant is peanut or tobacco.

A method for improving the aluminum resistance of plants by using peanut AhGSNOR genes is realized by increasing the expression level of endogenous AhGSNOR genes of peanuts or over-expressing exogenous AhGSNOR genes in other plants.

Furthermore, the overexpression of the exogenous AhGSNOR gene refers to that the AhGSNOR gene is transformed into a receptor plant for expression by using a plant expression vector through an agrobacterium-mediated or gene gun method.

Further, the AhGSNOR gene is introduced into a recipient plant cell, tissue or organ via a plant expression vector.

Further, the method specifically comprises the following steps:

a. obtaining a positive clone which contains a DNA fragment of the AhGSNOR gene;

b. constructing an AhGSNOR gene overexpression vector through the positive cloning;

c. transforming the AhGSNOR gene overexpression vector into agrobacterium;

d. the obtained agrobacterium is utilized to introduce the AhGSNOR gene into a target plant, and a plant which stably expresses transgenosis is screened out.

Further, finally, genetically stable transgenic plants are obtained through cultivation.

Further, the plant expression vector drives the expression of the AhGSNOR gene through a constitutive promoter.

Further, the constitutive promoter is a 35S promoter.

Alternatively, the method comprises the steps of:

a. obtaining a DNA fragment of the AhGSNOR gene;

b. constructing an AhGSNOR overexpression vector;

c. agrobacterium-mediated genetic transformation;

d. and (4) screening stably expressing transgenic plants.

Further, transgenic plants with enhanced aluminum resistance are cultivated.

Further, the method also comprises the step of carrying out aluminum stress treatment on the transgenic plant and evaluating the aluminum resistance of the plant.

The invention at least comprises the following beneficial effects:

the AhGSNOR gene derived from peanuts is introduced into plants such as peanuts or tobacco by adopting a genetic engineering method, so that the tolerance of the plants such as the tobacco or the peanuts to aluminum stress is obviously improved.

The AhGSNOR gene has positive regulation and control effects on the aspect of plant aluminum resistance, and provides a new idea for cultivating aluminum-resistant germplasm, particularly peanut germplasm.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a schematic diagram showing the construction of the expression vector pBI121-AhGSNOR of example 2;

FIG. 2 is a comparison of GSNOR activity of the roots of wild type plants and transgenic plants of the invention under aluminum stress in example 4;

FIG. 3 is a comparison of the relative root growth rates of wild type plants and transgenic plants of the invention under aluminum stress in example 4;

FIG. 4 is a comparison of the root cell death rates of wild type plants and transgenic plants of the invention under aluminum stress in example 4;

FIG. 5 is a comparison of the SOD activity of the roots of wild type plants and transgenic plants of the present invention under aluminum stress in example 4;

FIG. 6 is a comparison of root POD activity of wild type plants and transgenic plants of the present invention under aluminum stress in example 4;

FIG. 7 is a comparison of CAT activity in the root system of wild type plants and transgenic plants of the present invention under aluminum stress in example 4;

FIG. 8 is a comparison of the MDA content in the root system of the wild type plant and the transgenic plant of the present invention under aluminum stress in example 4.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

Example 1

And obtaining a DNA fragment of the AhGSNOR gene. The method comprises the following specific steps: 0.2g of peanut root tip was taken and total RNA was extracted using Trizol reagent (Invitrogen). RNA was reverse transcribed into cDNA using the RevertAID TM first Strand cDNA Synthesis Kit (Thermoscientific). Designing an upstream primer (5-CTCGAGATGGCAACTCAAGGTCAAGT-3', wherein the Xho I site is underlined) and a downstream primer (5 ″)GTCGACTGCATCTGTTGAAAGAACAC-3', wherein the Sal I site is underlined), and PCR was performed using the peanut cDNA as a template. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 90s for 35 cycles. The PCR product was recovered by gel using DNA purification recovery kit (Tiangen), the recovered product was ligated to pMD19-T vector, positive clones were selected and sequenced to obtain the desired DNA fragment (sequence shown in SEQ ID NO: 1). The positive clone was named pMD-AhGSNOR.

Example 2

Constructing an overexpression vector of the AhGSNOR gene. The method comprises the following specific steps: the pMD-AhGSNOR-positive cloning plasmid and the pBI121-EGFP vector plasmid (which are commercially available from publicly available sources) in example 1 were extracted, and double-digested with restriction enzymes XhoI and Sal I, respectively, and the digested fragments were recovered using a DNA purification recovery kit (Tiangen). The digested fragments were ligated with a DNA ligation kit (Takara) in a ligation reaction system of pMD-AhGSNOR gel recovery product 4. mu. L, pBI121-EGFP gel recovery product 1. mu.L under 16 ℃ for 3 h. The ligated product was transformed into DH5a competent cells, single colonies were resistance-screened using 100mg/L kanamycin, and the positive clones obtained were sequence-verified. The positive clone that was sequenced correctly was named pBI121-AhGSNOR (vector construction scheme is shown in FIG. 1). The pBI121-AhGSNOR plasmid was extracted and transformed into Agrobacterium strain EHA 105.

Example 3

The AhGSNOR gene is introduced into tobacco plants through an agrobacterium-mediated genetic transformation system. The method comprises the following specific steps:

(1) culturing the tobacco aseptic seedlings: soaking tobacco seeds in 75% ethanol for 1min, soaking in 0.1% mercuric chloride for 5min, and washing with sterile water for 3-5 times. The sterilized seeds were inoculated onto MS minimal medium with sterile forceps and cultured in an incubator at 26 ℃.

(2) Obtaining a leaf disc: when the sterile seedling induced by the seeds grows to the width of 1-1.5cm, the leaves are cut and the leaf back is scratched, and the leaf is inoculated into a solid culture medium containing 1.0mg/L6-BA and 0.2mg/L NAA and cultured in the dark for 2 d.

(3) Infection: 2mL of the pBI 121-AhGSNOR-containing Agrobacterium EHA105 of example 2 was pipetted and inoculated into 100mL of YEP liquid medium containing 25mg/L rifampicin, 25mg/L streptomycin and 100mg/L kanamycin, and cultured overnight at 200rpm at 28 ℃. The bacterial liquid is transferred to a sterilized centrifuge tube, centrifuged at 12000rpm and 4 ℃ for 10min, and the supernatant is discarded. The cells were resuspended in MS broth containing 2.5% sucrose. The leaf blade after dark culture for 2d is placed in the staining solution, shaken at 28 ℃ and 200rpm for 10min, and then clamped on sterile filter paper. After the bacterial liquid on the surface of the leaf is sucked dry, the leaf is inoculated into a culture medium containing 1.0mg/L6-BA and 0.2mg/L NAA, and is placed in an incubator at 26 ℃ in the dark for co-culture for 2 d.

(4) Bacteriostatic culture and resistance screening: after the co-culture is finished, the leaves are washed 3-5 times with sterile water. The leaves are inoculated in an antibacterial culture medium containing 1.0mg/L6-BA, 0.2mg/L NAA and 100mg/L timentin, and induced to differentiate to generate cluster buds. The leaf blade from which the cluster buds have been differentiated is transferred to a resistant medium containing 1.0mg/L6-BA, 0.2mg/L NAA, 100mg/L timentin and 100mg/L kanamycin, and the cluster buds having resistance are selected.

(5) Rooting induction and hardening seedling transplantation: inoculating the single bud which grows to 2-3cm and has greener leaves to 1/2MS solid culture medium containing 0.5mg/LNAA and 100mg/L timentin, and inducing the aseptic seedling to root. When the height of the seedling is 4-5cm, opening the cover, hardening the seedling for 3d, and transplanting the seedling into a nutrition cup containing a mixed matrix (seedling culture soil, perlite and river sand are 1: 1) for culture.

Example 4

Identification of transgenic tobacco and analysis of aluminum resistance of T1 generation tobacco. The method comprises the following specific steps:

(1) identification of transgenic tobacco: 0.2g of tobacco resistant shoot leaves obtained by the culture in example 3 was taken, and DNA was extracted by the CTAB method. PCR detection is carried out by using AhGSNOR gene specific primers (upstream primer: 5 '-ATGGCAACTCAAGGTCAAGT-3', downstream primer: 5 '-TGCATCTGTTGAAAGAACAC-3') to obtain transgenic tobacco plants.

(2) Acquisition and genetic stability detection of T1 generation of tobacco: when the height of the transgenic tobacco seedling is about 10cm, transplanting the transgenic tobacco seedling into a greenhouse for conventional culture. The harvested transgenic T0 generation seeds were sown on 1/2MS medium containing 100mg/L kanamycin, while wild type tobacco seeds were sown on antibiotic-free 1/2MS medium. When the second true leaf grows out of the seedling, the seedling with normal leaf color is selected and planted in a seedling plug tray containing a mixed matrix (seedling soil: perlite: river sand is 1: 1). When 6-7 leaves grow out, the root systems of the transgenic plant and the wild plant are cut, total RNA is extracted by using an RNA extraction kit (Kangji), and the total RNA is reversely transcribed into cDNA by using a HiFiScript cDNA first strand synthesis kit (Kangji). PCR detection is carried out on RNA by using AhGSNOR gene specific primers (an upstream primer: 5 '-ATGGCAACTCAAGGTCAAGT-3' and a downstream primer: 5 '-TGCATCTGTTGAAAGAACAC-3'), and the result shows that the transgenic tobacco can obtain an amplification strip with the size consistent with that of a target gene through detection, while the wild tobacco does not detect the target strip, so that the AhGSNOR gene is stably inherited in a T1 generation of the transgenic tobacco.

(3) Analysis of aluminum resistance of transgenic tobacco: when 6-7 leaves grow on the T1 generation transgenic plant or the wild tobacco plant, selecting a plant with uniform growth, cleaning the root system, and carrying out stress treatment for 24 hours under different aluminum treatment concentrations (0 mu mol/L, 50 mu mol/L, 100 mu mol/L and 200 mu mol/L). Taking root system for GSNOR activity, relative growth rate, cell death rate, antioxidase activity and MDA content determination. It is found that under the condition of no aluminum stress or aluminum stress, the activity of the root system GSNOR of the over-expressed plant is obviously higher than that of the wild plant (figure 2), the over-expressed AhGSNOR can relieve the inhibition of the aluminum stress on the tobacco root elongation (figure 3), and the root system cell death caused by the aluminum stress is reduced (figure 4). Under the condition of aluminum stress, the SOD activity of the root systems of the over-expressed plants and the wild plants has no obvious difference (figure 5), but the POD and CAT activities of the root systems of the over-expressed plants under the higher concentration aluminum treatment concentration are higher than those of the wild plants (figure 6 and figure 7). Overexpression of AhGSNOR is beneficial to the tobacco plants to resist the anti-oxidation stress caused by the aluminum stress and relieve the peroxidation of root system membrane lipid (figure 8). The above results indicate that overexpression of AhGSNOR is advantageous for improving the aluminum tolerance of plants.

While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art.

<110> Guangxi university

<120> method for improving plant aluminum resistance by applying peanut AhGSNOR gene

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Glu Pro Asn Lys Pro Leu Ser Ile Glu Asp Val Gln Val Ala Pro Pro

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Gln Ala Gly Glu Val Arg Val Lys Ile Leu Tyr Thr Ala Leu Cys His

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Thr Asp Ala Tyr Thr Trp Ser Gly Lys Asp Pro Glu Gly Leu Phe Pro

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Cys Ile Leu Gly His Glu Ala Ala Gly Ile Val Glu Ser Val Gly Glu

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Gly Val Thr Asn Val Gln Pro Gly Asp His Val Ile Pro Cys Tyr Gln

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Ala Glu Cys Gly Glu Cys Lys Phe Cys Lys Ser Gly Lys Thr Asn Leu

100 105 110

Cys Gly Lys Val Arg Ser Ala Thr Gly Val Gly Val Met Leu Ser Asp

115 120 125

Arg Lys Ser Arg Phe Ser Ile Asn Gly Lys Thr Ile Tyr His Phe Met

130 135 140

Gly Thr Ser Thr Phe Ser Gln Tyr Thr Val Val His Asp Val Ser Val

145 150 155 160

Ala Lys Ile Asp Pro Ala Ala Pro Leu Asp Lys Val Cys Leu Leu Gly

165 170 175

Cys Gly Val Pro Thr Gly Leu Gly Ala Val Trp Asn Thr Ala Lys Val

180 185 190

Glu Pro Gly Ser Ile Val Ala Val Phe Gly Leu Gly Thr Val Gly Leu

195 200 205

Ala Val Ala Glu Gly Ala Lys Ser Ala Gly Ala Ser Arg Ile Ile Gly

210 215 220

Ile Asp Ile Asp Ser Lys Lys Phe Asp Thr Ala Lys Asn Phe Gly Val

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Thr Glu Phe Ile Asn Pro Lys Glu His Asp Lys Pro Ile Gln Gln Val

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Ile Val Asp Leu Thr Asp Gly Gly Val Asp Tyr Ser Phe Glu Cys Ile

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Trp Gly Thr Ser Val Ile Val Gly Val Ala Ala Ser Gly Gln Glu Ile

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Ser Thr Arg Pro Phe Gln Leu Val Thr Gly Arg Val Trp Lys Gly Thr

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Ala Phe Gly Gly Phe Lys Ser Arg Ser Gln Val Pro Trp Leu Val Glu

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Leu Thr Leu Gly Glu Ile Asn Lys Ala Phe Asp Leu Met His Gly Gly

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Gly Cys Leu Arg Cys Val Leu Ser Thr Asp Ala

370 375

Claims

1. The application of the peanut AhGSNOR gene in improving the aluminum resistance of plants, wherein the AhGSNOR gene has a nucleotide sequence shown in SEQ ID NO. 1.

2. The use according to claim 1, wherein the nucleotide sequence shown in SEQ ID NO. 1 is the coding sequence of the AhGSNOR gene.

3. The use of claim 1, wherein the AhGSNOR gene encodes an amino acid sequence as set forth in SEQ ID NO. 2.

4. Use according to claim 1, wherein the plant is peanut or tobacco.

5. A method for improving the aluminum resistance of a plant by using the peanut AhGSNOR gene as claimed in any one of claims 1 to 3, which is realized by improving the expression level of endogenous AhGSNOR genes in peanuts or overexpressing exogenous AhGSNOR genes in other plants.

6. The method for improving the aluminum resistance of the peanut AhGSNOR gene according to claim 5, wherein the step of over-expressing the exogenous AhGSNOR gene is that the AhGSNOR gene is transformed into a receptor plant for expression by an agrobacterium-mediated or gene gun method by using a plant expression vector.

7. The method for improving the aluminum tolerance of a plant by using the peanut AhGSNOR gene according to claim 6, wherein the AhGSNOR gene is introduced into a cell, a tissue or an organ of a recipient plant through a plant expression vector.

8. The method for improving the aluminum resistance of the plants by using the peanut AhGSNOR gene according to claim 5, which is characterized by comprising the following steps:

c. transforming the AhGSNOR gene overexpression vector into agrobacterium;

9. The method for improving the aluminum tolerance of plants by using the peanut AhGSNOR gene according to claim 8, wherein genetically stable transgenic plants are obtained by final cultivation.