CN117947051A - Potato StCuRG gene, biological material and application of over-expressed StCuRG gene - Google Patents

Abstract

The invention discloses a potato StCuRG gene, a biological material and application of over-expressed StCuRG gene. The invention constructs a vector by using a strong promoter 35S and a potato StCuRG1 gene, and converts a potato commercial variety Desiree with good quality but not resistant to late blight, thereby obtaining a new transgenic potato variety with enhanced response to copper ion induced resistance and high resistance to late blight. Late blight resistance detection shows that compared with a wild type control, the transgenic potato with the StCuRG gene being over-expressed has obviously enhanced late blight resistance, which indicates that improving StCuRG1 gene expression can improve the resistance of the potato to the late blight. The gene can be applied to the genetic engineering improvement of potato late blight resistance.

Description

Potato StCuRG gene, biological material and application of over-expressed StCuRG gene

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a potato StCuRG gene, a biological material and application of over-expressed StCuRG gene.

Background

Late blight of potato caused by phytophthora infestans (Phytophthora infestans) is a first large oomycete disease that severely jeopardizes global potato production. Late blight causes a 10% -30% loss in potato yield in the general epidemic years, and the severe epidemic years can reach over 50% or even absolute production. Cultivation of late blight resistant varieties and improvement of efficient pesticide utilization are one of the main targets for potato breeding.

The copper preparation is a protective bactericide, has the advantages of wide sterilization range, safety to agricultural products and environment, short residual period, no drug resistance and the like, and is widely applied to the prevention and treatment of plant diseases such as late blight and the like. The traditional theory holds that the sterilization mechanism is the disease prevention principle of the copper preparation, wherein the heavy metal toxicity of copper ions directly interferes with the activity of intracellular enzymes of pathogenic microorganisms, and inhibits the growth of fungi and bacteria, thereby playing a role in sterilization. The use of copper formulations has been in the past two hundred years, and a large number of pathogenic microorganisms with copper ion tolerance have been found in production, but copper-ion-tolerant strains can still be effectively controlled by spraying copper formulations. Recent studies have revealed that very low concentrations (10 nM) of copper ions in the Arabidopsis thaliana-P.syringae system can increase the resistance of Arabidopsis thaliana to P.syringae DC 3000. The copper ions can activate plants to generate a series of disease-resistant reactions, such as promoting the accumulation of active oxygen substances, promoting the deposition of callose, up-regulating the expression of genes related to the course of disease, activating the phosphorylation of MAPK proteins, promoting the rapid release of ethylene and the like, and enhancing the resistance of arabidopsis to bacterial diseases. In the potato-late blight bacterium interaction system, copper ions activate ethylene synthesis faster and stronger, and inhibit transcription of abscisic acid synthesis related genes StABA, stNCED1 and the like, so that the level of abscisic acid is reduced, and the disease resistance of potatoes to late blight is enhanced. In the rice-Rice Stripe Virus (RSV) system, the trace element copper can enhance the antiviral ability of rice by inhibiting SPL 9. The function of the copper ions for resisting viruses depends on the pathway of SPL9-miR528-AO-ROS, and the copper ions can inhibit the protein accumulation level of SPL9 and the capability of SPL9 to combine with a miR528 promoter, so that the miR528 expression level is reduced, and the accumulation amount of Ascorbate Oxidase (AO) and the Reactive Oxygen Species (ROS) level are further enhanced; on the other hand, copper ions can directly influence the enzyme activity of AO, and when the copper ion binding site of AO is mutated, the AO mediated antiviral ability is also inhibited. Copper ions are directly applied exogenously or the copper ion transport gene (COPT) of the rice is knocked out by a gene editing technology, so that copper ion accumulation can be improved, SPL9-miR528-AO pathway is regulated and controlled to enhance the resistance of the rice to viruses, and a potential thought is provided for practical application of copper ion antivirus. Importantly, the above-mentioned antiviral pathway mediated by copper ions has broad-spectrum resistance to different rice viruses (RSV and RDV), suggesting a broad application prospect for copper ions.

However, no genetic engineering technology based on copper ion theory is disclosed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a potato StCuRG gene, a biological material and application of over-expressed StCuRG gene. The invention constructs a vector by using a strong promoter 35S and a potato StCuRG1 gene, and converts a potato commercial variety Desiree with good quality but not resistant to late blight, thereby obtaining a new transgenic potato variety with enhanced response to copper ion induced resistance and high resistance to late blight.

The technical scheme of the invention comprises the following aspects:

as a first aspect of the present invention, there is provided a potato StCuRG gene comprising at least one of the following nucleotide sequences:

a, a nucleotide sequence shown in SEQ ID NO. 1;

b, a nucleotide sequence shown as SEQ ID NO. 2;

c, a nucleotide sequence shown as SEQ ID NO. 3;

d, coding the nucleotide sequence corresponding to the amino acid sequence with equivalent function formed by artificial engineering or substitution, deletion or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO. 4.

As a second aspect of the present invention, there is provided a biological material comprising the potato StCuRG gene and a pCXSN vector.

As a third aspect of the invention, there is provided the use of an overexpressed potato StCuRG1 gene to enhance resistance of potatoes to late blight.

The application is characterized in that the biological material for over-expressing potato StCuRG gene is prepared, which comprises the following steps:

Step 1, extracting total RNA of potato variety Desiree, carrying out reverse transcription to obtain cDNA, taking the cDNA as a template, and using StCuRG specific primer F1:5'-GATGGAGAAGAATAAGATTATTAAGAG-3', R1:5'-TCATAAACACATCGATGGATCG-3' is primer amplification StCuRG1 coding region sequence, which is connected to pCXSN vector cut by XcmI to obtain vector pCXSN: stCuRG1;

Preferably, in the step 1, firstly, a normal potato variety Desiree is used as a material, a copper sulfate solution (10 mu M) is sprayed to treat the potato, leaves are collected after treatment, and a TRIZOL method is adopted to extract total RNA;

Step 2, pCXSN: stCuRG1 transforming Agrobacterium competent cells AGL1, and performing activation and culture to transform the vector into cultivar Desiree by a stem segment transformation method to obtain a transformed pCXSN: stCuRG1 Gene plants.

In the embodiment of the invention, the late blight resistance detection shows that compared with a wild type control, the transgenic potato with the StCuRG over-expressed gene has obviously enhanced late blight resistance.

The application is characterized in that the potato cells are modified by biological materials which over express the StCuRG gene of the potato.

The method provided by the invention specifically comprises the following steps: and (3) adopting a strong potato Desiree aseptic seedling grown for 21 d-28 d, cutting off axillary bud-free stem segments, and carrying out pCXSN (prestressed high-strength concrete) transformation: stCuRG1 of agrobacterium AGL1 bacteria liquid is fully contacted to obtain a conversion material, co-culture is carried out, and the co-cultured stem sections are subjected to callus differentiation, bud callus emergence and regeneration to obtain regenerated plants.

The use is also embodied in a method for enhancing plant resistance comprising overexpressing the potato StCuRG gene and applying a copper formulation.

Further, the copper formulation is a copper sulfate solution. The embodiment of the invention shows that the expression technology for inhibiting the gene weakens the control effect of the copper preparation, and the constitutive expression of the gene enhances the control effect of the copper preparation. The invention provides application of the gene in plant resistance improvement and copper preparation control.

In the embodiment of the invention, the plant is potato.

Compared with the prior art, the invention has the beneficial effects that:

in conclusion, the transgenic potato with the over-expressed StCuRG gene is obtained, and the late blight resistance detection shows that compared with a wild type control, the transgenic potato with the over-expressed StCuRG gene has obviously enhanced late blight resistance, which indicates that the improvement of StCuRG1 gene expression can improve the resistance of the potato to the late blight. The gene can be applied to the genetic engineering improvement of potato late blight resistance. The invention discloses application of a genetic engineering technology based on a copper ion theory, and provides a new idea for implementation of the theory.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a graph showing the relative expression levels of StCuRG gene transcription assays in copper ion-activated potatoes of example 1.

FIG. 2 is a graph showing the results of the generation and disease resistance characterization of StCuRG gene-silenced plants of example 2, wherein (a) is the silencing of StCuRG gene expression in potato using the VIGS technique; (b) Detecting cell necrosis of control pTV00 and StCuRG1 silencing materials after being treated by copper sulfate and magnesium sulfate and inoculated with late blight for 3 days by trypan blue staining, and taking magnesium sulfate as a control; (c) Statistical results of disease index for 3 days after inoculation of late blight for control pTV00 and StCuRG1 silencing materials treated with copper sulfate, magnesium sulfate, wherein: t-test, P <0.01 compared to control (pTV 00, mgSO ₄ treatment).

FIG. 3 is a construction diagram of StCuRG1 over-expression vector in example 4, in which (a) is CDS region amplification of StCuRG; (b) StCuRG A.tumefaciens PCR assay.

FIG. 4 is a diagram showing the stage of transformation of potato by the stem segment method in example 4, wherein (a) is differentiated callus; (b) is a budding callus; (c) regenerating potato seedlings.

FIG. 5 is a graph showing the molecular level detection of StCuRG a transgenic potato in example 4, wherein (a) is the DNA level detection of transgenic potato; (b) For transgenic potato RNA level detection, desiree was used as a control.

FIG. 6 is a graph of a resistance identification test of example 5, wherein (a) StCuRG is a 3-day disease condition of a transgenic potato and Desiree post-epidemic disease; (b) Statistical results of disease index for StCuRG days after inoculation of transgenic potatoes with desire for 3 days of late blight, wherein: t-test, P <0.05 compared to wild-type leaf: t-test, P <0.01 compared to wild type.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

In the present invention, the term "CDS sequence" refers to a Coding sequence (Coding sequence). The CDS is a DNA sequence corresponding to the protein sequence one by one, and the sequence does not contain other sequences not corresponding to the protein, and the CDS completely corresponds to the codon of the protein without considering the sequence change in the process of mRNA processing and the like.

In the present invention, the term "gene" refers to a nucleic acid fragment expressing a specific protein. "Gene" includes a region of DNA encoding a gene product, as well as all regions of DNA that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding and/or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, introns, and locus control regions.

The proteins of the invention may be altered in various ways, including amino acid substitutions, deletions, truncations and insertions. Methods for such operations are generally known in the art. Methods for mutagenesis and polynucleotide alteration are well known in the art.

In the present invention, the term "expression" refers to the biosynthesis of a gene product, including transcription and/or translation of said gene product. "expressing" or "producing" a protein or polypeptide from a DNA molecule refers to the transcription and translation of a coding sequence to produce a protein or polypeptide, while "expressing" or "producing" a protein or polypeptide from an RNA molecule refers to the translation of an RNA coding sequence to produce a protein or polypeptide.

Example 1

This example describes copper sulfate treatment to activate StCuRG gene transcription in potatoes.

Potato seeds Desiree of normal soil culture are used as materials, a copper sulfate solution (10 mu M) is sprayed by a handheld small sprayer to carry out spraying treatment on potatoes growing for 4 weeks, leaves are collected for 0h, 2h and 24h after the treatment, a TRIZOL method (purchased from Kagaku as century biotechnology Co., ltd.) is adopted to extract total RNA, reverse transcriptase MMLV (purchased from Aibo) is adopted to carry out reverse transcription on the total RNA into cDNA first strand, and the reaction conditions are that: 42 ℃ for 1h;65℃for 20min. Then, using this cDNA as a template, stEF a (PGSC 0003DMG 400023272) as an internal reference gene, and StEF a gene amplification primer was F3:5'-CAAGGATGACCCAGCCAAG-3', R3:5'-TTCCTTACCTGAACGCCTGT-3', using StCuRG gene-specific primer F4:5'-ATGGGTTCATTGATAGGAGTAA-3' and R4:5'-ATCTATGTCAGGTCCTTCAGC-3' real-time quantitative PCR was performed, reaction conditions: pre-denaturation at 95℃for 3min;95 ℃ for 10sec;60 ℃,10sec,72 ℃,30sec,40 cycles. The results are shown in FIG. 1.

Example 2

This example describes the generation and disease resistance identification of StCuRG gene-silenced plants.

Potato leaf cDNA was used as a template, and StCuRG gene-specific primers StCuRG-VIGS-F2: 5'-CGGGATCCAAAGATGGAGGAGTTCTCGC-3' (wherein the underlined sequence is the restriction enzyme BamHI recognition sequence), stCuRG1-VIGS-R2:5'-GGGGTACCGATGTGGTGTTGGCATTTCG-3' (wherein the underlined sequence is the recognition sequence of restriction enzyme KpnI), a StCuRG gene specific fragment was amplified, the PCR product and vector pTV00 were digested with restriction enzymes BamHI, kpnI, and then reacted at 16℃under the action of T4 DNA LIGASE, the ligation product transformed E.coli DH 5. Alpha. Competent cells (purchased from Shanghai Biotechnology Co., ltd.) to screen for positive clones with correct sequencing, designated TRV-CuRG1, and three sets of parallel experiments were TRV-CuRG1-1, TRV-CuRG1-2, and TRV-CuRG-1-3, respectively. The TRV-CuRG1 transformed Agrobacterium GV3101 competent cells, mixed with the TRV 2-containing GV3101 in equal proportion, were injected into Desiree leaves using a needleless syringe, and after four weeks, the expression level of StCuRG1 gene was examined, as shown in FIG. 2 (a), confirming that StCuRG1 gene was silenced.

TRV-CuRG1 leaf and control pTV00 leaf (n=10) were inoculated with potato late blight EC1 spore suspension, respectively, and left to stand in a dark place at 20℃in an incubator for 3 days, followed by phenotypic observation, statistics and photographing, and the results are shown in FIG. 2. The results showed that the TRV-CuRG1 gene-silenced potato leaf (TRV-CuRG 1) was more susceptible to late blight than the control (pTV 00) leaf, as shown in FIG. 2 (b), and the disease index was also higher than the control, as shown in FIG. 2 (c), indicating that the silenced StCuRG1 gene-expressed potato was more susceptible to late blight, and that copper ions on StCuRG1 gene-silenced potato material were unable to trigger resistance to late blight. It can be seen that inhibition of StCuRG gene reduces resistance of potato to late blight and attenuates copper formulation-induced plant disease resistance.

Example 3

This example describes the isolation and cloning of the potato StCuRG1 gene.

Potato seeds of normal soil culture are taken as materials, a copper sulfate solution (10 mu M) is sprayed by a handheld small sprayer to carry out spraying treatment on potatoes growing for 4 weeks, leaves are collected for 0h, 2h and 24h after the treatment, a TRIZOL method (purchased from century Biotechnology Co., ltd.) is adopted to extract total RNA, a reverse transcriptase SuperScript IV (purchased from ThermoFisher Scientific) is adopted to carry out reverse transcription on the total RNA to synthesize a cDNA first strand, and the reaction conditions are as follows: 50 ℃ for 15min;80 ℃ for 10min. Then, using this cDNA as a template, using StCuRG gene-specific primer F1:5'-GATGGAGAAGAATAAGATTATTAAGAG-3' and R1:5'-TCATAAACACATCGATGGATCG-3' PCR amplification was performed to amplify a DNA fragment containing the full-length coding frame of StCuRG gene, as shown in FIG. 3 (a), under the following reaction conditions: pre-denaturation at 95℃for 3min;95 ℃,10sec,55 ℃,10sec,72 ℃,1min,35 cycles; finally, the extension is carried out for 5min at 72 ℃. The PCR product obtained was reacted with XcmI (from NEB) treated pCXSN vector at 16℃under the action of T4 DNA LIGASE, and the ligation product transformed E.coli DH 5. Alpha. Competent cells (from Shanghai Biotechnology Co., ltd.) to select positive clones sequenced correctly, designated pCXSN: stCuRG1, the nucleotide sequence obtained by amplification is shown as SEQ ID NO. 1. The strong promoter 35S is positioned on the vector pCXSN, and the target gene expression is driven by the 35S strong promoter.

Example 4

This example describes the preparation of potato StCuRG1 over-expressing transgenic material.

PCXSN: stCuRG 1A 1 transformed Agrobacterium competent cells AGL1 as shown in FIG. 3 (b). Transformation of potato variety Desiree using Agrobacterium-mediated transformation, will be transformed with pCXSN: stCuRG1 of Agrobacterium AGL1 on LA solid medium containing antibiotics (Rif 50mg/L and Kan 50 mg/L), 28 ℃ culture for 48 hours, picking up monoclonal and in liquid LB medium (additionally adding antibiotics Rif 50mg/L and Kan 50 mg/L) expansion culture, 28 ℃ culture for 16 hours-18 hours at 200 r/min. When the OD600 value reaches 0.6-0.8, 100 mu L of bacterial liquid is added into 20mL of liquid MS0 culture medium, 21 d-28 d robust potato Desiree aseptic seedlings are selected to grow, axillary bud-free stem segments (about 0.5cm long) are cut and placed into agrobacterium suspension, about 30 stem segments are placed into each 20mL of bacterial suspension, the bacterial suspension is mixed on a shaking table at 24 ℃ and 42rpm under dark conditions, so that the explants are fully contacted with the bacterial liquid, after 20 minutes, a conversion material is taken out, surface bacterial liquid is wiped off by sterilizing filter paper, and the bacterial liquid is transferred into a co-culture medium M0, and co-culture is carried out for 3d at 18 ℃; transferring all the co-cultured stem segments to M1 callus differentiation culture medium, placing in artificial climate box at 21+ -1deg.C for 16h/d under 2000lx illumination intensity, selecting stem segments with good callus development after 12d, transferring to M2 differentiation culture medium for selective culture, and transferring 1 time every 14d later; the callus starts to grow regeneration buds when the transfer is about 3 times, and when the seedlings to be regenerated grow to about 2cm high, the seedlings are transferred to a rooting medium MR containing hygromycin and termeiding for rooting screening. When the roots grow well, the top stem segments of the regenerated plants with no obvious difference between the growth state and the non-transgenic plants are taken and transferred to a selective rooting culture medium again for screening and confirmation, and the process is shown in fig. 4 and comprises differentiated calli shown in (a) in fig. 4, budded calli shown in (b) in fig. 4 and regenerated potato seedlings shown in (c) in fig. 4.

Co-acquisition of the trans-pCXSN: stCuRG1 Gene plants (T0 generation) 7 plants. DNA was extracted from 7 plants and PCR was performed. The results show that 4 strains with common numbers of 2,3, 6 and 7 amplify StCuRG gene specific fragments with the length of about 1620bp, which proves that the target gene is successfully transferred into potato Desiree, and the positive rate reaches 57.14 percent (positive plants/inoculated stems are 4/7) as shown in (a) of fig. 5.

To further identify the transcript level of StCuRG gene in transgenic positive plants, leaves were collected for positive plants and Desiree grown for 4 weeks, total RNA was extracted by TRIZOL method (available from Kagaku Biotech Co., ltd.) and reverse transcribed into cDNA first strand by reverse transcriptase MMLV (available from Ebola) under the following reaction conditions: 42 ℃ for 1h;65℃for 20min. Then, using the cDNA as a template, stEF a as an internal reference gene and StEF a gene amplification primers as F3:5'-CAAGGATGACCCAGCCAAG-3', R3:5'-TTCCTTACCTGAACGCCTGT-3', using StCuRG gene-specific primer F4:5'-ATGGGTTCATTGATAGGAGTAA-3' and R4:5'-ATCTATGTCAGGTCCTTCAGC-3' real-time quantitative PCR was performed, reaction conditions: pre-denaturation at 95℃for 3min;95 ℃ for 10sec;60 ℃,10sec,72 ℃,30sec,40 cycles. As a result, as shown in FIG. 5 (b), stCuRG gene transcript levels in the transgenic positive plants of the over-expression groups OE-2, OE-3, OE-6 reached about 20-25 times that of the control Desiree, demonstrating that the target gene StCuRG1 was over-expressed in potato Desiree.

Example 5

This example describes the identification of StCuRG1 over-expressed transgenic potatoes for resistance to late blight.

Transgenic potato leaf blades and control Desiree leaf blades (n=10) were inoculated with potato late blight EC1 spore suspensions, respectively, and left to stand in an incubator at 18℃for 3 days, followed by phenotype observation and statistics of late blight disease index. The results show that transgenic potato leaves OE-2, OE-3, OE-6 overexpressing StCuRG1 are more disease resistant than control Desiree leaves, as shown in FIG. 6 (a); as shown in FIG. 6 (b), the over-expression groups OE-2, OE-3, OE-6 also had significantly lower disease indices than the control group. In conclusion, over-expression StCuRG of the gene improves the disease resistance of the potato to the late blight, and StCuRG1 over-expression of the gene improves the disease resistance of the potato to the late blight.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (3)

1. A potato StCuRG gene comprising at least one of the following nucleotide sequences:

a, a nucleotide sequence shown in SEQ ID NO. 1;

b, a nucleotide sequence shown as SEQ ID NO. 2;

c, a nucleotide sequence shown as SEQ ID NO. 3;

d, coding the nucleotide sequence corresponding to the amino acid sequence with equivalent function formed by artificial engineering or substitution, deletion or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO. 4.

2. A biological material comprising the potato StCuRG gene of claim 1 and a pCXSN vector.

3. Use of an overexpressed potato StCuRG gene to enhance resistance of a potato to late blight.