CN117660478A - Gene for improving resistance of potatoes to late blight and application thereof - Google Patents
Gene for improving resistance of potatoes to late blight and application thereof Download PDFInfo
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- CN117660478A CN117660478A CN202311534327.2A CN202311534327A CN117660478A CN 117660478 A CN117660478 A CN 117660478A CN 202311534327 A CN202311534327 A CN 202311534327A CN 117660478 A CN117660478 A CN 117660478A
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- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—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 biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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Abstract
The invention belongs to the technical field of plant genetic engineering, and discloses a gene for improving resistance of potatoes to late blight and application thereof, wherein the nucleotide sequence is shown in SEQ ID NO: 1. Gene StWRKY40 and potato WRKY transcription factor StWRKY40 coded by the gene are related to potato late blight resistance. Silencing the gene makes potatoes more susceptible to late blight, while over-expression of the gene with a strong promoter can increase resistance of potatoes to late blight. The gene StWRKY40 plays a positive regulation role in the defense reaction of the potato against late blight, and can be used for improving the disease resistance of potatoes and other crops to cultivate new varieties of disease-resistant transgenic plants. The gene provided by the invention can be used for improving the disease resistance quality of plants by using plant genetic engineering technology.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene for improving resistance of potatoes to late blight and application thereof.
Background
The country is the first country of potato planting and consumption in the world, and the potato planting history has been 400 years. Late blight of potato caused by phytophthora infestans (Phytophthora infestans) is a destructive disease in potato production, listed as the first disease of world food crops, and has caused irish familiarity in 1845-1850, resulting in death of more than 100 tens of thousands of people. Late blight generally occurs in potato main producing areas in China from the 80 s, the harm is in an ascending trend, the yield loss of potatoes is 10% -30% due to common epidemic years, the serious epidemic years can reach more than 50%, and even the serious epidemic years are out of production. Prevention and control of late blight has become one of the priority targets in potato production and breeding. In China, chemical agents are mainly used for preventing and treating late blight, wherein metalaxyl is most widely applied, and researches prove that the effective prevention and treatment of 99.9% can be realized by using the metalaxyl before the late blight occurs. However, long-term use of single chemical bactericides is prone to cause drug resistance of phytophthora infestans, such as metalaxyl in Shanxi, hebei and other places, and is not suitable for preventing and treating late blight. Cultivation of late blight resistant varieties is one of the main goals of potato breeding.
Mesotrione, a carotenoid synthesis inhibitor, targets p-hydroxyphenylpyruvate dioxygenase (HPPD) to block the synthesis of plastoquinone and tocopherol, thereby blocking the electron transfer process in photosynthesis and finally enabling plants to whiten and die. Carotenoids are mainly terpene pigments containing 40 carbon atoms, of which zeaxanthin is the precursor of abscisic acid. Abscisic acid is an important plant hormone, and not only participates in regulating the growth and development of plants, but also plays an important role in plant response, resistance to biotic stress, abiotic stress and the like. Treatment of potatoes with low concentrations of mesotrione, fludioxonil and the like does not result in crop death but induces salicylic acid signals dependent on antagonism by abscisic acid, activating salicylic acid dependent disease-resistant responses including the StWRKY40 gene (PGSC 0003DMG 402007388) (unpublished results).
The WRKY transcription factor is a plant-specific zinc finger transcription regulator, and the amino terminal of the WRKYGQK transcription factor is a highly conserved WRKYGQK sequence. The DNA binding domain of the WRKY transcription factor specifically binds to the TGAC (C/T) sequence (i.e., the W box) and regulates the expression of multiple plant disease, stress and senescence resistant genes. The WRKY gene is not expressed constitutively, and the expression has the characteristics of rapidness, transience and the like, is induced by pathogenic microorganisms, drought, low temperature, salicylic acid and the like, has tissue specificity and participates in various physiological processes in plants. More than half of the members of the WRKY family in arabidopsis are induced to be expressed by pathogenic microorganisms or salicylic acid treatment, and play an important role in plant disease resistance. For example, the AtWRKY70 gene deletion mutants enhance the resistance of plants to the necrotic nutritional pathogenic fungus Botrytis cinerea, whereas the deletion mutants of the WRKY7, WRKY11, WRKY17, WRKY48, WRKY38 and WRKY62 genes, etc., exhibit resistance to Pseudomonas syringae. AtWRKY40 in Arabidopsis is induced to be expressed by pathogenic microorganisms and interacts with AtWRKY18 and AtWRKY60 in plant disease-resistant reaction, wherein AtWRKY40 is a transcription repressor, and AtWRKY18 and AtWRKY60 are transcription activators. Therefore, the forward regulation and control effect of the StWRKY40 gene in the potato late blight resistance stimulated by carotenoid synthesis inhibitors is studied, the molecular mechanism of the transcription factor in the potato late blight resistance is revealed, and the transcription factor has important significance for cultivation of excellent resistant varieties, reasonable utilization of the resistant varieties and durable prevention and treatment of the potato late blight.
Through the above analysis, the problems and defects existing in the prior art are as follows: in China, chemical agents are mainly used for preventing and treating late blight, such as chlorothalonil, copper hydroxide, pyraclostrobin, mancozeb, zineb, mefenoxam, dimethomorph, mandipropamid, azoxystrobin, trifloxystrobin, famoxadone, fluazinam, cyazofamid and the like, wherein the most number of the fluazinam is registered, and then the mancozeb, azoxystrobin and cyazofamid are required to be applied for multiple times in one growing season, so that the cost of application is high, the cost of application is increased, the labor cost is increased inevitably for multiple times, and the phytophthora infestans is easy to generate drug resistance due to unreasonable use of the chemical agents for a long time, so that the drug effect of the bactericide is reduced or even fails. In addition, the irregular use of the chemical agent is easy to bring non-point source pollution, not only affects the soil and water environment, but also reduces the quality of agricultural products. The use of disease-resistant genes to cultivate disease-resistant varieties is the most economical and effective measure for preventing and treating potato late blight, and the cultivation of the disease-resistant varieties becomes one of main targets of potato breeding.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a gene for improving the resistance of potatoes to late blight and application thereof.
The invention is realized by a gene for improving the resistance of potatoes to late blight, wherein the gene StWRKY40 for improving the resistance of potatoes to late blight comprises an open reading frame with the whole length of the gene StWRKY40, and the open reading frame has a sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification.
The gene StWRKY40 is expressed by carotenoid inhibitors.
Gene StWRKY40 and potato WRKY transcription factor StWRKY40 coded by the gene are related to potato late blight resistance.
The gene StWRKY40 has positive regulation and control effects in the defense reaction of potato against late blight.
Furthermore, the strong promoter CaMV 35S is used for driving the expression of the gene StWRKY40 in the potato, and the overexpression of the gene can improve the resistance of the potato to late blight.
The gene StWRKY40 has a nucleotide sequence shown as SEQ ID NO:2, silencing the specific gene fragment can reduce the expression level of the gene StWRKY40, and the reduction of the gene can reduce the resistance of potatoes to late blight.
The invention also aims to provide an application of the gene for improving the resistance of the potato to the late blight in the cultivation of the potato anti-late blight variety, and a new disease-resistant transgenic variety is cultivated by improving the disease resistance of the potato and other crops by using the gene.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
first, compared with traditional disease-resistant breeding technology, the plant disease-resistant genetic engineering technology can break through reproductive isolation and distant hybridization incompatibility among species, realize directional improvement of target characters in a shorter time, and provide more comprehensive, continuous, broad-spectrum and specific protection for crops.
Through a plant disease resistance gene engineering technology, the invention discovers that the StWRKY40 gene plays a positive regulation role in the late blight resistance reaction of the potato, namely, the up-regulation of the StWRKY40 gene can improve the disease resistance of the potato to the late blight, and the inhibition of the StWRKY40 gene reduces the disease resistance of the potato to the late blight.
When the StWRKY40 gene is excessively expressed in a plant body, a series of expression of downstream disease resistance function genes can be activated in a transcription manner, so that a good disease resistance effect is provided for transgenic plants.
Secondly, the invention constructs a vector by using a strong promoter CaMV 35S and a potato StWRKY40 gene, and the StWRKY40 gene is expressed transiently on a potato commercial variety Desiree through agrobacterium GV 3101. The late blight resistance detection shows that compared with a wild type control, the potato material with the transiently over-expressed StWRKY40 gene has obviously enhanced late blight resistance, which indicates that the resistance of the potato to the late blight can be improved by improving the expression of the StWRKY40 gene. The resistance of potato materials with the StWRKY40 gene silenced by using a Virus-mediated gene silencing (Virus-Induced Gene Silencing, VIGS) technology to late blight is obviously weakened, which indicates that the StWRKY40 gene positively regulates the resistance of potatoes to late blight, and the gene can be applied to the genetic engineering improvement of potato late blight resistance.
Thirdly, the expected benefits and commercial value after the technical scheme of the invention is converted are as follows: the invention constructs a vector by using the strong promoter CaMV 35S and the potato StWRKY40 gene, converts the commercial potato variety Desiree with excellent quality and high yield, and internationally obtains the transgenic potato with the over-expressed StWRKY40 gene for the first time. Compared with a wild type control, the potato material over-expressing the StWRKY40 gene has obviously enhanced resistance to late blight, so that the transgenic potato is a new variety with high resistance to late blight.
The technical scheme of the invention fills the technical blank in the domestic and foreign industries: the technical scheme of the invention proves that the resistance of the potato to late blight can be improved by inducing StWRKY40 gene expression.
Whether the technical scheme of the invention solves the technical problems that people want to solve all the time but fail to obtain success all the time is solved: the technical scheme of the invention proves that the coding product transcription factor StWRKY40 of the StWRKY40 gene positively regulates and controls the disease resistance response of the potatoes. The potato variety with the StWRKY40 gene up-regulated expression cultivated or screened can be used as a candidate material to be applied to the work of potato disease-resistant varieties, and a new target is provided for potato breeding.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an analysis chart of the expression profile of StWRKY40 gene in a carotenoid inhibitor-activated potato provided by the example of the present invention;
FIG. 2 is a diagram showing the silencing of StWRKY40 gene expression in potato by VIGS technology according to an embodiment of the present invention; * : p <0.05 compared to control pTV 00;
FIG. 3 is a graph showing the onset of a control pTV00 and StWRKY40 gene silencing material 4 days after late blight inoculation provided in the examples of the present invention;
FIG. 4 is a graph showing the statistical result of the disease index of 4 days after the control pTV00 and StWRKY40 gene silencing material provided by the embodiment of the invention is inoculated with late blight; * : p <0.05 compared to control pTV 00;
FIG. 5 is a diagram showing the onset of potato material with transient expression of the control CK and StWRKY40 genes for 4 days after inoculation of late blight according to the examples of the present invention;
FIG. 6 is a graph showing the statistical result of the disease index of 4 days after the potato material is inoculated with the control CK and StWRKY40 genes in the embodiment of the invention; * *: p <0.01 compared to control CK;
FIG. 7 is a diagram of the transformation of potato by the stem segment method according to the present invention, including differentiated callus, budded callus and regenerated potato seedlings;
FIG. 8 is a DNA level detection chart of a transgenic potato with StWRKY40 overexpression provided by the embodiment of the invention; wherein N is a negative control and P is a positive control;
FIG. 9 is a diagram for detecting transcription level of StWRKY40 gene in a StWRKY40 overexpression transgenic potato provided by the embodiment of the invention; wild-type Desiree was used as a control and StEF1a gene was used as an internal reference. * *: p <0.01 compared to wild type;
FIG. 10 is a diagram showing the onset of a StWRKY40 gene over-expression transgenic potato material according to an embodiment of the present invention 4 days after late blight inoculation;
FIG. 11 is a graph showing statistical results of disease indexes of the StWRKY40 gene over-expression transgenic potato material 4 days after late blight inoculation; * *: p <0.01 compared to the wild type.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Based on the characteristics and functions of the gene StWRKY40 for improving the resistance of potatoes to late blight, the following two specific application embodiments and corresponding implementation schemes are provided:
application example 1 overexpression of the Gene StWRKY40 to enhance late blight resistance of Potato
1. Gene cloning and vector construction:
extracting total RNA from potato, reverse transcription synthesizing to obtain cDNA single chain, PCR amplifying to full-length open reading frame of gene StWRKY40 by using the cDNA as template, and inserting it into plant expression carrier with strong promoter CaMV 35S to drive gene expression.
2. Plant transformation:
transforming the constructed vector into potato cells or tissues by an agrobacterium-mediated method or other applicable transformation methods to obtain transgenic potato plants.
3. Disease resistance evaluation:
the resistance of transgenic potatoes to late blight was evaluated by field trials and late blight inoculation tests under laboratory conditions.
4. Expression analysis:
the expression of the gene StWRKY40 in transgenic potatoes is analyzed by molecular biological methods such as RT-PCR or Western blotting, and whether the increase of the expression is related to the enhanced disease resistance is verified.
Application example 2 reduction of expression of Gene StWRKY40 by Virus mediated Gene silencing (VIGS) technology to investigate its function in potato anti-late blight
1. Construction of VIGS vector:
a specific VIGS sequence for gene StWRKY40 was designed and inserted into a VIGS vector suitable for potato.
2. Plant transient expression:
and (3) transforming the constructed VIGS vector into potato cells or tissues by a proper transformation method to obtain potato plants with reduced expression of the gene StWRKY 40.
3. Functional analysis:
the resistance of the VIGS-interfered potatoes to the late blight and the influence of reduced expression of the gene StWRKY40 on the disease resistance of the potatoes are evaluated through field tests and late blight inoculation tests under laboratory conditions.
4. Expression analysis:
using RT-PCR or other related molecular biology techniques, it was verified whether VIGS successfully reduced expression of gene StWRKY40, and reduced the effect of expression on potato anti-late blight defense responses.
Through the two application embodiments, the effect and the mechanism of the gene StWRKY40 in improving the resistance of the potato to late blight can be explored and verified in practical application, and basic knowledge and technical support are provided for further disease resistance breeding and disease management.
Aiming at the problems existing in the prior art, the invention provides a gene for improving the resistance of potatoes to late blight and application thereof.
The gene for improving the resistance of the potato to the late blight provided by the embodiment of the invention, namely the gene StWRKY40 for improving the resistance of the potato to the late blight, comprises an open reading frame with the full length of the gene StWRKY40, and the open reading frame has the sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification.
Sequence table SEQ ID No.1 the invention separates the DNA sequence, CDS sequence and amino acid sequence of cloned StWRKY40 gene and encoded protein.
SEQ ID NO:1 StWRKY40 Gene CDS Sequence
StWRKY40 expression protein sequence
StWRKY40 gene DNA Sequence
SEQ ID NO:2 Gene fragment for silencing StWRKY40 Gene by VIGS
The invention verifies the expression profile of StWRKY40 gene under carotenoid inhibitor treatment, and the StWRKY40 gene is induced to be expressed by carotenoid inhibitors.
Since carotenoid inhibitor treatment enhances the resistance of potatoes to late blight, the gene StWRKY40 and the coded potato WRKY transcription factor StWRKY40 thereof are related to the resistance of potatoes to late blight.
The gene StWRKY40 has positive regulation and control effects in the defense reaction of potato against late blight. Further, the strong promoter CaMV 35S is used for driving the expression of the gene StWRKY40 in the potatoes, so that the resistance of the potatoes to late blight can be enhanced.
The expression of StWRKY40 gene in potato is silenced by Virus-mediated gene silencing (Virus-Induced Gene Silencing, VIGS) technology, and the disease resistance of potato to late blight is reduced. The gene StWRKY40 has a nucleotide sequence shown as SEQ ID NO:2, and silencing the specific gene fragment can reduce the expression quantity of the gene StWRKY 40.
The invention constructs a vector by using the strong promoter CaMV 35S and the potato StWRKY40 genes, and converts the commercial potato variety Desiree with excellent quality and high yield to obtain a new transgenic potato variety with high late blight resistance.
The present invention relates to the isolation and use of a coding region fragment comprising the StWRKY40 gene. Wherein, the coding region fragment of the StWRKY40 gene is shown in a sequence table SEQ ID NO:1 or substantially corresponds to SEQ ID NO:1, or a DNA sequence encoding the same.
The invention extracts total RNA of potato variety Desiree, obtains cDNA by reverse transcription, takes the cDNA as a template, stWRKY40-F: ATGTGTATATTCAAGGTGGC, stWRKY40-R: TCATCTTGAAGAAGAAGAAGG the primer amplifies the coding region sequence of StWRKY40, and is connected to the pCXSN vector subjected to Xcm I digestion to construct a vector pCXSN: stWRKY40, and transformed into Agrobacterium engineering bacteria AGL I. The vector was transformed into cultivar Desiree using the stem transformation method. Late blight resistance detection it was found that transgenic potatoes overexpressing the StWRKY40 gene were significantly more resistant to late blight than the wild-type control.
Example 1
This example describes the induction of transcription of potato WRKY transcription factor gene StWRKY40 by the carotenoid inhibitor mesotrione.
Potato seeds of normal soil culture are taken as materials, a carotenoid inhibitor mesotrione (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-shi Biotech Co., ltd.) is adopted to extract total RNA, reverse transcriptase MMLV (purchased from Eboltai) is adopted to carry out reverse transcription on the total RNA into a cDNA first strand, and the reaction conditions are as follows: 42 ℃ for 1h;65℃for 20min. Then, using this cDNA as a template and StEF1a (PGSC 0003DMG 400023272) as an internal reference gene, (StEF 1a gene amplification primer is F1:5'-CAAGGATGACCCAGCCAAG-3', R1: 5'-TTCCTTACCTGAACGCCTGT-3') using StWRKY40 gene-specific primer F2:5'-TCATTTGCACCAACATGCCC-3' and R2:5'-ACGAAGACGCCATTTGTTCG-3' real-time quantitative PCR was performed under the following 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 StWRKY40 gene-silenced plants.
Potato leaf cDNA was used as template, stWRKY40 gene specific primer F3:5' -CGGGATCCAATGGCGTCTTCGTTGTCCA-3' (wherein the underlined sequence is the recognition sequence for the restriction enzyme BamH I), R3:5' -GGGGTACACTCCCCAATACACAATAGAGCG-3' (wherein the underlined sequence is a restriction enzyme Kpn I recognition sequence), a StWRKY40 gene specific fragment was amplified, the PCR product and vector pTV00 were digested with restriction enzymes BamHI and KpnI, and then reacted at 16℃under the action of T4 DNA library, and the ligation product transformed E.coli DH 5. Alpha. Competent cells (purchased from Shanghai Weidi Biotechnology Co., ltd.) to select positive clones with correct sequencing, designated TRV-WRKY40. Transforming the competent cells of Agrobacterium GV3101 with TRV-WRKY40, mixing with GV3101 containing TRV2 in equal proportion, and using needlelessFour weeks later the expression level of the StWRKY40 gene was examined (fig. 2), confirming that the StWRKY40 gene was silenced.
TRV-WRKY40 leaf and control pTV00 leaf (n=10) were inoculated with potato late blight EC1 spore suspension, respectively, and left in a dark place in an incubator at 20℃for 4 days, followed by phenotypic observation, statistics and photographing, and the results are shown in FIG. 3. The results showed that StWRKY40 gene-silenced potato leaves (TRV-WRKY 40) were more susceptible to late blight than the control (pTV 00) leaves (fig. 3), and the disease index was also higher than the control (fig. 4), indicating that the StWRKY40 gene-silenced potato was more susceptible to late blight.
Example 3
This example describes the isolated cloning, transient expression and disease resistance identification of the potato WRKY transcription factor gene StWRKY 40.
Potato seeds of normal soil culture are taken as materials, a carotenoid inhibitor mesotrione (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 the company of 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 into a cDNA first strand, and the reaction conditions are as follows: 50 ℃ for 15min;80 ℃ for 10min. Then, using the cDNA as a template, using StWRKY40 gene-specific primer F4:5'-ATGGGAAACAAGTCTTTTTT-3' and R4:5'-TCAAAAAAGTAATCCTGAGA-3' PCR amplification is carried out to amplify DNA fragments containing the full-length coding frame of the StWRKY40 gene, and the reaction conditions are as follows: 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 Xcm I (from NEB) treated pCXSN vector at 16℃under the action of T4 DNA library, 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: stWRKY40, the nucleotide sequence obtained by amplification is shown as SEQ ID NO. 1.
pCXSN: stWRKY40 transformed Agrobacterium GV3101 competent cells, with needleless syringe injection Desiree leaf blade, 2 days later inoculated potato late blight EC1 spore suspension, in 20 ℃ incubator, light-proof standing, 4 days later phenotype observation, statistics and shooting. The results show that the potato leaf (StWRKY 40-OE) with the StWRKY40 gene expressed transiently is more resistant to late blight than the leaf of the control group (figure 5), and the disease index is higher than that of the control group (figure 6), which indicates that the resistance of the potato to late blight is improved by the StWRKY40 gene expressed transiently.
Example 4
This example describes the positive detection of potato genetic transformation and transgenic plants.
pCXSN: stWRKY40 transformed Agrobacterium competent cells AGL1. Transformation of potato variety Desiree using Agrobacterium-mediated transformation, will be transformed with pCXSN: agrobacterium AGL1 of StWRKY40 was activated on LA solid medium containing antibiotics (Rif 50mg/L and Kan 50 mg/L), cultured at 28℃for 48 hours, and the monoclonal was picked up and cultured in liquid LB medium (additionally, antibiotics Rif 50mg/L and Kan 50 mg/L) for expansion at 28℃for 16 to 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, 21-28 d robust potato Desiree aseptic seedlings are selected, axillary bud-free stem segments (about 0.5cm in length) 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 ℃ under the dark condition, the explants are fully contacted with the bacterial liquid, after 20 minutes, the transformation material is taken out, the bacterial liquid on the surface is wiped off by sterilizing filter paper, the bacterial liquid is transferred into a co-culture medium M0, and co-culture is carried out for 3d at 18 ℃ in a dark place; 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 medium again for screening and confirmation (FIG. 7).
Co-acquisition of the trans-pCXSN: stWRKY40 gene plant (T0 generation) 7 plants. Extracting DNA from 7 transgenic plants by adopting a CTAB method, taking the DNA as a template, and utilizing a StWRKY40 gene specific primer F5:5'-GGGAGCACAACCATCCTCAA-3' R5:5'-TGGACAACGAAGACGCCATT-3' PCR detection was performed. The results showed that 3 strains, commonly numbered OE-4, OE-5, OE-7, amplified a StWRKY40 gene-specific fragment of approximately 500bp in length, demonstrating successful transfer of the gene of interest into potato Desiree (FIG. 8).
To further identify the transcript level of the StWRKY40 gene in transgenic positive plants, leaves were collected separately from positive plants and desirees grown for 4 weeks, total RNA was extracted by the TRIZOL method (available from the company of the century biotechnology Co., ltd.) and reverse transcribed into cDNA first strand by the reverse transcriptase MMLV (available from erbtai) under the following reaction conditions: 42 ℃ for 1h;65℃for 20min. Then, using the cDNA as a template and StEF1a as an internal reference gene, (StEF 1a gene amplification primers are F1:5'-CAAGGATGACCCAGCCAAG-3', R1: 5'-TTCCTTACCTGAACGCCTGT-3'), and StWRKY40 gene-specific primers F2:5'-TCATTTGCACCAACATGCCC-3' and R2:5'-ACGAAGACGCCATTTGTTCG-3' real-time quantitative PCR was performed under the following reaction conditions: pre-denaturation at 95℃for 3min;95 ℃ for 10sec;60 ℃,10sec,72 ℃,30sec,40 cycles. As shown in FIG. 9, the transcription level of StWRKY40 gene in the transgenic positive plants reaches about 20-25 times that of the control Desiree, and the target gene StWRKY40 is proved to be over-expressed in the potato Desiree.
Example 5
This example describes resistance of StWRKY40 overexpressing transgenic potatoes to late blight.
Transgenic potato leaves and Desiree leaves with positive detection are respectively inoculated with potato late blight EC1 spore suspension, and kept stand in a 20 ℃ incubator in dark place for 4 days, and phenotypes are observed and the late blight disease indexes are counted. The results showed that transgenic potato leaves were more resistant to disease than control Desiree leaves (FIG. 10), and the disease index was lower than control (FIG. 11).
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (7)
1. A gene for improving the resistance of potatoes to late blight is characterized in that the gene StWRKY40 for improving the resistance of potatoes to late blight has a gene sequence shown in SEQ ID NO. 1.
2. The gene for increasing resistance of potato to late blight according to claim 1, wherein the gene StWRKY40 is expressed by induction of carotenoid inhibitors.
3. The gene for improving potato resistance to late blight according to claim 1, wherein the gene StWRKY40 and the potato WRKY transcription factor StWRKY40 encoded by the gene are related to potato resistance to late blight.
4. The gene for improving resistance of potato to late blight according to claim 1, wherein the gene StWRKY40 has an up-regulating effect in a defense reaction of potato against late blight.
5. The gene for improving the resistance of potatoes to late blight according to claim 1, wherein the gene StWRKY40 in the potatoes is driven by a strong promoter CaMV 35S, and the expression of the gene is over-expressed to improve the resistance of the potatoes to late blight.
6. The gene for improving resistance of potato to late blight according to claim 1, wherein the gene StWRKY40 has a nucleotide sequence as set forth in SEQ ID NO:2, and silencing the specific gene fragment can reduce the expression quantity of the gene StWRKY 40.
7. The use of a gene for improving resistance of potato to late blight in breeding of potato varieties with late blight resistance according to any one of claims 1 to 6, wherein the gene is used to improve disease resistance of potato and other crops, and breeding new varieties with disease resistance and transgene.
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| CN202311534327.2A Pending CN117660478A (en) | 2023-11-17 | 2023-11-17 | Gene for improving resistance of potatoes to late blight and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115976051A (en) * | 2022-11-16 | 2023-04-18 | 西北农林科技大学 | Potato StRTP7 gene and application thereof in disease-resistant breeding |
| CN117947051A (en) * | 2024-03-26 | 2024-04-30 | 山东蓬勃生物科技有限公司 | Potato StCuRG gene, biological material and application of over-expressed StCuRG gene |
-
2023
- 2023-11-17 CN CN202311534327.2A patent/CN117660478A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115976051A (en) * | 2022-11-16 | 2023-04-18 | 西北农林科技大学 | Potato StRTP7 gene and application thereof in disease-resistant breeding |
| CN115976051B (en) * | 2022-11-16 | 2024-04-30 | 西北农林科技大学 | Potato StRTP gene and application thereof in disease-resistant breeding |
| CN117947051A (en) * | 2024-03-26 | 2024-04-30 | 山东蓬勃生物科技有限公司 | Potato StCuRG gene, biological material and application of over-expressed StCuRG gene |
| CN117947051B (en) * | 2024-03-26 | 2024-06-18 | 山东蓬勃生物科技有限公司 | Potato StCuRG gene, biological material and application of over-expressed StCuRG gene |
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