CN116426528A - Inducible promoter responding to aphid piercing and sucking and application thereof - Google Patents
Inducible promoter responding to aphid piercing and sucking and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and discloses application of an inducible promoter responding to aphid piercing, which is at least one of the following: the application of Y1 in improving the sucking response sensitivity of aphids; application of Y2 in promoting exogenous gene expression under aphid sucking induction; use of Y3 to increase plant resistance to aphids; the promoter is PpTNL1pro and is a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1. Transient transgenic tobacco is treated by aphids, and the expression of exogenous gene LUC can be increased by the PpTNL1pro promoter under aphid treatment, which proves that the PpTNL1pro promoter is induced by aphid piercing. As a promoter induced by aphid piercing, the promoter region and the upstream regulatory sequence play an important role in genetic engineering and improving plant aphid resistance, and can provide help for realizing high-quality, high-efficiency and labor-saving cultivation of peach.
Description
Technical Field
The invention relates to an inducible promoter responding to aphid piercing and sucking and application thereof in the field of biotechnology.
Background
Peach (Prunus persica) is an important fruit tree species worldwide, wherein the cultivation area and yield of peach in China are the first place in the world. Along with the continuous aggravation of population aging, the labor cost is continuously increased, and labor-saving cultivation management is a trend of the peach industry in the future. In the peach cultivation management process, besides basic conditions such as soil, fertilizer, water and the like, the pest control is extremely important for the high-quality and safe production of the peaches.
Aphids are insect pests seriously harming the growth of crops, wherein green peach aphids (Myzus persicae Sulzer) can harm peach trees and fruits by taking juice of phloem at tender parts of the peach trees, the growth and development of the peach trees are seriously influenced, and the aphids can absorb food through a mouth gag and can also spread viruses to harm the growth of plants. The damaged peach trees have curled leaves and deformed fruits, and a large amount of pesticides are required to be sprayed for many times in order to prevent and treat aphids in production, so that the environment is irreversibly damaged, the commodity value of the peach is reduced, and the development requirement of a labor-saving cultivation mode of the peach is not met.
A promoter is a DNA sequence that binds specifically to a transcription factor and causes initiation of transcription of a gene in conjunction with RNA polymerase. The type of the promoter can be divided into a constitutive promoter and an inducible promoter, wherein the inducible promoter is a promoter which needs specific external stimulus (such as high temperature, drought, aphid and other biological and abiotic stress) to induce the promoter to perform transcription initiation activity, and the activity of the promoter is enhanced by responding to the external environment stimulus, so that the expression quantity of a target gene is improved, the stress resistance of plants is improved, and the growth and development of the plants are stabilized.
The aphid damage problem of peach is still very serious, however, no researchers have published aphid-induced promoters and their applications on peach. Along with the rapid development of transgenic technology, transgenic breeding becomes a powerful means for rapidly polymerizing target characters, but most of existing transgenic works construct vectors based on constitutive promoters (such as 35S promoters), and target genes are expressed in a large amount in each period of the growth and development of transgenic plants and cannot realize fixed-point timing expression, so that development of inducible promoters and application significance of the inducible promoters are great.
Disclosure of Invention
The technical problem to be solved by the invention is how to improve the resistance of plants to aphids, in particular to improve the resistance of peach trees to green peach aphids.
It is a first object of the present invention to provide the use of a promoter in at least one of the following:
the application of Y1 in improving the sucking response sensitivity of aphids;
application of Y2 in promoting exogenous gene expression under aphid sucking induction;
use of Y3 to increase plant resistance to aphids;
the promoter is PpTNL1pro and is a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1.
In the above application, the promoter PpTNL1pro may be synthesized artificially or may be obtained by synthesizing its coding gene and then biologically expressing it.
A second object of the present invention is to provide the use of biological materials;
the biomaterial is any one of the following B1) to B5):
b1 A nucleic acid molecule comprising the promoter of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
b5 A) a transgenic plant line comprising the nucleic acid molecule of B1) and its progeny, or a transgenic plant line comprising the expression cassette of B2) and its progeny, or a transgenic plant line comprising the recombinant vector of B3) and its progeny;
the application is any one or more of Z1-Z3:
use of Z1 to increase sensitivity of aphid piercing response;
application of Z2 in promoting exogenous gene expression under aphid sucking induction;
use of Z3 for increasing plant resistance to aphids.
In the above application, in the biological material, B1) the nucleic acid molecule is a DNA molecule whose nucleotide is SEQ ID No. 1.
Recombinant expression vectors containing the gene expression cassettes can be constructed using existing plant expression vectors.
In the above applications, the recombinant microorganisms in the biological material may be yeast, bacteria, algae and fungi.
In the above application, the plant is dicotyledonous plant, and specifically can be Prunus plant of Rosaceae (such as Prunus persica) or Nicotiana plant of Solanaceae (such as Nicotiana tabacum (Nicotiana tabacum L.)).
The invention also provides the promoter PpTNL1pro and the biological material.
In order to solve the technical problem, the invention also provides a plant agent which is used for improving the sensitivity of the plant to the piercing response of aphids and/or the resistance of the plant to the aphids.
The plant reagent provided by the invention contains the promoter PpTNL1pro or/and the biological material.
The active ingredient of the plant agent may be the promoter PpTNL1pro or/and the biological material related to the promoter PpTNL1pro, the active ingredient of the plant agent may further contain other biological ingredients or/and non-biological ingredients, and the other active ingredient of the plant agent may be determined by a person skilled in the art according to the plant's susceptibility to piercing response by aphids and/or the plant's resistance improving effect.
In order to solve the technical problems, the invention also provides a method for producing plants which are sensitive to aphid piercing response and/or plants which are strong in aphid resistance.
The method for producing the plant sensitive to the aphid piercing response and/or the plant with strong aphid resistance provided by the invention comprises the steps of introducing the promoter PpTNL1pro into a receptor plant to obtain a target plant with higher aphid piercing response sensitivity and/or higher aphid resistance than the receptor plant; the sensitivity to aphid penetration response and/or the plant resistance to aphids are the result of comparison with the recipient plant.
In the above method, the plant sensitive to aphid piercing response and/or plant with strong resistance to aphid may be a transgenic plant, or may be a plant obtained by conventional breeding techniques such as crossing.
In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.
The invention also provides application of the promoter PpTNL1pro in response to aphid piercing.
The invention also provides application of the promoter PpTNL1pro in preparation of products responding to aphid piercing.
The product may be a plant. The plant is dicotyledonous plant, and can be plant of Prunus genus of Rosaceae family (such as peach (Prunus persica)) or plant of Nicotiana genus of Solanaceae family (such as Nicotiana tabacum (Nicotiana tabacum L.)).
In order to improve aphid resistance of peach trees, the invention provides an inducible promoter PpTNL1pro promoter responding to aphid piercing, which is cloned from a promoter region of a peach PpTNL1 gene, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1. When the transient transgenic tobacco plants are treated by aphids, the PpTNL1pro promoter is found to increase the expression of exogenous gene LUC, so that the LUC luminous intensity of the transgenic plants is enhanced, and the PpTNL1pro promoter is proved to be induced by aphid piercing. As a promoter induced by aphid piercing, the promoter region and the upstream regulatory sequence play an important role in genetic engineering and improving plant aphid resistance, and can provide help for realizing high-quality, high-efficiency and labor-saving cultivation of peach.
Drawings
FIG. 1 shows the results of the intensity of LUC expression of the aphid-vaccinated treatments following transient transformation of tobacco leaves with LUCs initiated by different promoters in example 1 of the invention. and (3) a graph a shows the LUC strength result around the instant injection hole of the tobacco leaf. Panel b shows the vehicle information and aphid inoculation at the instant injection site of the tobacco leaf in panel a. Panel c is the relative values of LUC fluorescence intensity before and after a transient injection of PpTNL1pro:: LUC aphid into tobacco leaves in panel a. LUC represents a vector of which the PpTNL1pro promoter drives LUC, pGREEN0800 represents a vector of which negative control is not connected with the LUC, and aphids added on the right side of the leaf blades represent infected aphids after transient injection, and the aphid piercing and sucking test is simulated. Graph c the abscissa number 1 represents PpTNL1pro:: LUC and the number 2 represents PpTNL1 pro::: LUC post-transient injection infested aphids.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.
The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The peach variety 'Jinzhu No. 1' in the following examples was an examined variety, the examined number was Lu S-SV-APN-009-2021, which was publicly available from the Shandong agricultural university of applicant to repeat the present experiment.
The tobacco of Bentham (N.benthamiana) of the following examples is described in the following literature: "synergistic effect of tobacco vein banding leaf virus and potato virus X" and both infest the transcriptomic analysis of Benshi tobacco. The public is available from the applicant's Shandong university of agriculture to repeat the experiment.
Example 1
The invention takes the aphid-resistant germplasm 'golden bead No. 1' peach of a test base of Shandong agricultural university science and engineering college as a test material for subsequent cloning of a promoter PpTNL1pro.
1. DNA extraction
And extracting DNA of tender leaf tissue of 'golden bead No. 1' peach by adopting a conventional CTAB method, removing RNA, and ensuring that the total volume of DNA samples is not less than 15 mu l. Measuring OD value of DNA sample at 260nm and 280nm by ultraviolet photometer, calculating DNA content and OD 260/280 Is a ratio of (2). DNA sample purity OD 260/280 The value should be between 1.8 and 2.0, diluted to a concentration of 10 ng/. Mu.l.
2. Designing primer amplification to obtain promoter PpTNL1pro
The promoter PpTNL1pro reference sequence in the peach genome was downloaded using the peach reference genome v.2.0.a1 (https:// www.rosaceae.org/specie/prune_persona/genome_v2.0.a1), and primers were designed based on the sequence:
f1:5'-GCAGTTAACAAATTCATTGATGACTTAT-3' (SEQ ID NO. 2);
r1:5'-TAATTGATACTGGTTCAGATAAGACCAA-3' (SEQ ID NO. 3).
The DNA sample obtained in step 1 was used as a template, and PCR amplification was performed using a primer set consisting of the upstream primer F1 and the downstream primer R1, which were diluted to a final concentration of 0.5. Mu.M after synthesis by Biotechnology Co.
The PCR amplification system is shown in Table 1:
TABLE 1PCR amplification reaction Components
Wherein, primer Mix is to add 0.5. Mu.l of each of the upstream Primer F1 and the downstream Primer R1 for PCR amplification. dNTP Mix comprises 4 equal amounts of mixed nucleotides A, G, T and C.
The PCR amplification procedure was as follows: 94 ℃ for 15min;94 ℃ for 20s,56 ℃ for 30s and 72 ℃ for 1min, 45 cycles in total; 72 ℃ for 3min; preserving at 4 ℃.
And preparing 1% agarose gel, carrying out electrophoresis on the PCR reaction product, and if the band is bright and single, carrying out a gel cutting recovery test to recover a target fragment, wherein the fragment is the promoter PpTNL1pro. The nucleotide sequence of the promoter PpTNL1pro is shown in SEQ ID NO.1, see Table 2:
TABLE 2 promoter PpTNL1pro sequence information
3. Construction of recombinant vectors
pGreenII 0800-Luc (Shanghai Kangshen Biotechnology Co., ltd., product No. MF3732-5 UG) was used as the starting vector, which contains the Luciferase (LUC) gene.
The upstream primer F2 and the downstream primer R2 containing the homology arm of the vector pGreenII 0800-Luc were designed:
the upstream primer F2:5'-gggccccccctcgaggtcgacGCAGTTAACAAATTCATTGATGACTTAT (homologous arm of vector pGreenII 0800-Luc indicated by lowercase letters, specific sequence binding to positions 1-28 of SEQ ID NO.1 indicated by uppercase letters) -3';
downstream primer R2:5'-cgctctagaactagtggatccTAATTGATACTGGTTCAGATAAGACCAA (homologous arm of vector pGreenII 0800-Luc in lowercase and specific sequence binding to SEQ ID NO.1 from 1305 to 1332 in uppercase) -3'.
After the primers were synthesized by the Biotechnology company, they were diluted to a final concentration of 0.5. Mu.M.
The PCR amplification system was as shown in Table 1, using a Primer set consisting of F2 and R2, and a DNA molecule having a nucleotide sequence of SEQ ID NO.1 as a template, to amplify a promoter PpTNL1pro fragment (containing the promoter PpTNL1pro shown in SEQ ID NO.1 and the homology arm of vector pGreenII 0800-Luc), wherein Primer Mix was prepared by adding 0.5. Mu.l of each of the upstream Primer F2 and the downstream Primer R2 for PCR amplification. dNTP Mix comprises 4 equal amounts of mixed nucleotides A, G, T and C.
The PCR amplification procedure was as follows: 94 ℃ for 15min;94 ℃ for 20s,56 ℃ for 30s and 72 ℃ for 1min, 45 cycles in total; 72 ℃ for 3min; preserving at 4 ℃.
Preparing 1% agarose gel, carrying out electrophoresis on the PCR reaction product, if the strip is bright and single, carrying out gel cutting recovery test, recovering the target fragment, namely the promoter PpTNL1pro fragment containing partial pGreenII 0800-Luc sequence, for subsequent carrier construction based on homologous arms,
the kit used for constructing the vector of the homology arm is a homologous recombination kit C112 of Norwegian company, and the specific operation steps are carried out by installing the kit operation instructions. Finally, pGREEN0800-PpTNL1pro:: LUC vector was constructed by homologous recombination. LUC vector is recombinant vector obtained by replacing small fragment between gggccccccctcgaggtcgac and cgctctagaactagtggatcc in pGreenII 0800-Luc with promoter PpTNL1pro (shown as SEQ ID NO. 1) and keeping other sequences of vector pGreenII 0800-Luc unchanged, and driving LUC gene with promoter PpTNL1pro.
4. Verification of the promoter PpTNL1pro in response to aphid penetration
The experimental material used is a leaf of Nicotiana benthamiana (N.benthamiana), and the specific method is as follows:
4.1, recombinant vector pGREEN0800-PpTNL1pro, LUC was transformed into Agrobacterium tumefaciens GV3101 by freeze thawing.
(1) Competent cells were thawed in ice at-80 ℃.
(2) LUC was added to 100. Mu.L of Agrobacterium competent cells GV3101, and the bottom of the tube was stirred by hand and mixed well.
(3) Sequentially placing on ice for 5min, standing with liquid nitrogen for 5min, water-bathing at 37deg.C for 5min, and ice-bathing for 5min.
(4) mu.L of YEB liquid medium without antibiotics was added and cultured with shaking at 28℃for 2-3 hours.
(5) The bacteria were harvested by centrifugation at 6000rmp for 1min, 100. Mu.L of the supernatant was left to resuspend the pellet, the bacterial liquid was spread on YEB solid medium containing kanamycin (50 mg/mL) +streptomycin (50 mg/mL) +rifampicin (50 mg/mL), and the pellet was placed upside down in an incubator at 28℃for 2-3d of dark culture.
Identifying transformed positive agrobacterium; the identification method comprises the following steps: picking single colony with uniform growth, and carrying out colony PCR amplification by using a primer pair consisting of the primers F1 and R1; the identified reaction system is shown in the step 2, and the recombinant agrobacterium obtained by identification is named GV3101-pGREEN0800-PpTNL1pro:: LUC.
Meanwhile, a control recombinant agrobacterium is arranged, specifically pGreenII 0800-Luc is transformed into agrobacterium GV3101 to obtain recombinant agrobacterium which is called GV3101-pGreenII 0800-Luc.
4.2 transient transformation of tobacco leaves
(1) The recombinant Agrobacterium GV3101-pGREEN0800-PpTNL1pro:: LUC obtained in step 4.1 was resuspended to OD600 nm=0.6-0.8 to obtain recombinant Agrobacterium GV3101-pGREEN0800-PpTNL1 pro::: LUC resuspension.
And (3) re-suspending the recombinant agrobacterium GV3101-pGreenII 0800-Luc obtained in the step (4.1) to OD600 nm=0.6-0.8 to obtain recombinant agrobacterium GV3101-pGreenII 0800-Luc re-suspension.
Transient transformation was performed on tobacco leaves of Nicotiana benthamiana in 4 regions (see FIG. 1) in a cross, as follows:
the upper left and right regions were injected with recombinant Agrobacterium GV3101-pGREEN0800-PpTNL1 pro::: LUC.
The lower left and lower right regions were injected with recombinant Agrobacterium GV3101-pGreenII 0800-Luc.
A total of 3 replicates were set.
The tobacco after transient injection is dark cultured for 12 hours, then normally cultured for 2-3 days, and an aphid control group and an aphid treatment group are arranged on the next day, specifically as follows:
PpTNL1 pro::: LUC (upper left region): on the basis of LUC injection, aphid treatment was not carried out on the basis of the aforementioned recombinant Agrobacterium GV3101-pGREEN0800-PpTNL 1pro.
pTNL1pro:: LUC+ aphid (upper right region): on the basis of LUC injection, 10 aphids are inoculated for sucking treatment.
pGREEN0800:: LUC (lower left): on the basis of the injection of the recombinant Agrobacterium GV3101-pGreenII 0800-Luc, no aphid treatment was carried out.
pGREEN0800:: LUC+ aphid (lower right region): on the basis of the injection of the recombinant Agrobacterium GV3101-pGreenII 0800-Luc, 10 aphids were inoculated for sucking treatment.
After aphid inoculation for 2 days, a plant living body imager (name: plant living imaging system; model: lumazone PyLoN 2048B; manufacturer: roper Technologies) is used for observing the strength of LUC around the injection hole, and the result is shown in figure 1, and compared with the control pGREEN0800:: LUC, ppTNL1pro:: LUC has strong LUC signal; LUC signal was stronger after inoculation with aphids than before inoculation, and no change in the negative control indicated that aphids were able to activate the PpTNL1Pro promoter.
The PpTNL1pro promoter disclosed by the invention can rapidly respond to aphid piercing response, and is an important genetic resource for aphid resistance breeding of peach trees in the future.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (9)
1. Use of a promoter in at least one of the following:
the application of Y1 in improving the sucking response sensitivity of aphids;
application of Y2 in promoting exogenous gene expression under aphid sucking induction;
use of Y3 to increase plant resistance to aphids;
the promoter is PpTNL1pro and is a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1.
2. The application of biological materials;
the biomaterial is any one of the following B1) to B5):
b1 A nucleic acid molecule comprising the promoter of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
b5 A) a transgenic plant line comprising the nucleic acid molecule of B1) and its progeny, or a transgenic plant line comprising the expression cassette of B2) and its progeny, or a transgenic plant line comprising the recombinant vector of B3) and its progeny;
the application is any one or more of Z1-Z3:
use of Z1 to increase sensitivity of aphid piercing response;
application of Z2 in promoting exogenous gene expression under aphid sucking induction;
use of Z3 for increasing plant resistance to aphids.
3. The use according to claim 2, characterized in that: b1 The nucleic acid molecule is a DNA molecule with the nucleotide shown as SEQ ID NO. 1.
4. The promoter according to claim 1.
5. A biomaterial as claimed in claim 2 or claim 3.
6. A plant agent, characterized in that the active ingredient of the plant agent is the promoter PpTNL1pro according to claim 4 or/and the biological material according to claim 5, which is an agent that increases the plant's susceptibility to and/or resistance to aphids piercing response.
7. A method of producing a plant susceptible to and/or resistant to aphid penetration comprising introducing a promoter according to claim 4 into a recipient plant to produce a plant of interest that is susceptible to and/or resistant to aphid penetration more than said recipient plant.
8. Use of the promoter of claim 1 in response to aphid sucking.
9. Use of a promoter according to claim 1 for the preparation of a product responsive to aphid penetration.
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