CN115141838A - Construction of resveratrol synthase gene transformation peanut system - Google Patents
Construction of resveratrol synthase gene transformation peanut system Download PDFInfo
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Abstract
The invention belongs to the technical field of peanut molecular breeding, and particularly relates to construction of a resveratrol synthase gene conversion peanut system. The invention utilizes an agrobacterium infection method to transform the full-length sequence (1179 bp) of the resveratrol synthetase RS gene of the tiger creeper from Hubei province into peanuts, and molecular biological detection such as PCR, southern blot hybridization, northern hybridization and the like proves that the resveratrol synthetase gene RS is integrated into the genome of the peanuts. The invention improves the RS content in the peanut by using the transgenic technology, promotes the increase of the content of the downstream product resveratrol while improving the peanut resistance, provides a theoretical basis for researching the biological function and the regulation mechanism of the RS gene metabolite, and provides an effective way for improving the species resistance and increasing the metabolite yield.
Description
Technical Field
The invention belongs to the technical field of peanut molecular breeding, and particularly relates to construction of a resveratrol synthase gene conversion peanut system.
Background
Peanuts (Arachis Hypogaea L.) are the fourth largest oil crop in the world, are one of the important economic and oil crops in China, and have important positions in agriculture and even in the whole national economy. However, peanut cultivars are narrow in genetic base and poor in stress resistance, are particularly susceptible to various diseases and pests, and seriously affect the yield and quality of the peanut cultivars. With the development of biotechnology, the development of transgenic technology for improving peanut cultivars is receiving wide attention from researchers.
Resveratrol (Res) is a natural polyphenol phytoalexin, has significant anticancer activity, is mainly contained in giant knotweed rhizome, grapes and peanuts, has the effects of resisting oxidation, resisting proliferation, promoting apoptosis, resisting aging and regulating immunity, and is widely applied to the treatment of cancers and immune system diseases. Resveratrol was originally obtained from extracts of the root of veratrum pilosellum and has been obtained from over 70 plants. The research shows that: the rhizoma Polygoni Cuspidati, fructus Vitis Viniferae, and semen Arachidis Hypogaeae are higher in content. Resveratrol Synthetase (RS) is the key enzyme catalyzing the reaction of malonyl-CoA and coumaroyl-CoA, and the final product of the catalysis is resveratrol. The catalytic reaction of the RS enzyme is a series of condensation reactions and is also an important regulation enzyme influencing the biosynthesis of the resveratrol. Therefore, the RS gene has good application prospect in the aspect of the modification of plant and microorganism functions.
In recent years, researches on functions, development and utilization of resveratrol have been greatly advanced, but how RS genes participate in stress response and action mechanisms thereof are still to be deeply researched, and with the development of sustainable agriculture and the continuous demands of human beings on medicines and health care products, the yield of resveratrol is urgently improved by utilizing plant genetic engineering technology. With the continuous cloning and improvement of the resveratrol synthetase RS gene, the heritage improvement and new variety cultivation of crops, medicines and microorganisms by utilizing the RS gene are also carried out in sequence, and the Res can meet the requirements of Res in industries such as food, health care, medicine and the like by transferring the resveratrol synthetase RS gene to improve the self nutrition added value of a receptor plant and the Res yield in recombinant microorganisms. Therefore, the RS gene is transformed into the peanut through the gene transformation technology, so that the stress resistance of the peanut and the yield of the resveratrol are expected to be improved, and the economic value of the peanut is further improved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for constructing a resveratrol synthase gene conversion peanut system, which improves the stress resistance and the yield of resveratrol of peanuts and improves the economic value of the peanuts by transferring a resveratrol synthase RS gene.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention provides a construction method of a resveratrol synthase gene conversion peanut system, which comprises the following steps:
s1, transforming a plasmid pCAMBIA3300-RJ39-RS-Tnos into an agrobacterium-infected LBA4404, and culturing the screened positive colonies in a culture medium containing Rif, sm and Kan to a logarithmic growth phase to obtain an agrobacterium liquid;
s2, cutting the leaves of the aseptic peanut seedlings into small pieces, then infecting the small pieces with the agrobacterium liquid obtained in the step S1, putting the infected small pieces into an MS0 culture medium for co-culture, transferring the small pieces to a regeneration culture medium for differentiation culture, transferring the small pieces to a rooting culture medium for rooting culture after the differentiation culture, and finally transplanting the rooted peanut seedlings to a greenhouse for culture.
Resveratrol Synthase (RS) is a key enzyme in the synthetic pathway of Resveratrol (Res), and plays an important biological role in the aspects of plant metabolism, regulation and the like. The resveratrol synthetase RS gene is obtained by cloning from a Himalayan tiger in Chuanbei province, the nucleotide sequence of the resveratrol synthetase RS gene is 1179bp long, 392 amino acids are coded (Wangxianli, the regulation and control of resveratrol biosynthesis in transgenic plants [ D ] Shihui university, 2007; qianjin, research on strawberry genetic transformation by the resveratrol synthetase gene [ D ] Fujian agriculture and forestry university, 2010.), and the homology of the gene and a peanut resveratrol synthetase gene with GenBank accession number of FM955393 is 84.41%. In view of the important regulation and control effect of the promoter on the expression of the gene and the physiological and biochemical characteristics expressed by the receptor plant, the invention utilizes the pCAMBIA3300-RJ39-RS-Tnos plant expression vector constructed by the strawberry fruit specific promoter RJ39 to transform the peanut, aims to obtain the over-expression of the RS gene in the fruit of the peanut, and improves the economic value of the peanut by heterologous expression of the RS gene cloned by the parthenocarpic bacterium transferred into the Huperzia californica.
Preferably, liquid nitrogen freeze-thaw method is used to transform plasmid pCAMBIA3300-RJ39-RS-Tnos into Agrobacterium competent LBA 4404. The plasmid pCAMBIA3300-RJ39-RS-Tnos is constructed by placing a cloned RS gene under the control of a strawberry fruit specific promoter RJ39 and using a herbicide resistance gene (bar) as a selection marker.
Preferably, the peanut is peanut variety Luhua No. 18.
Further, when the transformation is performed by a liquid nitrogen freeze thawing method, the liquid nitrogen is frozen for 2min, and then the incubation is performed for 5min at 37 ℃.
Preferably, the culture medium containing Rif, sm and Kan is liquid CYM culture medium containing 50mg/L Rif,100mg/L Sm and 100mg/L Kan.
Preferably, the time for infecting the agrobacterium liquid is 15-30min. Further, the time for infecting the agrobacterium liquid is 20min.
Preferably, the co-culture is performed in a dark culture mode for 2-4d. Further, the co-cultivation time was 3d.
Preferably, after differentiation culture is carried out until the height of the seedling is 1.5-2.5cm, the seedling is transferred to a rooting culture medium for rooting culture. Further, after differentiation culture is carried out until the height of the seedling is 2cm, the seedling is transferred to a rooting culture medium for rooting culture.
Preferably, the rooting culture is culture until adventitious roots are formed. The time is about 5 weeks.
Preferably, the method also comprises the step of identifying the rooted peanut seedlings.
Further, PCR method is used for identification, and primers used in PCR such as SED ID NO;1 and SED ID NO;2, respectively.
Specifically, the reaction system (20. Mu.L) of PCR was: 1 μ L of template DNA; primer RS-F1 uL; primer RS-R1 mu L;10 × buffer2 μ L; dNTPs (2.5 mmol/L) 2. Mu.L; taq DNA polymerase (10000U/mL) 0.2. Mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min30s, extension at 72 ℃ for 1min30s, 30 cycles; then further extension at 72 ℃ for 10min.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a method for constructing a resveratrol synthase gene conversion peanut system, which utilizes an agrobacterium infection method to convert a Huperzia serrata resveratrol synthase RS gene full-length sequence (1179 bp) into peanuts, and molecular biological detection such as PCR, southern blot hybridization, northern hybridization and the like proves that a resveratrol synthase gene RS is integrated into a genome of the peanuts. The invention improves the RS content in the peanut by using the transgenic technology, promotes the increase of the content of the downstream product resveratrol while improving the peanut resistance, provides a theoretical basis for researching the biological function and the regulation mechanism of the RS gene metabolite, and provides an effective way for improving the species resistance and increasing the metabolite yield.
Drawings
FIG. 1 is a pCAMBIA3300-RJ39-RS-Tnos plant expression vector;
FIG. 2 shows PCR identification of pCAMBIA3300-RS-RJ39-Tnos transgenic peanuts (1-8: positive result of PCR identification of transgenic peanuts;: negative control with DNA of wild type peanuts as template; +: plasmid; pCAMBIA3300-RJ39-RS-Tnos as positive control with template; M: DL2000 marker);
FIG. 3 shows the results of PCR amplification and PCR-Southern hybridization of RS-transgenic peanuts (1;
FIG. 4 shows the results of Northern blotting analysis (1-4;: negative control (non-transgenic material); +: positive control).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, and is not intended to limit the present invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples, unless otherwise specified, were all conventional, the test materials used in the following examples, unless otherwise specified, were all commercially available in a conventional manner.
Example 1 construction of resveratrol synthase Gene-transformed peanut System
1. Experimental materials and methods
1.1 Experimental materials
(1) Peanut tissue culture and a culture medium for transferring RS genes:
MS0 minimal medium, pH 5.8, solid medium containing 0.8% agar;
an infection medium, MS +2.0 mg/L6-BA +0.1 mg/L2, 4-D;
regeneration medium, MS +3.0 mg/L6-BA +0.1 mg/L2, 4-D +350mg/L Cb;
enrichment medium, MS +1.0 mg/L6-BA +0.1mg/L IAA +350mg/L Cb;
screening a culture medium, wherein MS +0.1mg/L IBA +2mg/L PPT +350mg/L Cb;
rooting medium, MS +2.0mg/L IBA. The culture temperature of the aseptic seedling is 25-28 ℃, the illumination intensity is 1000-1200 Lx, and the illumination time is 14h/d.
And (3) annotation: MS: MS culture medium; 6-BA: 6-benzylaminopurine; 2,4-D:2, 4-dichlorophenoxyacetic acid; cb: carbenicillin; IAA: indole acetic acid; IBA: indolebutyric acid; PPT glufosinate.
(2) Bacterial strain and expression vector
Agrobacterium tumefaciens (Agrobacterium tumefaciens), LBA4404 (Rif R, smR), expression vector pCAMBIA3300, RS gene recombination vector pCAMBIA3300-RJ39-RS-Tnos (RJ 39 is fruit specific promoter) were constructed and stored by plant genetic engineering and protein engineering national key laboratories of Beijing university Life sciences.
(3) Primers, enzymes and reagents
Primers for amplification of the RS gene (synthesized by Shanghai Bioengineering technologies Co.):
RS-F：5’-CGGGATCCGCCATGGCTTCAGTTGAGAAATTTAG-3’(SED ID NO；1)；
RS-R：5’-GTGAGCTCGAAGGGTAAACCATTCTCTTTTAT-3’(SED ID NO；2)；
t4 DNA ligase, taq DNA polymerase, DNA restriction enzyme, pMD18T vector, d NTPs, gel recovery kit and the like are purchased from Takara corporation (Japan); kanamycin, streptomycin, rifampicin, and the like were purchased from biotechnology companies in Beijing ancient countries; sequencing was performed by Shanghai bioengineering, inc.
(4) Peanut
Peanut variety Luhua No. 18, purchased from Taobao.
1.2 Experimental methods
(1) Cultivation of aseptic peanut seedlings
The peanut of Luhua No. 18 is sterilized by 70% ethanol for 3 minutes, sterilized by 10% sodium hypochlorite for 15 minutes, washed by sterile water for three times and then inoculated in MS0 culture medium.
(2) Agrobacterium mediated transformation method
Agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA4404 (RifR, smR) and plasmid pCAMBIA3300-RJ39-RS-Tnos are used, a liquid nitrogen freeze-thaw method is used for transforming the plasmid containing the target gene into Agrobacterium tumefaciens sensitive LBA4404 (liquid nitrogen freeze-thaw for 2min, incubation for 5min at 37 ℃), and the screened positive bacteria are dropped into liquid CYM culture medium containing Rif (50 mg/L), sm (100 mg/L) and Kan (100 mg/L) and are cultured for 2d to logarithmic growth period under the oscillation at 28 ℃, thus obtaining the Agrobacterium tumefaciens bacterial liquid. Cutting the leaves of aseptic peanut seedlings into 0.5cm 2 And (3) infecting the small pieces with agrobacterium liquid diluted by 100 times by using an MS0 liquid culture medium for 20min, sucking the infected leaves with sterile filter paper to remove redundant agrobacterium liquid, putting the leaves into the MS0 culture medium for co-culture (dark culture) for 3d, transferring the co-cultured peanut leaf discs to a regeneration culture medium, transferring the peanut seedlings which are differentiated to a rooting culture medium after the peanut seedlings are about 2cm high, forming adventitious roots after about 5 weeks, and transplanting the rooted peanut seedlings to a greenhouse for culture.
(3) Identification and detection of RS-transgenic plants
1) PCR detection of RS gene transferred peanuts
Cutting leaves of the peanut with the transferred RS gene and the peanut with the non-transferred RS gene, extracting genome DNA of a transformant and a non-transformant by adopting a CTAB method, wherein a PCR reaction system (20 mu L) comprises the following steps: 1 μ L of template DNA; 1 mu L of primer RS-F; 1 mu L of primer RS-R; 10 × buffer2 μ L; dNTPs (2.5 mmol/L) 2. Mu.L; taq DNA polymerase (10000U/mL) 0.2. Mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min30s, extension at 72 ℃ for 1min30s, 30 cycles; then further extension for 10min at 72 ℃. And (3) taking the PCR product to perform electrophoresis on 0.7% agarose gel, and observing the size of the target gene by using a gel imager.
2) Southern blot hybridization detection of RS gene-transferred peanuts
Extracting the total DNA of young leaves of the peanuts by adopting a CTAB (cetyl trimethyl ammonium bromide) method, carrying out PCR (the same as the step 1), carrying out membrane conversion, preparing a probe, hybridizing and detecting.
Preparation and pretreatment of the probe: sucking 2. Mu.L of Agrobacterium LBA4404 plasmid DNA, and using ddH 2 Diluting the plasmid DNA by 10 times, treating the diluted plasmid DNA in boiling water for 15min to denature the plasmid DNA, adding 4 mu L of DIG solution subjected to precooling treatment into a denaturation system, centrifuging the solution at 5000rpm, and then putting the solution into a metal bath at 37 ℃ for pretreatment for 18h. After pretreatment, the centrifuge tube was transferred to a 70 ℃ water bath to terminate the pretreatment reaction for 15min, and the plasmid probe was prepared. The following operations are then carried out: (1) pre-hybridization: and (3) filling the prepared hybridization solution into a hybridization bottle for preheating, filling the hybridization bottle into a dried nitrocellulose filter membrane, and performing prehybridization for 2 hours at 42 ℃ in a hybridization furnace. (2) And (3) hybridization treatment: pouring out the pre-hybridization solution, adding the prepared probe, and performing hybridization treatment for 24 hours in a hybridization furnace at 50 ℃. (3) And (3) washing the membrane: and (3) taking out the hybridization filter membrane, rinsing the hybridization filter membrane for 3 times by using the membrane Washing solution, washing the hybridization filter membrane for 15min after full Washing, sealing the hybridization filter membrane in the sealing solution for 15min, and rinsing the hybridization filter membrane for 2 times by using the Washing buffer again, wherein each time lasts for 10min. Finally, the filters were placed in a Detection buffer for 15min before use. (4) Southern blot color reaction: the filter membrane is taken out and then is sucked dry by filter paper, and is placed into a Detection buffer containing a chromogenic reagent NBT/BCIP for chromogenic treatment, and is kept still for 24h in dark. And observing hybridization strips on the filter membrane after the color development is finished, washing the filter membrane for 15min by using TE buffer solution, and observing imaging and strip conditions.
3) Northern hybridization detection of RS transgenic plant
Extracting young leaf total RNA of transgenic and non-transgenic peanuts by a Trizol method, performing electrophoresis, transferring membranes, preparing probes, hybridizing and detecting.
Preparation and labeling of probes: mu.L of 10 XPCR buffer, primers RS-F and
50pmol each of RS-R, 1. Mu.L dNTP, 0.5. Mu.L Taq enzyme, 0.1. Mu.L DIG-labeled solution, 2. Mu.L template DNA. And after the reaction is finished, detecting the labeling effect of the prepared probe by electrophoresis and storing the labeling effect at-20 ℃ for later use. Boiled in water for 15 minutes before use, and quickly transferred to ice for use.
Description of the drawings: general Kit for testing DIG labeling efficiency has the advantage that the detection efficiency of DNA labeled by a PCR method is rarely measured, because the method has higher labeling efficiency, and is not greatly influenced by experimental conditions like a random primer method. The detection method of efficiency is to judge the probe amount of DIG on the label in the probe to be detected according to the gradient development intensity of the standard probe, and calculate the added probe amount instead of the total content of the probe according to the estimated probe amount containing DIG during hybridization.
Hybridization and detection: (1) pre-hybridization: the reverse side of the membrane is tightly attached to a hybridization bottle, 5mL of prehybridization liquid is added, and prehybridization is carried out for 3h at 42 ℃. (2) And (3) hybridization: the denatured probe (denaturation at 95-100 ℃ for 5min, ice bath for 5 min) was added to the prehybridization solution and hybridized at 42 ℃ for 16h. (3) Washing the membrane: discarding the hybridization solution, adding 2 XSSC/0.1% SDS, washing for 15min in a wet room, further washing for 15min X2 times in 0.2 XSSC/0.1% SDS,55 ℃. (4) Tabletting: the membrane was rinsed with double distilled water for a while and water was removed from the membrane by suction with filter paper. Wrapping nylon film with preservative film, setting in dark box, and pressing X-ray film in dark room. The test strips were placed in a cassette at-70 ℃ for about 7 days.
2. Results and analysis
2.1 PCR identification of transgenic peanuts
The resveratrol synthetase RS gene cloned from the Tilapia hospita from Sichuan province in the national key laboratory of Beijing university Life sciences college of plant genetic engineering and protein engineering has the gene sequence length of 1179bp and encodes 392 amino acids. Construction of pCAMBIA3300-RJ39-Tnos plant expression vector using strawberry fruit-specific promoter RJ39 (qian queu. Study of genetic transformation of strawberry by resveratrol synthase gene [ D ]. Fujian agroforestry university, 2010.) (fig. 1).
The plant expression vector pCAMBIA3300-RJ39-RS-Tnos is transformed into agrobacterium tumefaciens LBA4404, a screening marker gene is a herbicide resistance gene (bar), 18 peanut plants in Luhua are transformed by an agrobacterium-mediated leaf disc method to obtain 50 resistance strains, genome DNA is extracted, primers RS-F and RS-R are used for PCR amplification, the electrophoresis result shows that 18 strains are positive, and the transformation rate reaches 36%. The amplified band size of the positive peanut plant with the transferred RS gene is about 1.1kb, but the negative control does not. The PCR results of the partially RS-transgenic peanut plants are shown in FIG. 2.
2.2 Southern blot hybridization detection of transgenic peanuts
Randomly selecting 7 strains from 18 trans-RS positive strains to perform Southern blot hybridization detection, extracting the total DNA of young leaves of peanuts by adopting a CTAB method, performing PCR (polymerase chain reaction), transferring a membrane, preparing a probe, hybridizing and detecting. Results 6 lines were all transgenic peanut lines, one being an RS negative line (FIG. 3).
2.3 Northern hybridization detection of transgenic plants
Selecting total RNA of the RS gene-transferred peanuts which are identified to be positive by PCR, transferring the total RNA to a Hybond N + membrane after formaldehyde denaturing electrophoresis. The full-length RS gene segment marked by digoxin is used as a probe to be hybridized with the gene segment, and the transcription level of the RS gene in the transgenic peanut is detected through Northern hybridization. Northern blotting analysis results showed that the exogenous RS gene can be normally transcribed in the transgenic peanut leaf, but the non-transgenic material has no hybridization signal (FIG. 4).
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> Zhuhai science and technology institute
<120> construction of resveratrol synthase gene transformed peanut system
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> DNA/RNA
<213> RS-F(Artificial Sequence)
<400> 1
cgggatccgc catggcttca gttgagaaat ttag 34
<210> 2
<211> 32
<212> DNA/RNA
<213> RS-R(Artificial Sequence)
<400> 2
gtgagctcga agggtaaacc attctctttt at 32
Claims (10)
1. A construction method of a resveratrol synthase gene transformation peanut system is characterized by comprising the following steps:
s1, transforming a plasmid pCAMBIA3300-RJ39-RS-Tnos into an agrobacterium-infected LBA4404, and culturing the screened positive colonies in a culture medium containing Rif, sm and Kan to a logarithmic growth phase to obtain an agrobacterium liquid;
s2, cutting leaves of aseptic peanut seedlings into small pieces, then infecting the small pieces with the agrobacterium liquid obtained in the step S1, putting the small pieces into an MS0 culture medium for co-culture after infection, transferring the small pieces onto a regeneration culture medium for differentiation culture, transferring the small pieces onto a rooting culture medium for rooting culture after differentiation culture, and finally transplanting the rooted peanut seedlings to a greenhouse for culture.
2. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, wherein a liquid nitrogen freeze-thaw method is adopted when the plasmid pCAMBIA3300-RJ39-RS-Tnos is transformed into the Agrobacterium tumefaciens competent LBA 4404.
3. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 2, wherein when the transformation is performed by a liquid nitrogen freeze-thaw method, the transformation is performed by freezing the peanut for 2min with liquid nitrogen and then incubating the peanut for 5min at 37 ℃.
4. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, wherein the culture medium containing Rif, sm and Kan is a liquid CYM culture medium containing 50mg/L Rif,100mg/L Sm and 100mg/L Kan.
5. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, wherein the infection time of the agrobacterium liquid is 15-30min.
6. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, wherein the co-culture is performed in a dark culture mode for 2-4 days.
7. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, characterized in that the rooting culture is carried out by transferring to a rooting culture medium after differentiation culture until the height of the seedling is 1.5-2.5 cm.
8. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, wherein the rooting culture is culture until adventitious roots are formed.
9. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 1, further comprising a step of identifying rooted peanut seedlings.
10. The method for constructing the resveratrol synthase gene transformed peanut system according to claim 9, wherein the identification is performed by a PCR method, and primers used in the PCR are SED ID NO;1 and SED ID NO;2, respectively.
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