CN116926114A - Application of heat shock protein gene LbHSP90 in changing low temperature stress resistance of cut lily - Google Patents
Application of heat shock protein gene LbHSP90 in changing low temperature stress resistance of cut lily Download PDFInfo
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Abstract
The invention discloses a heat shock protein geneLbHSP90Application of gene in changing low temperature stress resistance of cut lilyLbHSP90Has the nucleotide sequence shown in SEQ ID NO. 1, and the nucleotide sequence shown in SEQ ID NO. 1 can code the amino acid sequence shown in SEQ ID NO. 2. The gene is cloned from lily by increasing in model plant Arabidopsis thaliana or lilyLbHSP90The expression level of the gene is improved to improve the low resistance of arabidopsis thaliana or lilyAbility to warm stress, thereby yieldingLbHSP90The gene plays a key role in the aspect of participating in the response of lily low-temperature stress, and can be used as a favorable gene resource for cultivating lily with long flowering phase. The invention is thus achieved byLbHSP90The gene overexpression has a key effect on improving the low-temperature stress resistance of the lily, and the invention provides a favorable gene resource for prolonging the cultivation of the cut-flower lily in the florescence.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a heat shock protein geneLbHSP90The application in changing the low temperature resistance of the cut lily.
Background
Low temperature stress has been an important limiting factor in plant growth and development and has resulted in a substantial decrease in crop yield. During long-term evolution, plants have generated a range of physiological and biochemical mechanisms to accommodate, suppress or eliminate the effects of abiotic stress, with the synthesis of new stress proteins being one of the most common.
Heat shock proteins can be classified into 5 families, i.e., sHSP (small molecule heat shock proteins), HSP60, HSP70, HSP90, and HSP100, according to molecular weight. HSP90 is an ATP-regulated dimeric chaperone consisting essentially of three highly conserved domains, a C-terminal domain of about 25 kDa that binds to a substrate, a middle domain of 35 kDa and an N-terminal domain of 12 kDa of the ATP binding site. HSP90 is an important component of cells at normal developmental stages, contributing to translocation, folding and degradation of proteins, functioning in important biological processes such as signal transduction and cell cycle. HSP90 is also involved in responding to a variety of abiotic and biotic stresses, such as the expression pattern of the cabbage BoHSP90, indicating that BoHSP90-2, boHSP90-3, boHSP90-7, boHSP90-9, boHSP90-10 and BoHSP90-11 are induced under low temperature stress.
The lily is taken as a very important fresh cut flower, and the cut flower lily is extremely easy to cause flower malformation and premature senility due to water loss and low temperature in cold chain transportation after picking and after picking, so that the quality of flowers is affected, and the commodity value of the flowers is reduced. Yunnan is the most important place of lily production in China, cultivation of lily with long flowering phase (stress resistance) is one of important means for prolonging ornamental period, and lily breeding for prolonging ornamental period by utilizing research on flower stress has not been reported yet. One of the main reasons is that the research mechanism for regulating and controlling the response of lily petals to stress resistance is not clear, and the breeding of new varieties of lily with prolonged flowering phase is severely restricted. Therefore, the research for researching the stress resistance response of the lily has important significance for cultivating the new lily variety with long ornamental period, andLbHSP90no study of participation in low temperature stress response in lily was reported.
Disclosure of Invention
The invention aims at providing a heat shock protein geneLbHSP90Use of lily in altering the low temperature resistance of cut flowers by increasing in the model plant Arabidopsis thaliana or lilyLbHSP90The expression level of the gene is used for improving the low temperature stress resistance of the arabidopsis thaliana or the lily, thereby obtainingLbHSP90The gene plays a key role in the aspect of participating in the response of lily low-temperature stress, and can be used as a favorable gene resource for cultivating lily with long flowering phase.
The invention provides a heat shock protein geneLbHSP90Application of gene in changing low temperature stress resistance of cut lilyLbHSP90Has the nucleotide sequence shown in SEQ ID NO. 1, the nucleotide sequence shown in SEQ ID NO. 1 can code the amino acid sequence shown in SEQ ID NO. 2, and the nucleotide sequence is enhanced in Arabidopsis thalianaHSP90The expression level of the gene is used for improving the low temperature stress resistance of the arabidopsis thaliana, and the application is verified by using plant arabidopsis thaliana or lily.
Verification Using Pattern plant Arabidopsis, genes were generated by constructing an overexpression vector in Pattern plant Arabidopsis and transforming Pattern plant Arabidopsis by Agrobacterium-mediated flower dippingLbHSP90In the overexpression of Arabidopsis, the transgenic Arabidopsis with lower temperature resistance than wild Arabidopsis is obtained, and Arabidopsis is a model plant which can verify the general biological law, thereby obtaining the geneLbHSP90Can improve the low temperature stress resistance of lily.
The specific procedure for validation using the model plant arabidopsis was as follows:
(1) Constructing an over-expression vector: (1) by means ofLbHSP90Gene sequence design primerLbHSP90F andLbHSP90-R andLbHSP90f', PCR amplification Using the yellow Tianba cDNA as templateLbHSP90Gene, obtaining the objectFragmentsLbHSP90A gene fragment;
primer: lbHSP90-F: GCTTTCGCGAGCTCGGTACCATGGCCGCCGCCGCCGACGAAGCCAAAG
LbHSP90-R: CGCCCTTGCTCACCATGGATCCTTTCATTTGGACTATCCCAGCAC
LbHSP90-F’: CTAGAAGGCCTCCATGGGGATCCGGCACAGCTCATTTCTTTATG
(2) The PC1300S-GFP vector (a commercially available vector ready for laboratory use) was treated with KpnI and BamHI double enzyme and recovered, and the PCR amplified product of step (1)LbHSP90The gene fragment is recombined to obtain a recombined product, the recombined product is transformed into escherichia coli, then a resistance plate is coated, a clone and a shaking bacterium are selected, and then a primer A580-seqR is used for preparing a recombinant vectorLbHSP90The positive cloning verification is carried out on the F' colony through a PCR technology, and then the sequencing verification is carried out to obtain the overexpression vector of LbHSP 90;
primer: a580—seqr: AGAAGATGGTGCGCTCCTG;
(2) Arabidopsis seed disinfection treatment
Soaking wild Arabidopsis seeds in 70% ethanol for 2min, spin-washing with 15% NaClO solution for 10 min, and rinsing with sterile water for 5 times;
(3) Sowing and transplanting
Resuspension the sterilized seeds with sterile water, uniformly dispersing on the surface of 1/2MS solid culture medium, inverting the culture dish, vernalizing for two days at 4C, culturing in dark at 25deg.C for one day, and culturing in a tissue culture chamber at 22deg.C. Transplanting the seedlings into nutrient soil when the seedlings grow to four leaves, placing the seedlings in a constant temperature environment at 22 ℃ for 16h of illumination and 8h of dark culture, and 7.5ml of nutrient solution L -1 Root irrigation, 7 days/times in seedling stage and 14 days/times in bud stage;
(4) Transformation of Arabidopsis plants with overexpression vectors
(1) Single colonies containing the over-expression vector were picked and inoculated into 5mL LB liquid medium (containing 50 mg.L) -1 Rif and 100 mg Kan), 200 rmp.min at 28 ℃ -1 Shake culturing overnight;
(2) transferring 2mL of the bacterial liquid into 100 mL of LB medium (containing 50 mg.L) -1 Rif and 100 mg Kan), 200mp.min at 28 ℃ -1 Shake culturing until OD600 value is about 1.0-1.5;
③6000 rmp·min -1 centrifuging for 10 min, and discarding supernatant:
(4) 1mL of 5% sucrose solution was used to resuspend the bacteria, 6000 rmp.min -1 Centrifuging for 2min, and discarding supernatant;
(5) repeating (4);
(6) adding 5% sucrose solution to resuspend the thallus until OD600 value is about 0.4-0.7;
(7) adding silwet77 surfactant in an amount of 0.05% by volume prior to soaking;
(8) winding water before soaking, cutting off pod, and soaking bud in the above solution for 1 min:
(9) inverting the plant, culturing in the dark for 14 hours, taking out, and culturing in normal growth to obtain transgenic arabidopsis;
(5) Identification of transgenic Arabidopsis thaliana
Cutting transgenic arabidopsis plant leaves, extracting DNA by using a CTAB method, and performing PCR detection by using a screening marker gene specific primer; carrying out PCR positive detection on 5 LbHSP90 transformed plants by using a specific primer of the Hpt gene, wherein the positive plants are transgenic Arabidopsis;
hpt gene primer: hpt557-F ACACTACATGGCGTGATTTCAT
hpt557-R: TCCACTATCGGCGAGTACTTCT
(6) Sowing of transgenic Arabidopsis thaliana
Soaking the seeds of the transgenic arabidopsis thaliana in the step (5) in 70% ethanol for 2min, spin-washing the seeds with 15% NaClO solution for 10 min, and rinsing the seeds with sterile water for 5 times; uniformly dispersing on the surface of a 1/2MS solid culture medium, inverting a culture dish, vernalizing for two days at 4 ℃, performing normal dark culture at 25 ℃ for one day, and then placing in a tissue culture chamber at 22 ℃ for normal culture; transplanting the seedlings into nutrient soil when the seedlings grow to four leaves, placing the seedlings in a constant temperature environment at 22 ℃ for 16h of illumination and 8h of dark culture, and 7.5ml of nutrient solution L -1 Root irrigation, 7 days/times in seedling stage and 14 days/times in bud stage;
(7) The material treatment and detection method comprises the following steps: sowing transgenic and wild arabidopsis seeds respectively, culturing for 4 weeks, transferring to a greenhouse, observing the growth conditions of wild and transgenic plants, and recording; after the arabidopsis thaliana of 4 weeks of age is subjected to low-temperature stress treatment, the wild arabidopsis thaliana is obviously wilted after being subjected to low-temperature treatment, and the transgenic arabidopsis thaliana plant is slow in growth vigor but is bright in leaf color and weak in wilting condition; after room temperature is restored, part of wild plants cannot be restored, and transgenic plants can be restored, so that the over-expression plants have strong low-temperature stress resistance, and the LbHSP90 gene can participate in the low-temperature resistance process of the plants, and the low-temperature resistance performance of the plants is improved.
Genes of the inventionLbHSP90Has a nucleotide sequence shown as SEQ ID NO. 1, and the gene has a length of 1728bp (shown as figure 1 of the specification).
（ATGGCCGCCGCCGCCGACGAAGCCAAAGCTAAGGGCAACGCCGCCTTCTCCGCCGGCAACTTCTCCGACGCGATCCGCCACTTCACCGCTGCCATCGACCTCGCCCCCACCAACCACGTCCTCTTCTCCAACCGCTCCGCCGCCCACGCCTCCCTCGGCAACTTCTCCGACGCCCTCTCCGACGCCCTCCGTACCGTCGAGCTCAAGCCCGACTGGTCCAAAGGGTACTCCCGCCTCGGCGCCGCTCACCTCGGCCTCGGCGACGCCGCTGCCGCCGCCGAAGCCTACTCGAAGGGGCTCGAGCTCGATCCGTCGAATGAAGTTCTCAAGGCTGGTCTTGCCGACGCCCGCAAAGCGGCGTCCGCCGCCTCCCGCTCTGCCGGCTCCGGCGGTGCTAACCCGTTCGGTCAAATGTTCAGCGATCCGACCCTCTGGGCCAAGCTGACGGCGAATCCTACTACCCGTGACTACCTCAAGCAGCCGGACTTTGTCCAGATGATACGGGAGGTGCAGCGAAACCCTAGCAGCATGAACTTGTACTTGACTGATCAGCGCATGATGGCGGTGCTTGGTGTGCTGCTCAATGTTAATATCAGTACCAAAGATCCAAGCGAGATGGAGAGGGATTTCCCGGAGGCGCCATCGGTTAAGCCGGAGGTGAAGCCAGAGGAGGTGAAGAAGGAGCCGAAGCCAGAGCCGGAGCCAGAACCTATGGAGGAGGATGAGAGTAAGGTGAAGAAGAGGGAGGCACAAAAGGAGAAGGAGGCGGGGAATGCGGCGTACAAGAAGAAGGATTTTGAGTCGGCCATTGAACACTATAGTAAGGCGATGGAGCTGGATGATACTGATATCTCCTACCTTACTAATAGGGCTGCTGTTTATCTGGAGATGGGAAAGTATGATGAATGTATAAAAGATTGCGACAGAGCTGTTGAGAGGGGAAGGGAACTTCGTTCTGACTTCAAGATGGTTGCAAGGGCCTTGACTAGAAAAGGAACTGCACTTGCTAAACTTGCTAAAACCTCCAAGGATTATGAGCCTGCTATTGAGACTTTCCAGAAAGCTCTTACTGAGCACCGGAACCCTGACACTCTAAAAAGGCTTAATGATGCAGAGAGGGCGAAGAAAGAGTTGGAGCAACAGGAGTATTATGACCCAAAATTAGCTGATGAGGAGCGTGAGAAAGGTAATGAGTTTTTCAAGGAGCAAAAGTACCCTGAAGCAATAAAACACTATACTGAAGCACTGAGAAGAAACCCAAAGGATGCAAGGCTTTACAGCAACAGAGCTGCATGTTACACTAAGTTGGCTGCCCTGCCTGAGGCACTGAAAGATGCAGAGAAGTGCATTGAGCTGGATCCATCTTTTGTGAAAGGATATGTGAGGAAAGGCACAGCTCATTTCTTTATGAAGGAGTATGACAAAGCTATCGAAACTTACCAGGAGGGCTTGAAGCTTGACAGCAGCAACCAGGAACTTCTTGATGGTATTCAGAAGTGTGTTGGACAAATGAACAGGACTAATCTTAGTCCCGAAGAGGCGAAAGAGAGACAGGCCAAAGCAATGCATGACCCGGAAATACAGAATATCTTGAGAGATCCGATAATGAGTCAGGTATTGATGGACCTGCAGGAGAACTCGAGGTCTGCACAGGAGCATTTGAAGAATCCCCAGGTGATGCATAAGCTACAAAAGCTCATGAGTGCTGGGATAGTCCAAATGAAATAA）
The nucleotide sequence shown in SEQ ID NO. 1 can code for the amino acid sequence shown in SEQ ID NO. 2.
SEQ ID NO:2：（MAAAADEAKAKGNAAFSAGNFSDAIRHFTAAIDLAPTNHVLFSNRSAAHASLGNFSDALSDALRTVELKPDWSKGYSRLGAAHLGLGDAAAAAEAYSKGLELDPSNEVLKAGLADARKAASAASRSAGSGGANPFGQMFSDPTLWAKLTANPTTRDYLKQPDFVQMIREVQRNPSSMNLYLTDQRMMAVLGVLLNVNISTKDPSEMERDFPEAPSVKPEVKPEEVKKEPKPEPEPEPMEEDESKVKKREAQKEKEAGNAAYKKKDFESAIEHYSKAMELDDTDISYLTNRAAVYLEMGKYDECIKDCDRAVERGRELRSDFKMVARALTRKGTALAKLAKTSKDYEPAIETFQKALTEHRNPDTLKRLNDAERAKKELEQQEYYDPKLADEEREKGNEFFKEQKYPEAIKHYTEALRRNPKDARLYSNRAACYTKLAALPEALKDAEKCIELDPSFVKGYVRKGTAHFFMKEYDKAIETYQEGLKLDSSNQELLDGIQKCVGQMNRTNLSPEEAKERQAKAMHDPEIQNILRDPIMSQVLMDLQENSRSAQEHLKNPQVMHKLQKLMSAGIVQMK）
The amino acid sequence comprises 575 amino acids, the relative molecular weight of the protein is 64.372kDa in theory, and the isoelectric point is 6.12.
Compared with the prior art, the invention has the beneficial effects that: in lily with participation in low temperature stress responseLbHSP90Gene is not reported, the invention is realized by over-expressing lilyLbHSP90The gene has a key effect on improving the low-temperature stress resistance of the lily, and the invention provides a favorable gene resource for cultivating the lily with strong stress resistance.
Drawings
FIG. 1 shows PCR amplificationLbHSP90Agarose gel electrophoresis of genes;
FIG. 2 shows LbHSP90 colony PCR assay;
FIG. 3 shows PCR positive detection of LbHSP90 transformed Arabidopsis;
FIG. 4 is an Arabidopsis phenotype of wild type and transgene under low temperature stress;
FIG. 5 WT (wild type) and OE-LbHSP90Phenotype of lily petal discs under low temperature stress;
FIG. 6 WT (wild type)) And OE-LbHSP90Under low temperature stressLbHSP90Is a factor (B) of the expression level of (C).
Detailed Description
The invention is further described below with reference to the drawings and examples.
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope 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, the disclosure of which is incorporated herein by reference as is commonly understood by one of ordinary skill in the art to which this invention pertains.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims. The methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional commercial reagents or reagents formulated according to conventional methods unless otherwise specified.
Example 1: the genes of this exampleLbHSP90Cloning from lily, geneLbHSP90Has the nucleotide sequence shown in SEQ ID NO. 1, the nucleotide sequence shown in SEQ ID NO. 1 can code the amino acid sequence shown in SEQ ID NO. 2 by causing in lilyLbHSP90Gene overexpression is performed to improve the low-temperature stress resistance of lily, and the embodiment uses mode plant Arabidopsis thaliana for verification, and the gene is enabled to be transformed by constructing an overexpression vector in the mode plant Arabidopsis thaliana and transforming the mode plant Arabidopsis thaliana through an agrobacterium-mediated flower dipping methodLbHSP90In the overexpression of Arabidopsis, the transgenic Arabidopsis which is more resistant to low temperature than the wild Arabidopsis is obtained, and Arabidopsis is a model plant which can verify the general biological law, thereby obtainingThe gene is producedLbHSP90Can improve the low temperature stress resistance of lily, and comprises the following specific steps:
1. construction of an over-expression vector and acquisition of transgenic plants:
(1) Constructing an over-expression vector: (1) by means ofLbHSP90Gene sequence design primerLbHSP90F andLbHSP90-R andLbHSP90f', PCR amplification Using the yellow Tianba cDNA as templateLbHSP90Gene, obtaining target fragmentLbHSP90A gene fragment;
primer: lbHSP90-F: GCTTTCGCGAGCTCGGTACCATGGCCGCCGCCGCCGACGAAGCCAAAG
LbHSP90-R: CGCCCTTGCTCACCATGGATCCTTTCATTTGGACTATCCCAGCAC
LbHSP90-F’: CTAGAAGGCCTCCATGGGGATCCGGCACAGCTCATTTCTTTATG
(2) The PC1300S-GFP vector (a commercially available vector ready for laboratory use) was treated with KpnI and BamHI double enzyme and recovered, and the PCR amplified product of step (1)LbHSP90The gene fragment is recombined to obtain a recombined product, the recombined product is transformed into escherichia coli, then a resistance plate is coated, a clone and a shaking bacterium are selected, and then a primer A580-seqR is used for preparing a recombinant vectorLbHSP90The positive cloning of the colony F' is verified by a PCR technology, and then sequencing verification is carried out to obtain an overexpression vector of LbHSP90, as shown in figure 2;
primer: a580—seqr: AGAAGATGGTGCGCTCCTG.
(2) Arabidopsis seed disinfection treatment
Soaking wild Arabidopsis seeds in 70% ethanol for 2min, spin-washing with 15% NaClO solution for 10 min, and rinsing with sterile water for 5 times;
(3) Sowing and transplanting
Resuspension the sterilized seeds with sterile water, uniformly dispersing on the surface of a 1/2MS solid culture medium, inverting a culture dish, vernalizing for two days at 4 ℃ and culturing in dark at 25 ℃ for one day, and then culturing in a tissue culture chamber at 22 ℃ normally; transplanting the seedlings into nutrient soil when the seedlings grow to four leaves, placing the seedlings in a constant temperature environment at 22 ℃ for 16h of illumination and 8h of dark culture, and obtaining 7.5mL of nutrient solution -1 Root irrigation, 7 days/times in seedling stage and 14 days/times in bud stage;
(4) Transformation of Arabidopsis plants with overexpression vectors
(1) Single colonies containing the over-expression vector were picked and inoculated into 5ml LB liquid medium (containing 50 mg.L) -1 Rif and 100 mg Kan), 200 rmp.min at 28 ℃ -1 Shake culturing overnight;
(2) transferring 2ml of the bacterial liquid into 100 ml of LB culture medium (containing 50 mg.L) -1 Rif and 100 mg Kan), 200mp.min at 28 ℃ -1 Shake culturing until OD600 value is about 1.0-1.5;
③6000 rmp·min -1 centrifuging for 10 min, and discarding supernatant:
(4) 1mL of 5% sucrose solution was used to resuspend the bacteria, 6000 rmp.min -1 Centrifuging for 2min, and discarding supernatant;
(5) repeating (4);
(6) adding 5% sucrose solution to resuspend the thallus until OD600 value is about 0.4-0.7;
(7) adding silwet77 surfactant in an amount of 0.05% by volume prior to soaking;
(8) winding water before soaking, cutting off pod, and soaking bud in the above solution for 1 min:
(9) inverting the plant, culturing in the dark for 14 hours, taking out, and culturing in normal growth to obtain transgenic arabidopsis;
(5) Identification of transgenic Arabidopsis thaliana
Cutting transgenic arabidopsis plant leaves, extracting DNA by using a CTAB method, and performing PCR detection by using a screening marker gene specific primer; PCR positive detection is carried out on 5 LbHSP90 transformed plants by using a specific primer of the Hpt gene, wherein the numbers of the 5 positive transformed plants (shown in figure 3) are 1, 2, 3, 4 and 5 respectively;
hpt gene primer: hpt557-F ACACTACATGGCGTGATTTCAT
hpt557-R: TCCACTATCGGCGAGTACTTCT
(6) Sowing of transgenic Arabidopsis thaliana
Soaking the seeds of the transgenic arabidopsis thaliana in the step (5) in 70% ethanol for 2min, spin-washing the seeds with 15% NaClO solution for 10 min, and rinsing the seeds with sterile water for 5 times; uniformly dispersing on the surface of a 1/2MS solid culture medium, inverting a culture dish, vernalizing for two days at 4 ℃, performing normal dark culture at 25 ℃ for one day, and then placing in a tissue culture chamber at 22 ℃ for normal culture; to be treatedTransplanting the seedlings into nutrient soil when the seedlings grow into four leaves, placing the seedlings in a constant temperature environment at 22 ℃, culturing the seedlings in the dark under 16h of illumination and 8h of illumination, and culturing the seedlings in 7.5mL of nutrient solution -1 Root irrigation, 7 days/times in seedling stage and 14 days/times in bud stage;
(7) The material treatment and detection method comprises the following steps: after the arabidopsis seeds are sown, after being cultured to 4 weeks of age, the arabidopsis seeds are transferred to a greenhouse for observing the growth of the WT (wild type) and the transgenic plants and recorded. The results of the low temperature stress treatment (4 ℃) for 10d on 4-week-old Arabidopsis thaliana are shown in FIG. 4, in which the plants of WT (wild type) Arabidopsis thaliana were significantly wilted after the low temperature treatment for 10d, while OE-LbHSP90The (transgenic) Arabidopsis plants grow slowly but the leaf color is weakened in wilting. After 14d recovery from room temperature, part of the WT (wild type) plants could not be recovered and OE-LbHSP90The plant can be recovered in large part. Description of OE-LbHSP90The (over-expressed) plant has stronger low-temperature stress resistance, and the LbHSP90 gene can participate in the low-temperature resistant process of the plant, so that the low-temperature resistant performance of the plant is improved.
Example 2: in this example, the heat shock protein gene was verified by lilyLbHSP90Improving the low temperature stress resistance of lily.
1. Material treatment and vacuum infection conversion: (1) taking lily planted in a greenhouse base of the Kunming lily as an infection material, taking lily with a bud period of about 9+/-1 cm long and a bud length which is consistent with a bud length which is picked within two hours as an experiment material, pruning lily branches under water by using sterilized scissors, keeping the overall length of the flower stems at 5+/-1 cm, and then placing the lily branches in distilled water for later use;
(2) the expression OE obtained in example 1LbHSP90The over-expression vector of (2) is resuspended in infiltration buffer solution, and then placed under the condition of no light at 22 ℃ for 5 hours to obtain infiltration solution; the immersion buffers are divided into two types: 1) 10mM MES was mixed with 10mM MgCl solution and adjusted to pH 5.6; 2) 200M acetosyringone solution;
(3) taking out the green bud stage lily prepared in the step (1), and then taking out a circular sheet with the diameter of 1cm from the inner petals by using a puncher;
(4) converting the wafer prepared in the step (3) by using the infection liquid obtained in the step (2) through a vacuum infiltration method (placing a beaker in a vacuum filter), vacuumizing to 0.07Mpa, maintaining for 5 minutes, and infiltrating under negative pressure; rapidly cleaning the soaked wafer with sterile water, placing the wafer in a culture dish containing filter paper, and incubating at 22 ℃ for 96 hours;
(2) The detection method comprises the following steps: experimental group (OE)LbHSP90) Placing the culture dish in a climatic chamber (temperature 4 ℃,14h illumination/10 h darkness, luminous flux density 400 mu mol.m-2.s-1, relative humidity 55%); control group (WT) placing the dishes in a climatic chamber (temperature 24 ℃ C., 14h illumination/10 h darkness, luminous flux density 400. Mu. Mol.m-2.s-1, relative humidity 55%) and spraying distilled water into the dishes at regular intervals every day to maintain the humidity of the dishes, observing the phenotype and detecting the materials on days 0,2,4 and 6LbHSP90Expression level. As shown in FIG. 5, after the low temperature treatment, the test group (OE-LbHSP90) The discs were vivid in color compared to the control (WT), while the control (WT) discs clearly wilted and were not intact. The test results are shown in FIG. 6, and the experimental group was started from day 4LbHSP90The expression level is significantly higher than that of the control group.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (7)
1. Heat shock protein geneLbHSP90Use of said gene for modifying the low temperature resistance of cut lilyLbHSP90Cloning from lily, geneLbHSP90Has the nucleotide sequence shown in SEQ ID NO. 1, the nucleotide sequence shown in SEQ ID NO. 1 can code the amino acid sequence shown in SEQ ID NO. 2 by causing in lilyLbHSP90The gene is over-expressed to improve the low temperature stress resistance of the lily, and the gene improves the low temperature stress resistance of the lily and is verified by using plant arabidopsis thaliana or lily.
2. The heat shock protein gene according to claim 1LbHSP90The application of the lily bulb with cut flowers for changing the low temperature resistance is characterized in that: verification Using Pattern plant Arabidopsis, genes were generated by constructing an overexpression vector in Pattern plant Arabidopsis and transforming Pattern plant Arabidopsis by Agrobacterium-mediated flower dippingLbHSP90In the overexpression of Arabidopsis, the transgenic Arabidopsis with lower temperature resistance than wild Arabidopsis is obtained, and Arabidopsis is a model plant which can verify the general biological law, thereby obtaining the geneLbHSP90Can improve the low temperature stress resistance of lily.
3. The heat shock protein gene according to claim 2LbHSP90The application of the lily bulb with cut flowers for changing the low temperature resistance is characterized in that: the specific procedure for validation using the model plant arabidopsis was as follows:
(1) Constructing an over-expression vector: (1) by means ofLbHSP90Gene sequence design primerLbHSP90F andLbHSP90-R andLbHSP90f', PCR amplification Using the yellow Tianba cDNA as templateLbHSP90Gene, obtaining target fragmentLbHSP90A gene fragment; (2) the PC1300S-GFP vector was digested with KpnI and BamHI, recovered, and amplified by PCR in step (1)LbHSP90The gene fragment is recombined to obtain a recombined product, the recombined product is transformed into escherichia coli, then a resistance plate is coated, a clone and a shaking bacterium are selected, and then a primer A580-seqR is used for preparing a recombinant vectorLbHSP90The positive cloning verification is carried out on the F' colony through a PCR technology, and then the sequencing verification is carried out to obtain the overexpression vector of LbHSP 90;
(2) Arabidopsis seed disinfection treatment
Soaking wild Arabidopsis seeds in 70% ethanol for 2min, spin-washing with 15% NaClO solution for 10 min, and rinsing with sterile water for 5 times;
(3) Sowing and transplanting
Resuspension the sterilized seeds with sterile water, uniformly dispersing on the surface of a 1/2MS solid culture medium, inverting a culture dish, vernalizing for two days at 4 ℃ and culturing in dark at 25 ℃ for one day, and then culturing in a tissue culture chamber at 22 ℃ normally; when the seedling grows to four leaves, the seedling is grown to four leavesTransplanting into nutrient soil, placing in a constant temperature environment at 22deg.C, culturing in the dark for 8 hr under 16 hr, and culturing in 7.5ml of nutrient solution -1 Root irrigation, 7 days/times in seedling stage and 14 days/times in bud stage;
(4) Transformation of Arabidopsis plants with overexpression vectors
(1) Picking single colony containing over-expression vector and inoculating into 5mL LB liquid medium, and 200 rmp.min at 28 DEG C -1 Shake culturing overnight;
(2) transferring 2mL of bacterial liquid into 100 mL of LB culture medium, and transferring the bacterial liquid into 200 m.min at 28 DEG C -1 Shake culturing until OD600 value is 1.0-1.5;
③6000 rmp·min -1 centrifuging for 10 min, and discarding supernatant;
(4) 1mL of 5% sucrose solution was used to resuspend the bacteria, 6000 rmp.min -1 Centrifuging for 2min, and discarding supernatant;
(5) repeating (4);
(6) adding 5% sucrose solution to resuspend the thallus until OD600 value is 0.4-0.7;
(7) adding silwet77 surfactant in an amount of 0.05% by volume prior to soaking;
(8) winding water before soaking, cutting off pod, soaking bud in the above solution for 1 min;
(9) inverting the plant, culturing in the dark for 14 hours, taking out, and culturing in normal growth to obtain transgenic arabidopsis;
(5) Identification of transgenic Arabidopsis thaliana
Cutting transgenic arabidopsis plant leaves, extracting DNA by using a CTAB method, and performing PCR detection by using a screening marker gene specific primer; carrying out PCR positive detection on the LbHSP90 transformed plant by using a specific primer of the Hpt gene, wherein the positive plant is transgenic Arabidopsis;
(6) Sowing of transgenic Arabidopsis thaliana
Soaking the seeds of the transgenic arabidopsis thaliana in the step (5) in 70% ethanol for 2min, spin-washing the seeds in 15% NaClO solution for 10 min, and rinsing the seeds with sterile water for 5 times; uniformly dispersing on the surface of a 1/2MS solid culture medium, inverting a culture dish, vernalizing for two days at 4 ℃, performing normal dark culture at 25 ℃ for one day, and then placing in a tissue culture chamber at 22 ℃ for normal culture; transplanting the seedlings into nutrient soil when the seedlings grow to four leaves, and placing the seedlings in the nutrient soilCulturing in a constant temperature environment at 22deg.C for 16 hr under light for 8 hr, and culturing in 7.5mL of nutrient solution -1 Root irrigation, 7 days/times in seedling stage and 14 days/times in bud stage;
(7) The material treatment and detection method comprises the following steps: sowing transgenic and wild arabidopsis seeds respectively, culturing for 4 weeks, transferring to a greenhouse, observing the growth conditions of wild and transgenic plants, and recording; after the arabidopsis thaliana of 4 weeks of age is subjected to low-temperature stress treatment, the wild arabidopsis thaliana is obviously wilted after being subjected to low-temperature treatment, and the transgenic arabidopsis thaliana plant is slow in growth vigor but is bright in leaf color and weak in wilting condition; after room temperature is restored, part of wild plants cannot be restored, and transgenic plants can be restored, so that the over-expression plants have strong low-temperature stress resistance, and the LbHSP90 gene can participate in the low-temperature resistance process of the plants, and the low-temperature resistance performance of the plants is improved.
4. The heat shock protein gene according to claim 1LbHSP90The application of the lily bulb with cut flowers for changing the low temperature resistance is characterized in that: transformation of lily target discs by vacuum infestation and interference of endogenous lily target discsLbHSP90 The method of gene expression was verified.
5. A heat shock protein gene according to claim 3LbHSP90The application of the lily bulb with cut flowers for changing the low temperature resistance is characterized in that: the specific gene sequence of the primer in the step (1) is as follows:
primer: lbHSP90-F: GCTTTCGCGAGCTCGGTACCATGGCCGCCGCCGCCGACGAAGCCAAAG
LbHSP90-R: CGCCCTTGCTCACCATGGATCCTTTCATTTGGACTATCCCAGCAC
LbHSP90-F’: CTAGAAGGCCTCCATGGGGATCCGGCACAGCTCATTTCTTTATG。
6. A heat shock protein gene according to claim 3LbHSP90The application of the lily bulb with cut flowers for changing the low temperature resistance is characterized in that: primer A580-seqR was designed based on the PC1300S-GFP vector backbone, AGAAGATGGTGCGCTCCTG.
7. Root of Chinese characterA heat shock protein gene according to claim 3LbHSP90The application of the lily bulb with cut flowers for changing the low temperature resistance is characterized in that: step (4)HptSpecific primer specific sequence of gene is
hpt557-F: ACACTACATGGCGTGATTTCAT
hpt557-R: TCCACTATCGGCGAGTACTTCT。
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