Use of heat shock protein gene LbHSP17.9 in changing drought resistance of cut lily

Abstract

This invention discloses a heat shock protein gene. LbHSP17.9 The gene's application in altering the drought resistance of cut lilies. LbHSP17.9 Having the nucleotide sequence shown in SEQ ID NO:1, which encodes the amino acid sequence shown in SEQ ID NO:2, and enhancing [the effect] in lilies. LbHSP17.9 To increase gene expression levels and improve the drought resistance of cut lilies; to construct LbHSP17.9 Gene overexpression vectors, after being transformed into target discs via vacuum infection, can interfere with the endogenous expression of genes within the target discs. LbHSP17.9 Gene expression enables LbHSP17.9 Gene overexpression in lilies is involved in responding to drought stress. LbHSP17.9 No genes have been reported. This invention utilizes the gene in lilies... LbHSP17.9 Gene overexpression plays a key role in enhancing the drought resistance of lilies, and this invention provides a valuable gene resource for the cultivation of long-flowering lilies.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of a heat shock protein gene LbHSP17.9 in altering the drought resistance of cut lilies. Background Technology

[0002] Environmental stresses on plants include abiotic and biotic stresses. Over long periods of evolution, plants have developed a series of physiological and biochemical mechanisms to adapt to, suppress, or eliminate the effects of abiotic stresses, among which the synthesis of new stress proteins is the most common. Heat shock proteins (HSPs), also known as heat stress proteins, are a class of stress proteins produced intracellularly by plants under high temperature or other abiotic environmental stressors. These heat shock proteins are expressed not only when plants experience high temperature stress but also when responding to various other environmental stresses, such as water stress, salt stress, osmotic stress, cold stress, and oxidative stress.

[0003] Heat shock proteins can be classified into five families based on molecular weight: sHSPs (small heat shock proteins), HSP60, HSP70, HSP90, and HSP100. The monomeric molecular weight of sHSPs ranges from 15 to 42 kDa, containing a conserved α-crystalline protein domain (ACD) flanked by an N-terminal domain (NTD) and a C-terminal extended domain (CTD). Structural studies have shown that sHSPs exist as monomers, dimers, and large polymers. As molecular chaperones, sHSPs function in an ATP-independent manner, aiding in protein folding, preventing protein degradation by forming complexes with denatured proteins, and preventing irreversible protein aggregation. They play a crucial role in the defense of organisms during physiological stress and are also involved in the regulation of many biological processes, such as the cell cycle and plant development.

[0004] Lilies (Lilium spp.) are perennial herbaceous plants belonging to the genus Lilium in the family Liliaceae. They possess extremely high ornamental, edible, and medicinal value and are recognized worldwide as one of the five major cut flowers. Cut lilies are highly susceptible to dehydration and low temperatures during post-harvest and subsequent cold chain transportation, leading to flower deformities and premature fading, affecting flower quality and reducing their commercial value. Researching the tolerance of cut lilies to low temperatures and water loss stress has significant practical implications for cut lily breeding, production, and sales.

[0005] Although HSP has been studied in plants such as Arabidopsis, tobacco, grape, and tomato, research in lilies is scarce. Summary of the Invention

[0006] The purpose of this invention is to provide an application of the heat shock protein gene LbHSP17.9 in altering the drought resistance of cut lilies. The LbHSP17.9 gene plays a key role in the response of lilies to drought stress and can serve as a beneficial gene resource for breeding long-flowering lilies. Enhancing the expression level of the LbHSP17.9 gene in lilies can improve the drought resistance of cut lilies.

[0007] This invention provides an application of the heat shock protein gene LbHSP17.9 in altering the drought resistance of cut lilies. The nucleotide sequence of the gene LbHSP17.9 is shown in SEQ ID NO:1. The nucleotide sequence shown in SEQ ID NO:1 can encode the amino acid sequence shown in SEQ ID NO:2. Enhancing the expression level of the LbHSP17.9 gene in lilies can improve the drought resistance of cut lilies.

[0008] The specific steps are as follows:

[0009] Primers LbHSP17.9-F, LbHSP17.9-R, and LbHSP17.9-F' were designed using the LbHSP17.9 gene sequence. The LbHSP17.9 gene was amplified by PCR using Huang Tianba cDNA as a template to obtain the target LbHSP17.9 gene fragment. The primers were designed using Primer Premier 5 software.

[0010] Primer: LbHSP17.9-F:GCTTTCGCGAGCTCGGTACCATGTCGATCATCCGCAGGAGCAACGTCT

[0011] LbHSP17.9-R: CGCCCTTGCTCACCATGGATCCGCCAGAGATATCGATGGATTTG

[0012] LbHSP17.9-F': CTAGAAGGCCTCCATGGGGATCCCTAACACCCGCATCGACTGG.

[0013] (2) The PC1300S-GFP vector (a commercially available vector in the laboratory) was double-digested with KpnI (restriction endonuclease, experimental reagent) and BamHI (restriction endonuclease, experimental reagent) and recovered. It was then recombined with the LbHSP17.9 gene fragment of the PCR amplification product in step (1) to obtain the recombinant product. The recombinant product was transformed into Escherichia coli, and then plated on an antibiotic plate. Clones were selected and cultured. Then, primer A580-seqR and LbHSP17.9-F' colonies were verified by PCR technology to confirm the positive clones. Then, sequencing was performed to verify the bacterial culture expressing OE-LbHSP17.9. Primer A580-seqR was designed based on the PC1300S-GFP vector backbone, and the gene sequence was AGAGATGGTGCGCTCCTG.

[0014] (3) The bacterial culture expressing OE-LbHSP17.9 was resuspended in the infiltration buffer, which was: ① 10 mM MES and 10 mM MgCl solution were mixed and the pH was adjusted to 5.6; ② 200 M acetylsuccinone solution, MES was morpholine ethanesulfonic acid, and then placed at 22℃ in the dark for 5 hours to obtain the infection solution;

[0015] (4) The dye solution is transferred to cut lilies through vacuum dyeing to improve the drought resistance of cut lilies.

[0016] The gene LbHSP17.9 of the present invention has the nucleotide sequence shown in SEQ ID NO:1 and the gene is 468b in length.

[0017] (ATGCGATCATCCGCAGGAGCAACGTCTTCGATCCCTTCTCCCTCGACCTCTTTGATCCATTTGAGGGTTTCGGGTTCCCCTTCTTTGGTGGCCCCACCGCCCTCCCGCGCCGCCGGTCGTGCCGGTGAGACCTCCGCCTTCGCTAACACCCGCATCGACTGGAAGGAGACCCTGAGGCCCATCTCTTCAAGGCCGATCTGCCGGGGGTCAAAAAGGAGGAGGTCAAGGTCG AGGTGGAGGACGGCCGCGTTCTGAAAATTAGCGGCGAACGGAAGAAGGAGCAGGAGGAGAAGACCGACACCTGGCACCGAGTCGAGCGCAGCAGCGGTCATTTCCTGCGACGCTTCCAGCTGCCGGAGAACGCTAAGGTGGATCAGGTGAAGGCGGCAATGGAGAACGGCGTCCCTCACTGTCACTGTCCCCAAGGAGGAGGCCAAGAAGCCGGAGGTCAAATCCATCGATATGA)

[0018] The nucleotide sequence shown in SEQ ID NO:1 encodes the amino acid sequence shown in SEQ ID NO:2. This amino acid sequence contains 153 amino acids, theoretically giving the protein a relative molecular weight of 17.749 kDa and an isoelectric point of 6.34.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: the LbHSP17.9 gene involved in drought stress response in lilies has not been reported. The present invention plays a key role in improving the drought stress resistance of lilies by overexpressing the LbHSP17.9 gene in lilies. The present invention provides a favorable gene resource for the cultivation of lilies with strong stress resistance. Attached Figure Description

[0020] Figure 1 Agarose gel electrophoresis image of PCR amplification of the LbHSP17.9 gene;

[0021] Figure 2 Phenotypic changes of petal discs in cut lily flowers under drought stress;

[0022] Figure 3 The relative expression level of the LbHSP17.9 gene in the petal discs of cut lily flowers was detected by qRT-PCR in the CK group.

[0023] Figure 4The relative expression level of the LbHSP17.9 gene in the petal discs of cut lily flowers was detected by qRT-PCR in a 5% PEG-simulated drought group. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from the specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0026] Unless otherwise defined, 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 pertains, and all materials disclosed herein and cited by them are incorporated herein by reference.

[0027] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalent techniques for specific embodiments of the invention described herein. These equivalents will be included in the claims. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0028] Example 1: This example uses lilies from the Kunming Lily Greenhouse Base as the research material. The specific steps are as follows:

[0029] (1) Material processing and vacuum inoculation transformation: ① Lilies grown in Kunming Lily Greenhouse Base were used as inoculation materials. Lilies with green buds that were harvested within two hours, with long and straight flower stems and uniform growth, and flower buds about 9±1cm long were used as experimental materials. The lily flower stems were trimmed underwater with sterilized scissors. After keeping the overall length of the flower stems at 5±1cm, they were placed in distilled water for later use.

[0030] ② The bacterial culture expressing OE-LbHSP17.9 was resuspended in infiltration buffer and then placed at 22°C in the dark for 5 hours to obtain the infection solution; there are two types of infiltration buffer: ① 10 mM MES and 10 mM MgCl solution were mixed and the pH was adjusted to 5.6; ② 200 M acetylsyl syringone solution;

[0031] ③Take out the green bud stage lily prepared in step ①, and then use a punch to remove a 1cm diameter disc from the inner petals;

[0032] ④ The infection solution obtained in step ② was vacuum-permeable to 0.07 MPa and maintained for 5 minutes to transform the discs prepared in step ③, and then immersed under negative pressure; the immersed discs were then quickly washed with sterile water and placed in a petri dish containing filter paper and incubated at 22°C for 96 hours.

[0033] (2) Stress treatment: During the drought treatment, the culture dishes were placed in an artificial climate chamber (temperature 24℃, 14h light / 10h darkness, light flux density 400μmol·m-2·s-1, relative humidity 55%). A 5% PEG6000 solution was sprayed into the culture dishes at regular intervals every day to keep the discs in a dry state. The control group (WT) had its culture dishes placed in an artificial climate chamber (temperature 24℃, 14h light / 10h darkness, light flux density 400μmol·m-2·s-1, relative humidity 55%). Distilled water was sprayed into the culture dishes at regular intervals every day to keep the discs moist. All materials were collected on days 0, 2, 4, and 6 of the treatment.

[0034] The lily variety mentioned in step (1) is Huangtianba. The vacuum permeation method involves placing a beaker in a vacuum filter and placing the lily cut flower discs obtained in step ④ into a dyeing solution that can submerge the discs for dyeing.

[0035] like Figure 2 As shown, continuous observation of the petal discs in two groups with four treatments revealed that in the two treatments simulating drought with 5% PEG, the untreated wild-type control group and the experimental group inoculated with OE-LbHSP17.9 both showed that the petal discs began to shrivel around the second day, and the shrivelation worsened over time. In contrast, the untreated wild-type control group showed more severe water loss and wilting than the experimental group inoculated with OE-LbHSP17.9. Meanwhile, the petals in the OE-LbHSP17.9 inoculated group exhibited more vibrant color.

[0036] The expression level of the LbHSP17.9 gene in the above samples was detected by qRT-PCR. RNA was extracted after sampling and reverse transcribed to obtain cDNA. Using Lilyactin as an internal control, the relative expression level of the LbHSP17.9 gene under drought treatment was analyzed by RT-PCR, with the CK group (untreated lilies) as a control. The RNA extraction method is as follows:

[0037] (1) Total RNA was extracted from lily petal discs after drought treatment on days 0, 2, 4 and 6 for later use.

[0038] (2) The RNA extraction steps are as follows:

[0039] 1) Quickly transfer each tissue (approximately 0.2g / part) that has been cryogenically frozen to a mortar pre-cooled with liquid nitrogen, and grind the tissue with a pestle until it is ground into powder (with no visible particles);

[0040] 2) Then add 1 ml of TRIzol to completely cover the powdered sample. Let it stand at room temperature until it melts, then continue grinding until the lysis buffer is clear. Transfer the homogenate to a centrifuge tube and let it stand at room temperature for 5 minutes. Then centrifuge at 12000 rpm at 4°C for 5 minutes. Carefully aspirate the supernatant and transfer it to a new centrifuge tube.

[0041] 3) Add 200 μl of chloroform, tighten the sample cap, shake vigorously by hand for 15 seconds, and let stand at room temperature for 5 minutes; centrifuge at 4℃ and 120,000 r / min for 15 minutes;

[0042] 4) Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix by inverting the tube, and let stand at room temperature for 10 min; centrifuge at 4℃ and 12000 r / min for 10 min.

[0043] 5) Carefully discard the supernatant and keep the precipitate. Slowly add 1 ml of 75% ethanol (pre-cooled) along the wall of the centrifuge tube, centrifuge at 4°C and 12000 r / min for 5 min, and carefully discard the ethanol.

[0044] 6) Repeat step 5

[0045] 7) Dry at room temperature for 2-5 minutes, add 35 μl of RNase-free water to fully dissolve the RNA and obtain an RNA solution.

[0046] (3) First-strand cDNA was synthesized using total RNA as a template according to the instructions of the Thermo Scientific RevertAid First-Strand cDNA Synthesis Kit. The reaction system and operation process were as follows: 5 μl of total RNA was taken, and 1 μl of Oligo(dT) 18 Primer and 6 μl of Water were added in sequence. After mixing, the mixture was centrifuged for 25 s, heated in a metal bath at 65 ℃ for 8 min, and then rapidly cooled on ice for 1 min. Then, 4 μl of 5x Reaction Buffer, 1 μl of Ribolock RNase Inhibitor, 2 μl of 10 mMdNTPMix, and 1 μl of Revert Aid M-MulURT were added in sequence. After mixing, the mixture was centrifuged for 25 s, heated in a metal bath at 42 ℃ for 60 min, and then heated at 70 ℃ for 5 min to terminate the reaction. The synthesized first-strand cDNA was stored at -20 ℃ for later use.

[0047] (4) PCR amplification system: Table 1 Reagents and dosages used

[0048]

[0049] (5) Forward and reverse primers were designed using Primer Premier 5.0 software. The sequences were: LbHSP17.9-F: 5'-CTTTCAATCACGATGTCG-3', LbHSP17.9-R: 5'-AGAATGGAGCTTCAGCCA-3'. The PCR reaction program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 60℃ annealing for 30 s, 40 cycles, with 3 replicates per sample. The relative expression level was calculated using the 2-ΔΔCT method. The PCR products were analyzed by agarose gel electrophoresis. A specific amplification band was observed at approximately 400 bp. Figure 1 As shown, lane M is the DL 1000 DNA Marker; lane 1 is the LbHSP17.9 gene; purified according to the gel extraction kit (9672, Takara) for later use.

[0050] (6) Add A to the purified product fragment using the blunt-end A-addition reagent, and ligate it to pMD20-Tvector (6019, Takara) by TA cloning. Transform the ligation product into Escherichia coli DH5α. Select 2-3 positive clones from LB culture plates containing ampicillin (100 mg / L) for sequencing analysis. The results show that Huangtianba Lily LbHSP17.9 is as shown in SEQ ID NO.1, with a sequence length of 468 bp, encoding 153 amino acids. Theoretically, the relative molecular weight of the protein is 17.749 kDa, and the isoelectric point is 6.43.

[0051] (7) The LbHSP17.9 gene sequence is

[0052] (SEQ ID NO.1):ATGCGATCATCCGCAGGAGCAACGTCTTCGATCCCTTCTCCCTCGACCTCTTTGATCCATTTGAGGGTTTCGGGTTCCCCTTCTTTGGTGGCCCCACCGCCCTCTCCCGCGCCGCCGGTCGTGCCGGTGAGACCTCCGCCTTCGCTAACACCCGCATCGACTGGAAGGAGACCCCTGAGGCCCATCTCTTCAAGGCCGATCTGCCGGGGGTCAAAAAGGAGGAGGTCAAGGTCGAGGTGGAGGACGGCCGCGTTCTGAAAATTAGCGGCGAACGGAAGAAGGAGCAGGAGGAGAAGACCGAACCTGGCACCGAGTCGAGCGCAGCAGCGGTCATTTCCTGCGACGCTTCCAGCTGCCGGAGAACGCTAAGGTGGATCAGGTGAAGGCGGCAATGGAGAACGGCGTCCTCACTGTCACTGTCCCCAAGGAGGAGGCCAAGAAGCCGGAGGTCAAATCCATCGATATGA

[0053] (8) The amino acid sequence is

[0054] (SEQ ID NO:2):MRSSAGATSSIPSPSTSLIHLRVSGSPSLVAPPPSPAPPVVPVRPPPSLTPASTGRRPLRPISSRPICRGSKRRRSRSRWRTAAFKLAANGRRSRRRRPTPGTESSAAAVISCDASSCRRTLRWIRRRQWRTASSLSLSPRRRPRSRRSNPSI

[0055] The test results are as Figure 3 、 4As shown, analysis of the 5% PEG simulated drought treatment revealed no significant difference in LbHSP17.9 gene expression levels between the control and experimental groups before the second day. Starting on the second day, influenced by drought, LbHSP17.9 gene expression levels increased significantly in both groups, but the experimental group showed a significantly higher level than the control group. After the second day, LbHSP17.9 gene expression levels decreased significantly between the two groups, but the difference was highly significant. Analysis of the three treatments cultured in distilled water showed similar results to the 5% PEG simulated drought treatment. Before the second day, there was no significant difference in LbHSP17.9 gene expression levels between the experimental and control groups. However, on the second day, the LbHSP17.9 gene expression level in the experimental group was significantly higher than that in the control group. These results demonstrate that transient overexpression of the LbHSP17.9 gene enhances the stress resistance of lilies. This proves that this gene plays a crucial role in the drought stress resistance of lilies and provides a valuable gene resource for the cultivation of long-flowering lilies.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A heat shock protein gene LbHSP17.9 Its application in modifying the drought resistance of cut lilies is characterized by... The gene LbHSP17.9 The nucleotide sequence is shown in SEQ ID NO:

1. The nucleotide sequence shown in SEQ ID NO:1 encodes the amino acid sequence shown in SEQ ID NO:2, which enhances the effect in lilies. LbHSP17.9 To increase gene expression levels and improve the drought resistance of cut lilies.

2. The heat shock protein gene according to claim 1 LbHSP17.9 Its application in modifying the drought resistance of cut lilies is characterized by: The specific steps are as follows: (1) Utilization LbHSP17.9 Gene sequence design primers LbHSP17.9 -F and LbHSP17.9 -R and LbHSP17.9 -F', PCR amplification was performed using Huang Tianba cDNA as a template. LbHSP17.9 Genes, to obtain the target fragment LbHSP17.9 Gene fragments; (2) The PC1300S-GFP vector was digested with KpnI and BamHI and recovered, and then compared with the PCR amplification product from step (1). LbHSP17.9 Gene fragments were recombined to obtain recombinant products, which were then transformed into E. coli. Antibiotic plates were then plated, clones were selected, and the bacteria were shaken. Primers A580-seqR were then used... LbHSP17.9 -F' colonies were validated as positive clones using PCR, followed by sequencing to confirm the expression of OE-. LbHSP17.9 The bacterial culture, primer A580-seqR was designed based on the PC1300S-GFP vector backbone, and the gene sequence is AGAGATGGTGCGCTCCTG; (3) Expressing OE- LbHSP17.9 The bacterial culture was resuspended in the infiltration buffer and then placed at 22°C in the dark for 5 hours to obtain the infection solution; (4) The dye solution is transferred to cut lilies through vacuum dyeing to improve the drought resistance of cut lilies.

3. The application of the heat shock protein gene LbHSP17.9 according to claim 2 in altering the drought resistance of cut lilies, characterized in that: In step (1), primers were designed using the software Primer Premier 5.

4. The heat shock protein gene according to claim 2 LbHSP17.9 Its application in modifying the drought resistance of cut lilies is characterized by: The specific gene sequences of the primers in step (1) are as follows: Primer: LbHSP17.9 -F: GCTTTCGCGAGCTCGGTACCATGTCGATCATCCGCAGGAGCAACGTCT LbHSP17.9 -R: CGCCCTTGCTCACCATGGATCCGCCAGAGATATCGATGGATTTG LbHSP17.9 -F’: CTAGAAGGCCTCCATGGGGATCCCTAACACCCGCATCGACTGG。 5. The heat shock protein gene according to claim 2 LbHSP17.9 Its application in modifying the drought resistance of cut lilies is characterized by: The soaking buffer in step (3) is of two types: ① 10mM MES and 10mM MgCl2 mixed and then adjusted to pH 5.6; ② 200M acetylsuccinone solution.

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