A rose heat shock protein family gene RcHSP70-4 and its application in delaying plant flowering

Abstract

The present invention provides a rose heat shock protein family gene RcHSP70‑4, whose nucleotide sequence is shown in SEQ ID No.1, and whose amino acid sequence is shown in SEQ ID No.2. The present invention discloses the application of the rose heat shock protein family gene RcHSP70‑4 in delaying plant flowering, and the RcHSP70‑4 protein is localized in the cell nucleus, and overexpression in Arabidopsis can delay flowering. The present invention helps to understand the mechanism of plant flowering regulation and provides a theoretical basis and reference for genetic engineering breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of a rose family gene RcHSP70-4 gene and its encoded protein in delaying plant flowering. Background Art

[0002] Flowering is the hub of plant transformation from vegetative growth to reproductive growth. It plays a core role in plant growth and development and is mediated by complex genetic mechanisms. In recent years, the molecular mechanism of plant flowering induction has been continuously studied, revealing the molecular mechanisms of multiple flowering regulation pathways, such as photoperiod pathway, vernalization pathway, gibberellin pathway, temperature pathway, autonomous pathway, age pathway, etc. These signal pathways are both independent and interconnected, forming a complex regulatory network with coexistence of activation and inhibition, positive feedback and negative feedback.

[0003] Heat shock proteins (HSPs) are widely present in organisms. As molecular chaperones, they participate in protein folding, transport, degradation, and assembly of protein complexes. According to their structure, function, and molecular weight, heat shock proteins can be divided into six families: HSP20 (sHSP), HSP40 (DnaJ), HSP60, HSP70, HSP90, and HSP100. Heat shock proteins play an important role in plant responses to biotic and abiotic stresses. In addition, heat shock proteins also play an important role in the growth and development of plants. Many studies have shown that heat shock proteins can regulate plant flowering. In Arabidopsis, HSP90 interacts with key flowering regulators (LFY, SOC1, and AGL24), which is essential for the transition from the vegetative stage to the reproductive stage and the morphology of flowers. HSP101 can promote flowering under non-stress conditions, and this promotion process depends on the flowering-related genes FLC and SVP. Heat shock proteins play an important role in regulating plant flowering, but there are no reports on heat shock proteins in roses. Summary of the invention

[0004] The purpose of the present invention is to provide a rose heat shock protein family gene RcHSP70-4 gene and its application in delaying plant flowering.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] A rose flowering period regulating gene RcHSP70-4, whose nucleotide sequence is shown in SEQ ID No.1.

[0007] A protein encoded by a rose flowering period regulating gene RcHSP70-4, the amino acid sequence of which is shown as SEQ ID No.2.

[0008] The full-length CDS sequence of RcHSP70-4 was amplified using RcHSP70-4-F and RcHSP70-4-R primers, and RcHSP70-4 was constructed into the pCAMBIA1305-GFP vector by homologous recombination. The pCAMBIA1305-RcHSP70-4-GFP recombinant vector was transformed into Agrobacterium GV3101 and transiently transformed into Nicotiana benthamiana leaves. It was found that the RcHSP70-4 protein was localized in the cell nucleus. RcHSP70-4 was constructed into the pCAMBIA2300-35S vector by homologous recombination, and the recombinant vector pCAMBIA2300-35S-RcHSP70-4 was transformed into Agrobacterium GV3101. RcHSP70-4 was transformed into Arabidopsis thaliana by Agrobacterium-mediated floral organ infection. PCR identification was performed using DNA as a template to determine the positive plants. Through the phenotype observation of RcHSP70-4 transgenic Arabidopsis thaliana, it was found that the introduction of RcHSP70-4 significantly delayed the flowering period of Arabidopsis thaliana.

[0009] Beneficial Effects

[0010] The invention discloses an application of a rose heat shock protein family gene RcHSP70-4 in regulating the flowering period of plants, which is helpful to understand the flowering regulation mechanism of plants and provides a theoretical basis and reference for genetic engineering breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the PCR amplification diagram of RcHSP70-4 gene;

[0012] Figure 2 This is the subcellular localization map of RcHSP70-4 protein in Nicotiana benthamiana;

[0013] Figure 3 PCR identification of RcHSP70-4 transgenic Arabidopsis;

[0014] Figure 4 This is the phenotype of RcHSP70-4 transgenic Arabidopsis;

[0015] Figure 5 The flowering time statistics of RcHSP70-4 transgenic Arabidopsis;

[0016] In the figure: Bright field: bright field photo; GFP: green fluorescent protein; Merged: bright field and GFP superposition. DETAILED DESCRIPTION

[0017] The present invention will be further described in detail and completely below with reference to specific embodiments and drawings, but the present invention is not limited thereto.

[0018] Example 1 Cloning of RcHSP70-4 gene

[0019] 1. Extraction of total RNA from plants

[0020] Total RNA was extracted using a polysaccharide and polyphenol plant total RNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd.), and the specific steps were carried out according to the instructions.

[0021] 2. cDNA Synthesis

[0022] The first strand of cDNA was synthesized using the reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novogene Biotech Co., Ltd.), and the specific steps were carried out according to the instructions.

[0023] 3. Gene cloning

[0024] Specific primers for RcHSP70-4 gene cloning were designed using Snapgene software. The primer sequences are as follows:

[0025] RcHSP70-4-F: 5'-ATGGCCGGAAAAGGAGAG-3'

[0026] RcHSP70-4-R: 5'-TTAATCGACTTCTTCAATCTTGGGACC-3'

[0027] The full-length CDS sequence of the RcHSP70-4 gene was cloned using the high-fidelity PCR enzyme KOD FX (TOYOBO). A 50 μL reaction system was prepared for PCR amplification, including 25 μL 2x PCR buffer for KOD FX, 10 μL 2 mM dNTPs, 1.5 μL each of the primers RcHSP70-4-F and RcHSP70-4-R, 2 μL template cDNA, and 10 μL ddH 2 O. The reaction conditions were: 94°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 68°C extension for 2 min, 40 cycles (denaturation-extension). The PCR products were detected by 1% agarose gel electrophoresis ( Figure 1), the target gene band was cut out, and the target gene band was purified using FastPure Gel DNA Extraction Mini Kit (Nanjing Novozyme Biotechnology Co., Ltd.). The purified DNA fragment was connected to the cloning vector pTOPO-TA / Blunt Simple Vector (Beijing Polymer Biotechnology Co., Ltd.), and transformed into Fast-T1 Escherichia coli competent cells (Nanjing Novozyme Biotechnology Co., Ltd.), and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. Sequencing showed that the CDS region of the RcHSP70-4 gene contained 1950 nucleotides and encoded 649 amino acids.

[0028] The nucleotide sequence of the RcHSP70-4 gene is as follows:

[0029] ATGGCCGGAAAAGGAGAGGGACCAGCGATCGGGATTGACCTCGGC

[0030] ACCACCTACTCTTGCGTTGGAGTGTGGCAGCACGATCGTGTCGAGATCA

[0031] TCGCCAACGACCAGGGCAACAGAACCACGCCGTCTTATTGTTGCCTTTAC

[0032] TGATACTGAGCGTTTGATCGGGTGACGCGGCCAAGAACCAGGTCGCCATG

[0033] AACCCCGTCAACACCGTCTTCGATGCAAAGCGGTTGATTGGTAGAAGGT

[0034] TCAGTGACCCAACTGTTCAGGGTGATATTAGGCACTGGCCCTTCAAGGT

[0035] CATTGCAGGTCCTGCTGACAAGCCCATGATTGTGGTTTCATACAGAGGT

[0036] GAAGAGAAACAATTTGCTGCTGAGGAGATCTCGTCTATGGTTCTCATCA

[0037] AGATGCGGGAGATTGCAGAGGCCTATCTCGGCTCAACGGTGAAAAATGC

[0038] AGTTGTACTGTGCCTGCTTACTTCCAATGACTCGCAGCGTCAGGCTACGA

[0039] AGGATGCTGGAGTCATTGCTGGTCTTAATGTGATGCGTATTATCAATGAG

[0040] CCCACTGCTGCAGCCATAGCTTATGGTCTCGACAAGAAGGCAACCAGTG

[0041] TTGGAGAGAGAATGTCTTGATTTTCGATCTTGGTGGAGGAACTTTTGAT

[0042] GTCTCTCTACTTACAATTGAAGAGGGTATCTTTGAAGTCAAAGCTACTGC

[0043] TGGTGATACTCATTGGGAGGAGAGGATTTTGATAACAGGATGGTGAATC

[0044] ACTTTGTTCAAGAGTTCAAGAGGAAGCACAAGAAGGATATTAGTGGAAA

[0045] TCCAAGAGCTCTTAGGAGATTGAGAACAGCTTGTGAGAGGGCGAAGAG

[0046] GACTCTCTCGTCCACTGCTCAGACAACCATTGAGATTGACTCTTTGTATG

[0047] AGGGTGTTGATTTCTATTCATCTATCACCCGTGCAAGATTTGAAGAGCTG

[0048] AACATGGATTTATTTAGGAAGTGCATGGAGCCGGTTGAGAAGTGTTTGA

[0049] GAGATGCAGAGATGGACAAGAGCAGTGTCCATGATGTTGTGCTTGTGGG

[0050] TGGTTCTACTAGGATTCCAAAGGTGCAACAATTGCTGCAAGACTTCTTC

[0051] AATGGAAAGGAGTTGTGCAAAAGCATCAACCCGGATGAAGCTGTCGCTT

[0052] ATGGTGCTGCGGTGCAAGCTGCTATTTTGAGTGGTGAGGGCAATGAGAA

[0053] GGTGCAGGACCTATTGCTCCTAGATGTCACTCCTCTGTCCTTGGGATTGG

[0054] AGACTGCTGGAGGTGTTATGACAGTGTTGATCCCAAGGAATACTACCGT

[0055] TCCGACTAAGAAGGAGCAGGTGTTTTCTACATATTCAGACAACCAACCT

[0056] GGTGTCTTGATTCAGGTTTTTGAGGGTGAGAGGACGAGAACCCGCGATA

[0057] ACAACTTGTTGGGCAAATTTGAGCTCTCTGGCATTCCTCCAGCTCCAAG

[0058] GGGTGTGCCCCAGATTACTGTTTGCTTCGACATTGATGCCAATGGCATCC

[0059] TCAATGTCTCTGCGGAGGACAAGACCACTGGACAGAAGAATAAGATTAC

[0060] CATCACAAACGACAAGGGAAGACTATCCAAGGAGGATATCGAGAAGAT

[0061] GGTACAGGAGGCTGAGAAATACAAGTCGGAAGATGAGGAGCACAAGAA

[0062] GAAGGTGGAGTCCAAGAATGCTCTGGAGAATTACGCCTACAACATGCGG

[0063] AACACAATTAAAGATGACAAGATTTCTTCAAAGCTTGATGCAGCCGACA

[0064] AGAAGAAGATAGAGGATGCAATTGAACAGGCAATTCAATGGCTTGATGG

[0065] CAACCAACTTGCAGAGGCAGAGGAGTATGATGATAAGGTGAAGGAGCT

[0066] TGAAAGCATTTGCAACCCGATCATTGCCAAGATGTACCAGGGTGGTGCT

[0067] GCTCCTGACATGACTGGCGGCATGGATGAAGACGTTCCTCCTGCTGGTG

[0068] GCAGTGGTGCTGGTCCCAAGATTGAAGAAGTCGATTAA

[0069] The amino acid sequence encoded by the RcHSP70-4 gene is as follows:

[0070] MAGKGEGPAIGIDLGTTYSCVGVWQHDRVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVAMNPVNTVFDAKRLIGRRFSDPTVQGDIRHWPFKVIAGPADKPMIVVSYRGEEKQFAAEEISSMVLIKMREIAEAYLGSTVKNAVVTVPAYFNDSQRQAT KDAGVIAGLNVMRIINEPTAAAIAYGLDKKATSVGEKNVLIFDLGGGTFDVSLLTIEEGIFEVKATAGDTHLGGEDFDNRMVNHFVQEFKRKHKKDISGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLYEGVDFYSSITRARFEELNMDLFRKCMEPVE KCLRDAEMDKSSVHDVVLVGGSTRIPKVQQLLQDFFNGKELCKSINPDEAVAYGAAVQAAILSGEGNEKVQDLLLLDVTPLSLGLETAGGVMTVLIPRNTTVPTKKEQVFSTYSDNQPGVLIQVFEGERTRTRDNNLLGKFELSGIPPAPRGVPQITVCFDI DANGILNVSAEDKTTGQKNKITITNDKGRLSKEDIEKMVQEAEKYKSEDEEHKKKVESKNALENYAYNMRNTIKDDKISSKLDAADKKKIEDAIEQAIQWLDGNQLAEAEEYDDKVKELESICNPIIAKMYQGGAAPDMTGGMDEDVPPAGGSGAGPKIEEVD

[0071] Example 2 Subcellular localization analysis

[0072] 1. Construction of GFP fusion expression vector

[0073] (1) Preparation of linearized vector:

[0074] The pCAMBIA1305-GFP vector was double-digested with restriction endonucleases XbaI and BamHI (NEB), and the linearized vector was recovered.

[0075] (2) Insert the fragment to obtain:

[0076] Design homology arm primers for pCAMBIA1305 vector with XbaI and BamHI restriction sites. The primer sequences are:

[0077] pCAMBIA1305-RcHSP70-4-F:

[0078] 5'-aagtccggagctagctctagaATGGCCGGAAAAGGAGAGG-3'

[0079] pCAMBIA1305-RcHSP70-4-R:

[0080] 5'-gcccttgctcaccatggatccATCGACTTCTTCAATCTTGGGACC-3'

[0081] The RcHSP70-4 plasmid cloned in Example 1 was used as a template, and high-fidelity PCR enzyme KOD FX (TOYOBO) was used for PCR cloning, and the homologous sequence of the pCAMBIA1305-GFP vector was added to RcHSP70-4. After the PCR was completed, the product was purified. The PCR reaction system, reaction procedure, and product DNA purification were the same as in Example 1.

[0082] (2) Homologous recombination reaction:

[0083] The linearized vector and the inserted fragment were subjected to homologous recombination reaction using ClonExpress IIOne Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.) to obtain the pCAMBIA1305-RcHSP70-4-GFP recombinant plasmid, which was then transformed into Fast-T1 competent Escherichia coli (Nanjing Novogene Biotech Co., Ltd.). After cultivation, the single clone was identified by bacterial liquid PCR and sequenced, and the recombinant plasmid was extracted using FastPure Plasmid Mini Kit (Nanjing Novogene Biotech Co., Ltd.).

[0084] 2. Subcellular localization observation

[0085] The pCAMBIA1305-RcHSP70-4-GFP recombinant plasmid and pCAMBIA1305-GFP plasmid were respectively transferred into Agrobacterium GV3101, and the positive single colonies were picked and cultured overnight in YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif. The next day, 100 μL of the bacterial solution was inoculated into 10 mL YEP liquid medium containing 50 mg / L Kana, 25 mg / L Rif, 10 mM MES and 40 μM AS, and the bacteria were shaken until the OD 600 At about 1.0, collect the cells and use 10 mM MgCl 2 Resuspend to OD 600The concentration of Agrobacterium tumefaciens was 1.0, 200 μM AS was added, and the mixture was allowed to stand in the dark for 3 hours. The two Agrobacterium species were injected into tobacco leaves, cultured in the dark for 24 hours, and cultured under normal conditions for 2-3 days. The lower epidermis of the tobacco leaves was torn off, and the fluorescent part of the GFP fusion protein was observed using a confocal microscope and photographed and recorded. Figure 2 , the fluorescence signal of RcHSP70-4-GFP protein is mainly concentrated in the cell nucleus.

[0086] Example 3 Genetic transformation of RcHSP70-4 into Arabidopsis thaliana and phenotypic analysis

[0087] 1. Expression vector construction

[0088] (1) Preparation of linearized vector:

[0089] The pCAMBIA2300-35S vector was double-digested with restriction endonucleases SacI and XbaI (NEB), and the linearized vector was recovered.

[0090] (2) Insert the fragment to obtain:

[0091] Design homology arm primers of pCAMBIA2300-35S vector with SacI and XbaI restriction sites. The primer sequences are:

[0092] pCAMBIA2300-RcHSP70-4-F:

[0093] 5'-gagaacacgggggacgagctcATGGCCGGAAAAGGAGAGG-3'

[0094] pCAMBIA2300-RcHSP70-4-R:

[0095] 5'-tgcctgcaggtcgactctagaTTAATCGACTTCTTCAATCTTGGGA-3'

[0096] The RcHSP70-4 plasmid cloned in Example 1 was used as a template, and high-fidelity PCR enzyme KOD FX (TOYOBO) was used for PCR cloning, and the homologous sequence of the pCAMBIA2300-35S vector was added to RcHSP70-4. After the PCR was completed, the product was purified. The PCR reaction system, reaction procedure, and product DNA purification were the same as in Example 1.

[0097] (2) Homologous recombination reaction:

[0098] The method steps are the same as those of the vector construction part in Example 2.

[0099] 2. Arabidopsis genetic transformation

[0100] The pCAMBIA2300-35S-RcHSP70-4 recombinant plasmid and the pCAMBIA2300-35S plasmid were transferred into Agrobacterium GV3101, and the positive single colonies were picked and cultured overnight in YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif. The next day, 100 μL of the bacterial solution was inoculated into 5 mL of YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif for overnight shaking culture. 5 mL of the overnight cultured bacterial solution was added to 200 mL of YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif, and the Agrobacterium was cultured at 28°C and 200 rpm until the OD 600 Collect the cells and resuspend them in 5% sucrose to adjust the OD 600 The concentration of Silwet L-77 was between 0.8 and 1.0, and 0.03% of Silwet L-77 was added. The Arabidopsis inflorescence was immersed in the infection solution for 30 seconds, and the Arabidopsis was cultured in the dark for 24 hours after the bacterial solution was wiped dry.

[0101] 3. Screening of positive transgenic Arabidopsis

[0102] After the infected Arabidopsis plant seeds matured, they were T0 generation seeds. The seeds were sown on 1 / 2MS medium containing 50 μg / ml Kan to select positive transgenic seedlings, and the DNA of positive Arabidopsis plants was extracted and PCR identification was performed using RcHSP70-4-F and RcHSP70-4-R primers, such as Figure 3 , through PCR identification, RcHSP70-4 positive transgenic Arabidopsis plants were successfully obtained. Through qRT-PCR analysis, three strains with higher expression levels (OE-1, OE-2, OE-3) were selected for further observation. Repeat the above steps until the T3 generation is screened. OE-1, OE-2, OE-3 and pCAMBIA2300-35S empty-loaded Arabidopsis (EV) were sown at the same time and cultured in the same environment. The culture environment was an artificial climate chamber with a temperature of 25°C and a photoperiod of 16h / 8h. Figure 4 and Figure 5 As shown, the flowering time of EV is 27.7 days, while the earliest flowering time of RcHSP70-4 transgenic Arabidopsis is 36.3 days. The introduction of RcHSP70-4 significantly delays the flowering time of Arabidopsis.

Claims

1. A rose heat shock protein family gene RcHSP70-4 The use of the invention in delaying the flowering of Arabidopsis thaliana is characterized in that Rose heat shock protein family genes RcHSP70-4 The nucleotide sequence is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: Rose heat shock protein family genes RcHSP70-4 The construct was incorporated into a plant overexpression vector and genetically transformed into Arabidopsis thaliana to delay flowering.

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