Rosa chinensis heat shock protein family gene RcHSP70-8 and application thereof in delaying plant flowering

Abstract

The invention provides a Chinese rose heat shock protein family gene RcHSP70-8, the nucleotide sequence of the Chinese rose heat shock protein family gene RcHSP70-8 is shown as SEQ ID No. 1, and the amino acid sequence of the Chinese rose heat shock protein family gene RcHSP70-8 is shown as SEQ ID No. 2. The invention discloses an application of the Chinese rose heat shock protein family gene RcHSP70-8 in delaying flowering of plants, and the RcHSP70-8 protein is positioned in a cell nucleus and can delay flowering through overexpression in arabidopsis thaliana. The invention is helpful for enriching the research on the flowering regulation and control mechanism of the Chinese rose, and has important significance for genetic engineering breeding.

Description

Technical Field The present invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of the rose family gene RcHSP70-8 gene and its encoded protein in delaying plant flowering. Background Art Rose, a perennial woody plant belonging to the genus Rosa in the Rosaceae family, is ranked first among the world's four major cut flowers and one of my country's ten traditional famous flowers. Known as the "Queen of Flowers," it possesses both high commercial value and unique cultural connotations. Roses have three flowering phenotypes: once-flowering (OF), continuous flowering (CF), and occasional re-blooming (OR). Continuous-flowering roses do not require vernalization, requiring minimal nutrient accumulation. Under favorable environmental conditions, they continuously complete the floral transition and bloom continuously. This continuous flowering habit of roses makes them an excellent candidate for studying the mechanisms regulating flowering. Exploring the genetic and molecular mechanisms of rose flowering holds important implications for the targeted regulation of rose quality traits and molecular breeding. Heat shock proteins (HSPs) are a highly conserved class of proteins found in a wide variety of organisms. In plants, they are primarily involved in stress tolerance. Studies on the expression patterns of HSP70 in heat-tolerant and heat-intolerant rose varieties revealed low or no HSP70 expression in heat-intolerant rose varieties, while HSP70 was highly expressed in heat-tolerant varieties. This suggests that this gene can be induced by high temperatures and enhance heat tolerance in rose varieties. Research on heat shock proteins has also revealed their involvement in regulating flowering in plants, but studies investigating the regulation of rose flowering by heat shock proteins have yet to be reported. Summary of the Invention The purpose of the present invention is to provide a rose heat shock protein family gene RcHSP70-8 gene and its application in delaying plant flowering. The object of the present invention is achieved according to the following technical solutions: A rose flowering period regulatory gene RcHSP70-8, the nucleotide sequence of which is shown in SEQ ID No.1. A protein encoded by the rose flowering period regulating gene RcHSP70-8, the amino acid sequence of which is shown in SEQ ID No. 2. The full-length CDS sequence of RcHSP70-8 was amplified using RcHSP70-8-F and RcHSP70-8-R primers, and RcHSP70-8 was constructed into the pCAMBIA1305-GFP vector by homologous recombination. The pCAMBIA1305-RcHSP70-8-GFP recombinant vector was transformed into Agrobacterium GV3101 and transiently transformed into Nicotiana benthamiana leaves. It was found that the RcHSP70-8 protein was localized in the cell nucleus. RcHSP70-8 was constructed into the pCAMBIA2300-35S vector by homologous recombination, and the recombinant vector pCAMBIA2300-35S-RcHSP70-8 was transformed into Agrobacterium GV3101. RcHSP70-8 was transformed into Arabidopsis thaliana by Agrobacterium-mediated floral organ infection. PCR identification was performed using DNA as a template to determine positive plants. Through observation of the phenotype of RcHSP70-8 transgenic Arabidopsis thaliana, it was found that the introduction of RcHSP70-8 significantly delayed the flowering period of Arabidopsis thaliana. Beneficial effects The present invention discloses an application of a rose heat shock protein family gene RcHSP70-8 in regulating the flowering period of plants, which helps to enrich the research on the rose flowering regulation mechanism and is of great significance for genetic engineering breeding. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the PCR amplification diagram of RcHSP70-8 gene; Figure 2 This is the subcellular localization map of RcHSP70-8 protein in Nicotiana benthamiana; Figure 3 PCR identification of RcHSP70-8 transgenic Arabidopsis; Figure 4 The phenotype of RcHSP70-8 transgenic Arabidopsis thaliana is shown; Figure 5 Flowering time statistics of RcHSP70-8 transgenic Arabidopsis thaliana. In the figure: Bright field: bright field photo; GFP: green fluorescent protein; Merged: bright field and GFP superposition. DETAILED DESCRIPTION The present invention will be further fully described below in conjunction with specific embodiments and drawings, but will not be limited thereto. Example 1 Cloning of RcHSP70-8 gene 1. Extraction of plant total RNA Total RNA was extracted using a polysaccharide and polyphenol plant total RNA extraction kit (Nanjing Novozymes Biotechnology Co., Ltd.), and the specific steps were carried out according to the manufacturer's instructions. 2. cDNA Synthesis The first strand of cDNA was synthesized using the reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozymes Biotechnology Co., Ltd.), and the specific steps were carried out according to the manufacturer's instructions. 3. Gene Cloning Specific primers for RcHSP70-8 gene cloning were designed using Snapgene software. The primer sequences are as follows: RcHSP70-8-F: 5'-ATGGCTGAGGCAGCATACA-3' RcHSP70-8-R: 5'-CTAGATCCTCTTCACTGATACCAAGTC-3' The full-length CDS sequence of the RcHSP70-8 gene was cloned using the high-fidelity PCR enzyme KOD FX (TOYOBO). PCR amplification was performed in a 50 μL reaction system containing 25 μL 2x PCR buffer for KOD FX, 10 μL 2 mM dNTPs, 1.5 μL each of primers RcHSP70-8-F and RcHSP70-8-R, 2 μL template cDNA, and 10 μL ddH2O. Reaction conditions were: 94°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, and 68°C extension for 2 min, for 40 cycles (denaturation-extension). PCR products were detected by 1% agarose gel electrophoresis ( Figure 1 ), the target gene band was excised and purified using the FastPure Gel DNA Extraction Mini Kit (Nanjing Novozymes Biotech Co., Ltd.). The purified DNA fragment was ligated into the pTOPO-TA / Blunt Simple Vector cloning vector (Beijing Polymer Biotech Co., Ltd.), transformed into Fast-T1 competent Escherichia coli (Nanjing Novozymes Biotech Co., Ltd.), and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. Sequencing revealed that the CDS region of the RcHSP70-8 gene contains 1716 nucleotides, encoding 571 amino acids. The nucleotide sequence of the RcHSP70-8 gene is as follows: ATGGCTGAGGCAGCATACACAGTGGCATCTGATAGTGAAACCAGTC GGGAGGAGAAATCCTCATGCACCTTTCCTGAAACAACAATTGGAATTGA CATTGGCACTTCACAATGCAGGGTTGCGATCTGGAATGGCTCCCAGGTT GAGATCCTTAAAAACAGTAGGAACCAGAAGATGATGAGTTCATATGTTA CATTCAAGGATGGAACTCCTTCTGGGGAACTAAGCAATCAACTCTCTGC AGATCATGATATGTTATCCGGAGCAGCAATCTTCAACATGAAACGCTTAA TTGGCCGAGTTGACACGGATCCAGTTGTCCACTCTAGCAAAAGCCTTCC ATTTCTAGTACAAACATTGGACATTGGTGTCCGCCCATTTATTGCAGCCTT GGTTGGAAATTTCTGGAGATCCACCACTCCTGAAGAAGTTCTGGCAATA TTTTTGGTGGAACTTAGGGAAATGGCAGAAATACAGTTAAAGCGGCCCA TAAAAAATGTTGTTCTGACTCTGCCAGTTTCATTCAGCCGATTCCAGCTG ACTCGGATTGAGAGAGCATGTGCCATGGCTGGCCTTCATATCCTGAAACT GATGCCAGAGCCAACTGCAGTGGCATTGTTATATACCTTATATGCAAAAC AAAGTGGATTTCAGTCAACAGATAGGAATGTTCTAATCTTCAATATGGGT GCTGGATACTGTGATGTCGCCGTAAGTGTTACAGCTGAAGAGGCACCTC GAGGAATTTCTGAAATAATTGCCTTAACAGGAAGTGCCATTGGAGGAGA AGACTTGCTTCAGAATATGATGCGTCATCTGTTGCCAAATGCAGAGAATC TTTTGACAAGTCATGGAGTTGATGAAATCAAATCAAAGGGGTTGCTTCG AGTTGCGACCCAGAATGCAATCCACAGCCTCTCCTCCCAAAATTCTGTT CAAATTGATGTTGACTTGGGAAATGGGATGAAAATCTGCAAGGTTGTGG ACCGAGAAGAATTTGAGGAAGTTAATCGGGCCGTATTTGATGAGTGTGC TAGCCTCATAATCCAGTGTTTGCATGATGCAAAGGTCAAACCTGAGGATT TGAACGATGTAGTACTTGTAGGTGGATGTTCATACATTCCAAAGATAAAA AATCTTGTCATGAACGTATGCAAGAAAGAGGAGCTTTACGAAGGGATGA ACCCATTGGAAGCTGCTGTCTCTGGAGCAGCACTACAAGGATACGTAGC TTCTGGTGTTCATGATCCTTTTGAATCCGTGGATGTGCTATCTATTCAGGT CACACCTCTCTCAGTTGGAATTCGAGCTGACGGAAACAACTTTGTGCCT ATCATACCTCGAAACACCACAATACCAGCCCAAAGAGAGATGCTTTTCA CAACATCCCATGATAATCAAGCCGAAGCACTTGTAATTGTTTATGAAGGT GATGGCGAGAAGCTGGAAGAAAATCATTTGCTGGGATACTTCAAAATTG AGGGACTTCCTCCAGCACCCAAAGGAGTTCCACAAGTACGAGTAATGAT GGACATTGATGCTTCAAACGTGCTGAGAGTTTTGGCTGCTGTGTTAATGC CAGGTTCTCATGTTCCCATAAATCCTGTCCTGGACGTGAGGATGCCAACG GTTGACGATGGTCATGGCTGGTGCGCGGAAGCGTTGCACAGAGCTTATG GTGCTACTCAAGACTTGGTATCAGTGAAGAGGATCTAG The amino acid sequence encoded by the RcHSP70-8 gene is as follows: MAEAAYTVASDSETSREEKSSCTFPETTIGIDIGTSQCRVAIWNGSQVEILKNSRNQKMMSSYVTFKDGTPSGELSNQLSADHDMLSGAAIFNMKRLIGRVDTDPVVHSSKSLPFLVQTLDIGVRPFIAALVGNFWRSTTPE EVLAIFLVELREMAEIQLKRPIKNVVLTLPVSFSRFQLTRIERACAMAGLHILKLMPEPTAVALLYTLYAKQSGFQSTDRNVLIFNMGAGYCDVAVSVTAEEAPRGISEIIALTGSAIGGEDLLQNMMRHLLPNAENLLTSHG VDEIKSKGLLRVATQNAIHSLSSQNSVQIDVDLGNGMKICKVVDREEFEEVNRAVFDECASLIIQCLHDAKVKPEDLNDVVLVGGCSYIPKIKNLVMNVCKKEELYEGMNPLEAAVSGAALQGYVASGVHDPFESVDVLSIQV TPLSVGIRADGNNFVPIIPRNTTIPAQREMLFTTSHDNQAEALVIVYEGDGEKLEENHLLGYFKIEGLPPAPKGVPQVRVMMDIDASNVLRVLAAVLMPGSHVPINPVLDVRMPTVDDGHGWCAEALHRAYGATQDLVSVKRI Example 2 Subcellular localization analysis 1. Construction of GFP fusion expression vector (1) Preparation of linearized vector: The pCAMBIA1305-GFP vector was double-digested with restriction endonucleases XbaI and BamHI (NEB), and the linearized vector was recovered. (2) Insertion fragments to obtain: Design homology arm primers for the pCAMBIA1305 vector with XbaI and BamHI restriction sites. The primer sequences are: pCAMBIA1305-RcHSP70-8-F: 5'-gcccttgctcaccatggatccATGGCTGAGGCAGCATACACA-3'

[0038] pCAMBIA1305-RcHSP70-8-R: 5'-aagtccggagctagctctagaGATCCTCTTCACTGATACCAAGTCTTG-3' Using the RcHSP70-8 plasmid cloned in Example 1 as a template, PCR cloning was performed using the high-fidelity PCR enzyme KOD FX (TOYOBO). Homologous sequences from the pCAMBIA1305-GFP vector were added to RcHSP70-8. After completion of PCR, the product was purified. The PCR reaction system, reaction procedure, and product DNA purification were the same as in Example 1. (2) Homologous recombination reaction: The linearized vector and insert fragment were homologously recombined using the ClonExpress II One Step Cloning Kit (Nanjing Novozymes Biotech Co., Ltd.) to obtain the pCAMBIA1305-RcHSP70-8-GFP recombinant plasmid, which was then transformed into Fast-T1 competent Escherichia coli (Nanjing Novozymes Biotech Co., Ltd.). After culture, the single clones were identified by bacterial liquid PCR and sequencing, and the recombinant plasmid was extracted using the FastPure Plasmid Mini Kit (Nanjing Novozymes Biotech Co., Ltd.). 2. Subcellular Localization Observation The pCAMBIA1305-RcHSP70-8-GFP recombinant plasmid and the pCAMBIA1305-GFP plasmid were transformed into Agrobacterium GV3101, and 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 to OD 600 At around 1.0, collect the cells and resuspend them in 10 mM MgCl2 to an OD 600 The concentration of Agrobacterium 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. Figure 2 , the fluorescence signal of RcHSP70-8-GFP protein is mainly concentrated in the cell nucleus. Example 3 Genetic transformation of RcHSP70-8 into Arabidopsis thaliana and phenotypic analysis 1. Expression vector construction (1) Preparation of linearized vector: The pCAMBIA2300-35S vector was double-digested with restriction endonucleases SacI and XbaI (NEB), and the linearized vector was recovered. (2) Insertion fragments to obtain: Design homology arm primers of the pCAMBIA2300-35S vector with SacI and XbaI restriction sites. The primer sequences are:

[0050] pCAMBIA2300-RcHSP70-8-F: 5'-gagaacacgggggacgagctcATGGCTGAGGCAGCATACACA-3' pCAMBIA2300-RcHSP70-8-R: 5'-tgcctgcaggtcgactctagaCTAGATCCTCTTCACTGATACCAAGTCT-3' Using the RcHSP70-8 plasmid cloned in Example 1 as a template, PCR cloning was performed using the high-fidelity PCR enzyme KOD FX (TOYOBO). Homologous sequences from the pCAMBIA2300-35S vector were added to RcHSP70-8. After completion of PCR, the product was purified. The PCR reaction system, reaction procedure, and product DNA purification were the same as in Example 1. (2) Homologous recombination reaction: The method steps are the same as those for the vector construction in Example 2. 2. Arabidopsis genetic transformation The pCAMBIA2300-35S-RcHSP70-8 recombinant plasmid and the pCAMBIA2300-35S plasmid were transformed into Agrobacterium GV3101, respectively. 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 and cultured overnight with shaking. 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 Between 0.8 and 1.0, collect the cells and resuspend them in 5% sucrose and 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 solution was wiped dry and the Arabidopsis was cultured in the dark for 24 hours. 3. Screening of positive transgenic Arabidopsis 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 screen positive transgenic seedlings, and the DNA of positive Arabidopsis plants was extracted and identified by PCR using RcHSP70-8-F and RcHSP70-8-R primers, as shown in Figure 2. Figure 3 , through PCR identification, RcHSP70-8 positive transgenic Arabidopsis plants were successfully obtained, and 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 was 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 RcHSP70-8 transgenic Arabidopsis is 34 days. The introduction of RcHSP70-4 significantly delays the flowering time of Arabidopsis.

Claims

1. A rose heat shock protein family gene RcHSP70-8, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

1.

2. The rose heat shock protein family gene RcHSP70-8 as described in claim 1, whose encoded amino acid sequence is shown in SEQ ID No.

2.

3. Use of the rose heat shock protein family gene RcHSP70-8 as claimed in claim 1 in delaying plant flowering.

4. The use according to claim 3, characterized in that: The application is to construct the rose heat shock protein family gene RcHSP70-8 into a plant expression vector, and genetically transform Arabidopsis thaliana to delay the flowering of Arabidopsis thaliana.

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