Rosa chinensis RcHOP3 gene and application thereof in delaying plant flowering

Abstract

The application provides a Rosa chinensis RcHOP3 gene, the nucleotide sequence of which is shown in SEQ ID No. 1, and the amino acid sequence of which is shown in SEQ ID No. 2. The application discloses the application of the Rosa chinensis RcHOP3 gene in delaying plant flowering, and overexpression in Arabidopsis thaliana can delay flowering. The application helps to expand the understanding of the flowering regulation mechanism of plants, and provides effective gene resources for genetic engineering breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the rose RcHOP3 gene and its encoded protein in delaying plant flowering. Background Technology

[0002] Flowering is a crucial step in plant development, a complex process regulated by both endogenous genetic programs and external environmental factors. To adapt to diverse growth environments, plants have evolved sophisticated and interconnected signaling networks to precisely regulate flowering. Based on model plant studies, several pathways regulating flowering induction have been identified, primarily including the photoperiod pathway, vernalization pathway, gibberellin pathway, autonomous pathway, age pathway, and ambient temperature pathway. These pathways sense different endogenous signals and external environmental factors, ultimately inducing flowering through the regulation of the expression of flowering integrators.

[0003] HOP (HSP70-HSP90 organizing proteins), also known as stress-inducible proteins (Sti), mediate the interaction between HSP70 and HSP90 and play a crucial role in plant responses to biotic and abiotic stresses. The HOP3 protein can interact with and stabilize the auxin receptor TIR1 (TRANSPORT INHIBITOR PROTEIN 1), thereby positively regulating auxin signal transduction. Simultaneously, HOP3 positively regulates plant defense responses against biotic infections such as gray mold and two-spotted spider mites through its interaction with the jasmonic acid signaling pathway receptor COI1 (CORONATINE INSENSITIVE 1). Furthermore, HOP3 is involved in developmental processes such as pollen germination. While HOP3 plays a vital role in plant growth and development, its role in flowering has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a rose RcHOP3 gene and its application in delaying flowering in plants.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A rose flowering regulatory gene RcHOP3 Its nucleotide sequence is shown in SEQ ID No. 1.

[0006] A rose flowering regulatory gene RcHOP3 The encoded protein has the amino acid sequence shown in SEQ ID No. 2.

[0007] Amplification was achieved using RcHOP3-F and RcHOP3-R primers. RcHOP3The full-length CDS sequence was obtained and homologous recombination was used to... RcHOP3 Constructed into the pCAMBIA2300-35S vector, the recombinant vector pCAMBIA2300-35S- RcHOP3 Transformed into Agrobacterium GV3101, and then subjected to Agrobacterium-mediated floral organ infection. RcHOP3 Transformed Arabidopsis thaliana, positive plants were identified by PCR using DNA as a template, and the results were obtained through... RcHOP3 Phenotypic observation of transgenic Arabidopsis thaliana revealed RcHOP3 The introduction of this species significantly delayed the flowering of Arabidopsis thaliana.

[0008] The present invention has the following effects: This invention discloses the application of the rose RcHOP3 gene in regulating plant flowering, which helps to expand our understanding of the mechanism of plant flowering regulation and provides effective gene resources for genetic engineering breeding. Attached Figure Description

[0009] Figure 1 RcHOP3 Gene PCR amplification diagram; Figure 2 RcHOP3 PCR identification of transgenic Arabidopsis thaliana; Figure 3 RcHOP3 Phenotypic diagram of transgenic Arabidopsis thaliana; Figure 4 RcHOP3 Statistics on flowering time of transgenic Arabidopsis thaliana; Figure 5 RcHOP3 Number of rosette leaves in transgenic Arabidopsis thaliana during flowering; Figure 6 AtFT , AtTFL1 and AtSVP exist RcHOP3 Expression levels in transgenic Arabidopsis thaliana. Detailed Implementation

[0010] The technical solutions provided by the present invention will be described in detail below with reference to specific embodiments and accompanying drawings, but this is not intended to limit the present invention.

[0011] Example 1 RcHOP3 Cloning of genes I. Extraction of Total RNA from Plants Total RNA was extracted using a polysaccharide and polyphenol plant total RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.), following the instructions.

[0012] II. cDNA Synthesis The first strand of cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.), following the instructions.

[0013] III. Gene Cloning Designed with Snapgene software RcHOP3 The specific primers for gene cloning are as follows: RcHOP3-F: 5'-ATGGCCGACGAAGCCAA-3' RcHOP3-R: 5'-TCACTTAATCTGGACAATTCCAGCAC-3' The full-length CDS sequence of the RcHOP3 gene was cloned using the high-fidelity PCR enzyme KOD FX101 (TOYOBO). A 50L reaction system was prepared for PCR amplification, including 25L of 2x PCR buffer for KOD FX, 10L of 2mM dNTPs, 1.5L each of primers RcHOP3-F and RcHOP3-R, 2L of cDNA template, and 10L of ddH2O. The reaction conditions were: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 30 s, and 68℃ extension for 2 min, for 40 cycles (denaturation-extension). The 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 Novizan Biotechnology Co., Ltd.). The purified DNA fragment was ligated into the pTOPO-TA / Blunt Simple Vector (Beijing Jumei Biotechnology Co., Ltd.) and transformed into DH5α E. coli competent cells (Shanghai Weidi Biotechnology Co., Ltd.), then sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Sequencing revealed... RcHOP3 The gene's CDS region contains 1734 nucleotides, encoding 577 amino acids.

[0014] RcHOP3 The gene nucleotide sequence is as follows: RcHOP3 The amino acid sequence encoded by the gene is as follows: MADEAKAKGNAAYSAGDYTAAITHFTEAINLAPTNHVLYSNRSASYASLHKYSDALSDAKKTVELKPDWVKGYSRLGSAHHGLGHFDDAVSAYKKGLEIDPNNAALKEGLAESQSAAARAARARAPPPTNLFGDAFSGPQMWAK LTADPSTRAFMQQPDFVNMMQEIQKNPSNLNLYLKDQRVMQALGVLLNVKLQGGASEDDDMPDFPSERTRSAEAAAEPQKKEEKKPEPMEVREEEKEARKRKAAEALKEKEAGNAAYKKKDFDTAIQHYTKAMEIDDEDISYLLN RAATYLEMGQYDECIEDCDKAVEKGREVRADFKMIAKALTRKGTAIVKTAKTSKDYEPAIEIFQKALTEHRNPDTLKLNDAEKAKKDLEQQEYFDPKLADEEREKGNEFFKQQKYPEAIGHYTESLRRNPKDPKAYSNRAACY TKLGAMPEGLKDAEKCIELDPTFSKGYTRKGAVQFFMREYDKALETYQEGLKHDPGNQDLLDGVRRCVEQINKASRGDLSADELKERQAKGMQDPEIQNILSDPVMRQVLVDFQENPKAAQEHSKNPMVMAKIQKLVSAGIVQIK Example 2 RcHOP3 Genetic transformation of Arabidopsis thaliana and phenotypic analysis I. Construction of Expression Carrier (1) Preparation of linearized carrier: The pCAMBIA2300-35S vector was digested with the restriction endonucleases SacI and XbaI (NEB), and the linearized vector was recovered.

[0015] (2) Obtaining by inserting a fragment: Design primers for the homologous arms of the pCAMBIA2300-35S vector containing SacI and XbaI restriction sites. The primer sequences are as follows: pCAMBIA2300-RcHOP3-F: 5'gagaacacgggggacgagctcATGGCCGACGAAGCCAAA3' pCAMBIA2300-RcHOP3-R: 5'tgcctgcaggtcgactctagaTCACTTAATCTGGACAATTCCAGC3' Cloned in Example 1 RcHOP3 Using plasmids as templates, PCR cloning was performed using the high-fidelity PCR enzyme KOD FX101 (TOYOBO). RcHOP3 After adding the homologous sequence of the pCAMBIA2300-35S vector, the product was purified after PCR. The PCR reaction system, reaction procedure, and product DNA purification were the same as in Example 1.

[0016] (2) Homologous recombination reaction: The linearized vector and insert fragment were subjected to homologous recombination using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain pCAMBIA2300-35S- RcHOP3 After recombinant plasmids were transformed into competent DH5 coli cells, single clones were identified by culture PCR and sequenced. If the sequences were correct, recombinant plasmids were extracted using the FastPure Plasmid Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0017] II. Genetic transformation in Arabidopsis thaliana pCAMBIA2300-35S- RcHOP3 The recombinant plasmid and pCAMBIA2300-35S plasmid were transformed into Agrobacterium GV3101. 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 L of bacterial culture was inoculated into 5 mL of YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif and cultured overnight with shaking. The 5 mL of overnight culture was added to 200 mL of YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif and cultured at 28 ℃ with shaking at 200 rpm until the OD600 was between 0.8 and 1.0. After collecting the bacterial cells, they were resuspended in 5% sucrose and the OD600 was adjusted to between 0.8 and 1.0. 0.03% Silwet L-77 was added. The Arabidopsis inflorescences were immersed in the inoculum for 30 seconds, and after the inoculum was wiped dry, the Arabidopsis were cultured in the dark for 24 hours.

[0018] III. Screening of positive transgenic Arabidopsis thaliana After the seeds of infected Arabidopsis thaliana plants matured, they became T0 generation seeds. These seeds were sown on 1 / 2 MS medium containing 50 g / ml Kan to screen for positive transgenic seedlings. DNA was extracted from the positive Arabidopsis thaliana plants and identified by PCR using RcHOP3-F and RcHOP3-R primers. Figure 2 Successfully obtained through PCR identification. RcHOP3 Positive transgenic Arabidopsis plants were selected, and three lines (OE-1, OE-2, and OE-3) with high expression levels were chosen for further observation using qRT-PCR analysis. This process was repeated until the T3 generation. OE-1, OE-2, and OE-3 were simultaneously sown with pCAMBIA2300-35S transgenic Arabidopsis (EV) and cultured in the same environment: a climate chamber with a temperature of 25 ℃ and a photoperiod of 16 h / 8 h. Phenotypic analysis showed that compared to EV, RcHOP3 The introduction of this species significantly delayed the flowering time of Arabidopsis thaliana and increased the number of rosette leaves. Figure 3-5 Further analysis of gene expression levels revealed that, compared to EVs, RcHOP3 Flowering integrator in transgenic lines AtFT Expression was significantly reduced, and flowering inhibitory factor AtTFL1 and AtSVP The expression of [something] increased significantly ( Figure 6 ).

Claims

1. A type of rose RcHOP3 The application of genes in delaying flowering in Arabidopsis thaliana is characterized by, The rose RcHOP3 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, roses RcHOP3 Genes were constructed into plant overexpression vectors and genetically transformed into Arabidopsis thaliana to delay flowering.

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