A CmPRR3 gene for regulating chrysanthemum flowering and its application

Abstract

The present invention discloses a CmPRR3 gene for regulating the flowering of chrysanthemum and its application, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the coding region of the CmPRR3 gene is shown in SEQ ID NO: 1. The present invention discloses the chrysanthemum CmPRR3 gene sequence and the amino acid sequence encoded by it. The function and application of the gene in regulating flowering in chrysanthemum are reported for the first time. The results of qPCR analysis showed that the gene was significantly up-regulated by short-day induction; after constructing an overexpression vector and an artificial interference vector, chrysanthemum was transformed by Agrobacterium, and the transgenic plants were identified. The flowering period of the CmPRR3 over-expressing transgenic chrysanthemum strain was significantly delayed, and the CmPRR3 artificial interference transgenic chrysanthemum strain bloomed early. The present invention provides valuable gene resources for the cultivation of early-flowering chrysanthemum varieties, and is of great significance for analyzing the molecular mechanism of chrysanthemum photoperiod regulation of flowering.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a CmPRR3 gene for regulating chrysanthemum flowering and an application thereof. Background Art

[0002] Chrysanthemum (Chrysanthemum morifolium Ramat.) boasts a rich variety of flower shapes and colors, boasting economic value for ornamental, edible, and medicinal uses. Flowering is a crucial trait for chrysanthemum to fulfill its industrial value. However, most chrysanthemum varieties are typical short-day plants, and flowering can only be induced under the short-day conditions of autumn. This is attributed to the crucial role of photoperiod in regulating the flowering period of chrysanthemum. Chrysanthemums bloom at a concentrated time, and harvesting is primarily manual. This concentrated marketing effort requires significant labor and can easily lead to missed harvest times. Frost damage during the harvest period for tea-producing chrysanthemums can lead to flower damage. Early-flowering varieties are crucial for addressing this industry issue. Biotechnology-based approaches to identify key genes regulating chrysanthemum flowering can assist in molecular breeding to develop new early-flowering chrysanthemum varieties. Therefore, investigating the molecular mechanisms underlying rhythmic genes in the chrysanthemum photoperiod pathway holds important implications for understanding the regulation of flowering under varying light conditions.

[0003] PRR family proteins are characterized by a conserved Pseudo-Receiver (PR) domain at the N-terminus and a CONSTANS, constant-like, and TIMING OF CAB EXPRESSION 1 (TOC1 / PRR1) (CCT) domain at the C-terminus. Through these two domains, PRR proteins directly or indirectly interact with other circadian clock network genes, forming feedback regulatory loops. In Arabidopsis thaliana, TOC1 / PRR1, PRR5, PRR7, and PRR9 can transcriptionally repress the expression of CCA1 and LATE ELONGATEDHYPOCOTYL (LHY). CCA1 and LHY can also feedback on the promoters of PRR7 and PRR9 to promote their expression, forming a feedback regulatory loop. Three proteins, ELF3, ELF4, and LUX ARRHYTHMO (LUX), interact to form the Evening Complex (EC). Studies have found that CCA1 / LHY negatively regulates the EC complex, which in turn feedback-represses the expression of PRR9, PRR7, PRR5, and TOC1. In Arabidopsis, the prr9, prr7, and prr5 triple mutant exhibits a late-flowering phenotype compared to the wild type and is insensitive to photoperiod. The toc1-2 mutant has also been found to have an early-flowering phenotype under short-day conditions, which switches to a pronounced late-flowering phenotype under long-day conditions. In sorghum, SbPRR37 has been found to inhibit flowering under long-day conditions, while in rice, PRR37 inhibits flowering under long-day conditions by downregulating Hd3a expression. OsPRR73 has also been found to regulate flowering. In barley and wheat, the Arabidopsis AtPRR7 homolog, PHOTOPERIOD 1, regulates flowering time through a photoperiod pathway. These findings suggest that in other species, such as rice and Arabidopsis, PRR rhythmic genes form a feedback loop with other circadian clock network genes to maintain circadian rhythmicity and participate in the regulation of flowering. However, the functions and regulatory mechanisms of PRR proteins in chrysanthemum, which regulate flowering in response to circadian rhythms, have not been clearly characterized. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to overcome the deficiencies of existing research, and provides a function and application of a chrysanthemum PRR protein CmPRR3 in regulating chrysanthemum flowering.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] In a first aspect, the present invention provides a chrysanthemum CmPRR3 gene for regulating flowering, the nucleotide sequence of the coding region of which is shown in SEQ ID NO: 1.

[0007] The second aspect of the present invention provides the protein encoded by the chrysanthemum CmPRR3 gene, whose amino acid sequence is shown in SEQ ID NO: 2.

[0008] The third aspect of the present invention provides an expression vector containing the chrysanthemum CmPRR3 gene.

[0009] Preferably, the expression vector is a plant pORE-R4 overexpression vector.

[0010] The fourth aspect of the present invention proposes the use of the above-mentioned chrysanthemum CmPRR3 gene in breeding late-flowering chrysanthemum varieties.

[0011] Preferably, the CmPRR3 gene is transferred into the target chrysanthemum strain via a plant expression vector.

[0012] In a fifth aspect, the present invention provides an interfering primer for interfering with the expression of the CmPRR3 gene. There are four interfering primers, and their sequences are shown in SEQ ID NOs: 11 to 14.

[0013] The sixth aspect of the present invention provides an artificial interference vector for interfering with the expression of the CmPRR3 gene, which contains the interference primer according to claim 7.

[0014] The seventh aspect of the present invention proposes the use of the above-mentioned artificial interference vector for interfering with the expression of the CmPRR3 gene in the cultivation of early-flowering chrysanthemum varieties.

[0015] Preferably, the artificial interference vector is introduced into the target chrysanthemum strain.

[0016] The beneficial effects of the present invention are:

[0017] The present invention discloses the chrysanthemum CmPRR3 gene sequence and the amino acid sequence it encodes. This is the first report of the gene's function and application in regulating flowering in chrysanthemum. qPCR analysis results showed that the gene's expression was significantly upregulated by short-day induction. Overexpression and artificial interference vectors were constructed, and chrysanthemums were transformed using Agrobacterium. Identification of transgenic plants revealed that CmPRR3-overexpressing transgenic chrysanthemum lines had significantly delayed flowering, while CmPRR3-interference transgenic chrysanthemum lines had premature flowering.

[0018] The present invention provides valuable gene resources for the cultivation of early-flowering chrysanthemum varieties and is of great significance for analyzing the molecular mechanism of chrysanthemum photoperiod regulation of flowering. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Phylogenetic analysis of chrysanthemum CmPRR3 and comparison of amino acid sequences of ERF098 with other species;

[0020] Figure 2The expression of CmPRR3 in chrysanthemum under continuous long-day and short-day conditions;

[0021] Figure 3 This is the gel image of PCR identification of chrysanthemum CmPRR3 overexpression and artificial interference transgenic chrysanthemum;

[0022] Figure 4 The fluorescence quantitative results of chrysanthemum CmPRR3 overexpression and artificial interference transgenic lines;

[0023] Figure 5 This is the flowering phenotype of the chrysanthemum CmPRR3 transgenic line;

[0024] Figure 6 Statistical data on flowering period of chrysanthemum CmPRR3 transgenic lines. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0026] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0027] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0028] The present invention screened out CmPRR3, which is induced to express by short day, by performing transcriptome sequencing analysis on the leaves of chrysanthemum 'Shenma' treated with long day and short day. The applicant cloned the CmPRR3 gene using the leaves of chrysanthemum 'Shenma' as the material. The cDNA of the gene is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2. After constructing an overexpression vector and an artificial interference vector, chrysanthemum was transformed by Agrobacterium, and the transgenic plants were identified. The flowering period of the CmPRR3 overexpressing transgenic chrysanthemum strain was significantly delayed, and the CmPRR3 artificial interference transgenic chrysanthemum strain bloomed early. The gene of the present invention can be applied to the genetic improvement of early flowering chrysanthemum varieties through genetic engineering methods.

[0029] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.

[0030] Example 1: Acquisition of CmPRR3 gene sequence and its encoded protein sequence

[0031] Based on the transcriptome data of Chrysanthemum 'Shenma', full-length gene primers were designed, and the full-length cDNA of the CmPRR3 gene was cloned by PCR and other techniques. The ORF analysis of the gene cDNA sequence was performed using the biological software BioXM 2.6 ORF query. The conserved domains of the protein sequence were predicted based on NCBI's rpsblast. The amino acid homology and phylogenetic tree analysis of CmPRR3 were performed between CmPRR3 and PRR proteins of other plants.

[0032] PRR3 forward primer (PRR3-F): 5'-TTTCTTCTTTCTGAGGAGTCCTTGA-3' (SEQ ID NO: 3)

[0033] PRR3 reverse primer (PRR3-R): 5'-GTATGTGCCACTTCTCGACGAA-3' (SEQ ID NO: 4)

[0034] The present invention uses the cDNA of chrysanthemum 'Shenma' as a template for high-fidelity PCR amplification. The amplification system and procedure are carried out according to the instructions of the high-fidelity enzyme (Scottie, AF0802). The amplified target band is recovered by gel recovery, the product is ligated to the T vector and transformed into Escherichia coli DH5α, and the E. coli containing the target fragment is sequenced. Figure 1 As shown, the amplified CmPRR3 open reading frame is 2010bp in length (SEQ ID NO: 1) and encodes 670 amino acids (SEQ ID NO: 2). Phylogenetic tree analysis of the CmPRR3 protein and comparison with PRR3 homologous proteins from other species revealed that CmPRR3 is closely related to APRR3 from Asteraceae. CmPRR3 contains a phosphor receptor domain (REC domain) and a conserved CCT motif. Figure 1 .

[0035] Example 2: CmPRR3 gene expression analysis

[0036] According to the cDNA sequence of CmPRR3, real-time fluorescence quantitative PCR primers were designed:

[0037] PRR3-RT-F:5'-TTGGTTCAATGGGAGAGGTTTT-3'(SEQ ID NO: 5)

[0038] PRR3-RT-R:5'-CAACCAGTACCTTCCATGCTTCT-3' (SEQ ID NO: 6)

[0039] The internal reference primer used was sunflower HaEF1α, and the primer sequence is as follows:

[0040] HaEF1α-F:5'-GGTGACAACGTTGGGTTCAA-3'; (SEQ ID NO: 7)

[0041] HaEF1α-R:5'-GAGGTGTGGCAATCGAGAAC-3' (SEQ ID NO: 8)

[0042] Analysis of gene expression patterns was performed using 20-leaf-old 'Shenma' plants cultured under long-day conditions of 16h / 8h (light / dark). Samples were collected every 4 hours for 48 hours, beginning at the start of each day. For floral induction, plants at 20 leaves were placed under a short-day (8h / 16h) condition. Samples were collected every 4 hours after 3 days of short-day treatment. Plants at 20 leaves were placed under continuous light and continuous darkness for 3 days, followed by 4-hour sampling. The third fully expanded leaf was sampled from all plants, with three plants pooled at each time point. Three biological replicates were used for the experiment. RNA was then extracted using a plant RNA extraction kit (Huayueyang, 0416-50gk) according to the manufacturer's instructions and reverse-transcribed into cDNA using a reverse transcription kit (Scottje, AG0304). Sample addition and reaction program were set according to the instructions of 2×SYBR Green qPCR Mix (Sybril, AH0104), and the real-time fluorescence quantitative instrument was a Bio-Rad CFX96 PCR instrument.

[0043] The results are as follows Figure 2 As shown in Figure 3, CmPRR3 expression has a clear periodic rhythm under short-day and long-day conditions, and the expression level increases under short-day conditions ( Figure 2 ).

[0044] Example 3: Construction of CmPRR3 overexpression and artificial interference vector

[0045] High-fidelity PCR was used to amplify the full-length gene. Primer sequences were designed based on the pORE-R4 multiple cloning site. XhoI and SmaI restriction sites were introduced at both ends of the CmPRR3 sequence. The target fragment and vector were double-digested and then ligated and transformed to construct a plant pORE-R4 overexpression vector.

[0046] Primers with added restriction enzyme cutting sites:

[0047] PRR3-R4-F:5'-CCCTCGAGATGAGGAGTGTTGGAGTGA-3' (SEQ ID NO: 9)

[0048] PRR3-R4-R:5'-CCCCCGGGGGAATCCGCATCCTTCCCA-3' (SEQ ID NO: 10)

[0049] Log in to the microRNA primer design website (wmd3.weigelworld.org), upload the target gene fragment, obtain the sequence of amicroRNA, further obtain 4 gene-specific artificial interference primers and use pBSK-miR319 as a template and miR319 as a backbone to construct an artificial interference vector.

[0050] Artificial interference vector primers:

[0051] PRR3-ImiR:5'-gaTGTTAGATACCAAGTTTGCTGtctctcttttgtattcc-3'(SEQ ID NO: 11)

[0052] PRR3-IImiR:5'-gaCAGCAAACTTGGTATCTAACAtcaaagagaatcaatga-3' (SEQ ID NO: 12)

[0053] PRR3-IIImiR:5'-gaCAACAAACTTGGTTTCTAACTtcacaggtcgtgatatg-3'(SEQ ID NO: 13)

[0054] PRR3-IVmiR:5'-gaAGTTAGAAACCAAGTTTGTTGtctacatatatattcct-3' (SEQ ID NO: 14)

[0055] Example 4: Identification of CmPRR3 transgenic chrysanthemum lines

[0056] The constructed plant expression vector was transformed into Agrobacterium EHA105 and transformed into chrysanthemum 'Shenma' by the leaf disc infection method. After obtaining resistant strains, the expression of positive strains and CmPRR3 in transgenic strains was detected at the DNA and RNA levels. Figure 3 As shown in the figure, 3 overexpression strains of CmPRR and 3 interference strains were identified at the DNA level. At the same time, the RNA of the gene-grabbing strains was extracted and reverse transcribed and then subjected to fluorescence quantitative analysis. The results showed that the expression level of the overexpression strains was significantly increased, and the expression level of the overexpression strains was 7-9 times that of the wild type control. The expression level of the interference strains was reduced by 0.5 times, and the expression level was significantly decreased. Figure 4 shown.

[0057] Example 5 Statistical Observation of Flowering Phenotypes of CmPRR3 Transgenic Chrysanthemum Lines

[0058] The chrysanthemum transgenic strain obtained in Example 4 was transplanted to the Hefei High-Tech Agricultural Park. The wild type and transgenic strains were planted in a greenhouse in July. The natural flowering period was observed and statistically analyzed. The observation and statistics were conducted every 3 days before and after flower bud differentiation. The observation and statistics were conducted every 5 days after bud formation to determine the stages of flower bud development, visible color stage, early opening stage, open flower stage, and senescent flower stage. The flowering phenotype is shown in Figure 5 Compared with the wild type, the CmPRR3 overexpression line flowered about 28 days later ( Figure 6 ), the interference strains flowered 7 days earlier than the wild type ( Figure 6 ), CmPRR3 has the function of regulating chrysanthemum flowering.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A chrysanthemum with regulated flowering CmPRR3 A gene characterized by The nucleotide sequence of its coding region is shown in SEQ ID NO:

1.

2. The chrysanthemum according to claim 1 CmPRR3 The protein encoded by the gene is characterized in that Its amino acid sequence is shown in SEQ ID NO:

2.

3. Containing the chrysanthemum according to claim 1 CmPRR3 Gene expression vector.

4. The expression vector according to claim 3, characterized in that It is a plant pORE-R4 overexpression vector.

5. The chrysanthemum according to claim 1 CmPRR3 Application of genes in breeding late-flowering chrysanthemum varieties.

6. The use according to claim 5, characterized in that Will CmPRR3 The gene is transferred into the target chrysanthemum strain via a plant expression vector.

7. The use according to claim 6, characterized in that The plant expression vector is introduced by the leaf disc infection method.

8. A distraction CmPRR3 The artificial interference vector of gene expression is characterized in that By using interfering primers, pBSK-miR319 was used as a template and miR319 as a backbone to construct; There are 4 interfering primers, and their sequences are shown in SEQ ID NOs: 11-14.

9. The interference according to claim 8 CmPRR3 Application of artificial interference vectors for gene expression in breeding early-flowering chrysanthemum varieties.

10. The use according to claim 9, characterized in that The artificial interference vector according to claim 8 is introduced into the target chrysanthemum strain.

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