Polianthes tuberose PtMYB86 gene and application of polianthes tuberose PtMYB86 gene in regulation and control of flower blooming and anthocyanidin synthesis

Abstract

The invention discloses a polianthes tuberose PtMYB86 gene and application thereof in regulation and control of flower blooming and anthocyanidin synthesis, and belongs to the technical field of molecular biology. The nucleotide sequence of the polianthes tuberose PtMYB86 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein coded by the polianthes tuberose PtMYB86 gene is as shown in SEQ ID NO. 2. The invention finds that the PtMYB86 gene has double functions in polianthes tuberosa: on one hand, the PtMYB86 gene is instantaneously over-expressed in polianthes tuberosa petals, so that the expansion of the petals can be obviously promoted, and the problem of'stiff flowers' that flower buds are difficult to open in the cultivation, production and transportation processes of cut flowers and potted flowers of the polianthes tuberosa is effectively solved; on the other hand, the color of the petals can be obviously changed by transient overexpression of the PtMYB86 gene in the petals of the tuberose, so that the flower color becomes light and white from red / pink, and evidence and a key target gene are provided for clarification of a molecular mechanism of dynamic change of the flower color of the pink variety of the tuberose.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a tuberose PtMYB86 gene and application thereof in regulating flower opening and anthocyanin synthesis. BACKGROUND

[0002] Tuberose (Polianthes tuberosa), also known as night-blooming flower or moon-blooming flower, is an important economic flower with ornamental, spice and medicinal values, belonging to the Asparagaceae (traditional classification) or Agavaceae (recent classification). The flower stem of tuberose is slender, the flower color is bright, and the flower fragrance is pleasant, which has very high ornamental and economic values. The tuberose can be used as ground cover and pot flower, and is particularly suitable for cut flowers. In order to prolong the shelf life of cut flowers, the flowers are usually harvested at the bud stage and then transported by low-temperature cold chain. However, the cut flowers of tuberose often appear the phenomenon of "stiff flowers" in which the flower opening process is blocked and the flowers are stagnant at the bud stage during the cold chain transportation due to the reasons such as the separation from the mother plant and the long-term low temperature. This seriously affects the commercial value of the flowers. Even in the field cultivation, some plants also have the problem of difficult flower bud expansion due to temperature fluctuations or extreme climate. At present, the molecular mechanism of petal expansion and flower opening of tuberose is not clear.

[0003] In addition, the single-petal pink variety of tuberose has significant dynamic changes in flower color during the flower opening process: from green at the flower bud stage, to bright red at the red bud stage, to purple red at the initial opening stage (the top of the petals starts to become slightly lighter), and finally to light red or white at the full bloom stage. This process is closely related to the synthesis and degradation of anthocyanin in petals. As a natural active substance with anti-aging and antioxidant functions, anthocyanin not only determines the ornamental traits of tuberose, but also endows it with potential medical and cosmetic values. However, at present, the molecular regulation mechanism of flower opening of tuberose, the genetic basis of dynamic changes in flower color, and especially the key regulatory genes of anthocyanin metabolism have not been reported, which greatly limits the research progress of anthocyanin regulation and flower color improvement of tuberose. Therefore, it is of great significance to clarify the molecular mechanism of flower opening and anthocyanin synthesis of tuberose and find the key genes that can actively regulate the process, for improving the industrial value of tuberose.

[0004] MYB transcription factor family is one of the largest transcription factor families in plants, and the members of R2R3-MYB subfamily play a core role in plant growth and development, secondary metabolism (especially anthocyanin synthesis) and stress response. Previous studies have shown that MYB86 gene as an important member of R2R3-MYB family has significant differences in function under different plant species or different genetic backgrounds: in purple potato, grape and peach flower, MYB86 gene is confirmed to negatively regulate anthocyanin synthesis and inhibit petal coloration; while in Osmanthus fragrans, the homologous MYB gene is involved in the regulation of flower opening process and promotes flower organ development. This species-specific or genetic background-dependent functional differentiation indicates that the function of MYB86 gene cannot be simply inferred by cross-species homologous sequences, and direct functional verification must be carried out for specific species.

[0005] At present, the gene sequence and function research of tuberose are extremely weak, which seriously restricts the process of molecular breeding. Especially the molecular mechanism of petal expansion, flower opening and anthocyanin regulation is still blank, and the expression pattern and biological function of MYB86 gene in tuberose are also unclear. Therefore, isolating and identifying the key genes regulating these traits and elucidating their unique functions in tuberose have become the core problems to be solved in the field. SUMMARY

[0006] The purpose of the present application is to provide a tuberose PtMYB86 gene and its application in regulating flower opening and anthocyanin synthesis, so as to solve the problems existing in the prior art. It is found that PtMYB86 gene has a dual function in tuberose: on the one hand, transient overexpression of PtMYB86 gene in tuberose petals can significantly promote petal expansion and accelerate flower opening speed, effectively overcoming the "stiff flower" problem in field cultivation and cut flower production; on the other hand, transient overexpression of PtMYB86 gene in tuberose petals can significantly accelerate the degradation of anthocyanin in petals, resulting in the change of flower color from red / pink to light and white, which provides direct evidence and key target gene for elucidating the molecular mechanism of dynamic change of flower color in tuberose pink varieties.

[0007] To achieve the above purpose, the present application provides the following scheme:

[0008] The present application provides a tuberose PtMYB86 gene, and the nucleotide sequence of the tuberose PtMYB86 gene is shown as SEQ ID NO. 1.

[0009] The present application also provides a protein encoded by the tuberose PtMYB86 gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

[0010] The present application also provides a recombinant vector comprising the tuberose PtMYB86 gene.

[0011] The present invention also provides a recombinant microorganism comprising the recombinant vector.

[0012] The present invention also provides the application of the tuberose PtMYB86 gene, the protein, the recombinant vector, or the recombinant microorganism in regulating the opening process of tuberose flowers, by overexpressing the tuberose PtMYB86 gene to promote the opening of tuberose flowers.

[0013] Furthermore, promoting the opening of tuberose flowers includes promoting the expansion of tuberose petals.

[0014] This invention also provides the application of the tuberose PtMYB86 gene, the protein, the recombinant vector, or the recombinant microorganism in regulating anthocyanin content in tuberose flowers. Overexpression of the tuberose PtMYB86 gene reduces the anthocyanin content; the anthocyanins include proanthocyanidins and anthocyanins.

[0015] The present invention also provides a method for promoting the opening of tuberose flowers, comprising increasing the expression level of the tuberose PtMYB86 gene in tuberose or its flowers, the nucleotide sequence of the tuberose PtMYB86 gene being shown in SEQ ID NO.1.

[0016] Furthermore, promoting the opening of tuberose flowers includes promoting the expansion of tuberose petals.

[0017] The present invention also provides a method for promoting the degradation of anthocyanins in tuberose flowers, comprising increasing the expression level of the tuberose PtMYB86 gene in tuberose or its flowers, wherein the nucleotide sequence of the tuberose PtMYB86 gene is shown in SEQ ID NO.1; the anthocyanins include proanthocyanidins and anthocyanins.

[0018] The present invention discloses the following technical effects:

[0019] This invention is the first to clone the PtMYB86 gene from tuberose and determine its encoded protein sequence, filling a research gap in this gene in tuberose. Through experimental verification, this invention reveals that the PtMYB86 gene has a dual function in tuberose: on the one hand, transient overexpression of the PtMYB86 gene in tuberose petals significantly promotes petal expansion and accelerates flower opening, effectively overcoming the "stiff flower" problem described in the prior art; on the other hand, transient overexpression of the PtMYB86 gene in tuberose petals significantly accelerates the degradation of anthocyanins in the petals, causing the flower color to fade from red / pink to white, providing direct evidence and a key target gene for elucidating the molecular mechanism of the dynamic color change in pink tuberose varieties.

[0020] The identified PtMYB86 gene provides a key target gene for molecular breeding. By regulating the expression of the gene, the opening process of the cut flower of Tuberose can be actively intervened, the normal opening is promoted, and the commercial value is improved. Meanwhile, the gene provides a new technical path and gene resource for directional improvement of flower color of Tuberose and other ornamental plants (for example, cultivating a variety with more stable flower color or a specific fading mode). BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 PtMYB86 amino acid conservative sequence analysis of Tuberose;

[0023] Figure 2 Expression amount of PtMYB86 gene in different tissues of Tuberose;

[0024] Figure 3 pCY-H05251-PtMYB86 expression vector map;

[0025] Figure 4 Whole phenotype of Agrobacterium tumefaciens containing the target gene and empty vector respectively after 2 days of infection;

[0026] Figure 5 Phenotype of Tuberose flower after 2 days of infection of Agrobacterium tumefaciens containing the target gene and empty vector respectively;

[0027] Figure 6 Flower color content mass spectrum identification quantitative result graph of petals before and after transient overexpression of PtMYB86 gene;

[0028] Figure 7 Expression difference of laccase PtLAC gene and anthocyanin synthesis key gene in petals before and after transient overexpression of PtMYB86 gene;

[0029] Figure 8 Expression difference of cell expansion related genes in petals before and after transient overexpression of PtMYB86 gene. DETAILED DESCRIPTION

[0030] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, an intermediate value of is specifically contemplated. Each smaller range of values of any of the stated values or of an intermediate value in the stated ranges is also contemplated. The upper and lower limits of these smaller ranges can independently be included or excluded in the ranges.

[0032] 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art.

[0033] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in its application to the details of construction and the arrangements of components set forth in the description or illustrated in the drawings.

[0034] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0035] The inventors found a transcript annotated as MYB86 by analyzing the whole flower opening and the corresponding changes in flower color and fragrance of Tuberose, which has a complete coding region, high similarity with homologous genes, and a significantly increased expression in the process of flower opening (from bud to early and full bloom) and the corresponding changes in flower color (from bright to faded). It is possible that it is an important regulatory gene for regulating petal opening and anthocyanin changes. However, the functional characteristics of MYB86 gene in Tuberose are still unknown.

[0036] In the specific embodiment of the present application, through the spatial and temporal expression analysis of the tuberose PtMYB86 gene in different tissue organs and different development stages, it is found that the expression amount of the gene is significantly increased in the young leaves, growing leaves, mature leaves, stem tips, initial flowering and fruit tissues of tuberose, therefore, the tuberose PtMYB86 gene is constructed on a vector by the method of homologous recombination and is over-expressed in the flower petals of tuberose, it is found that the gene can obviously promote the expansion of the flower petals and the opening of the flowers, and can also regulate the synthesis of the anthocyanidins, thus it is inferred that the tuberose PtMYB86 gene is an important functional gene for regulating the opening of the flowers and the metabolism of the anthocyanidins of tuberose.

[0037] Further, the transient over-expression of the PtMYB86 gene mainly appears two aspects of phenotype differences, first, the color fading of the flower petals and the change of the anthocyanidins, second, the promotion of the opening of the flowers, the acceleration of the expansion of the flower petals and the increase of the diameter of the flowers. The color fading of the flower petals in the first phenotype is probably caused by the change of the anthocyanidins and other pigments, according to the existing research, the color of the flowers mainly depends on two pigments in the flower petals: anthocyanidins and carotenoids. The carotenoids make the flower petals present yellow or orange color; and the anthocyanidins make the flowers present red, blue or purple color. The MYB86 gene as a transcription factor will cause the expression of the related downstream genes and thus produce the change of the anthocyanidin content, firstly, the laccase gene LAC (laccase, Laccase) is positively regulated by the MYB transcription factor and over-expressed to cause the degradation of the anthocyanidins, secondly, the expression amount of the key genes for the synthesis of the anthocyanidins will also change, including the chalcone synthase (CHS, chalcone synthase) is a key enzyme in the anthocyanidin synthesis pathway; the flavanone 3-hydroxylase (F3H, flavanone 3-hydroxylase) catalyzes the flavanone to generate flavonol, which is a key enzyme in the anthocyanidin metabolic pathway; the anthocyanidin synthase (ANS, anthocyanidin synthase), also known as the leucoanthocyanidin dioxygenase (LDOX, leucoanthocyanidin dioxygenase), catalyzes the oxidation of the colorless proanthocyanidins to generate the colored anthocyanidins; the anthocyanidin reductase (Anthocyanidin reductase, ANR for short) is an enzyme that plays a key role in the secondary metabolism of plants and mainly participates in the biosynthesis of flavonoids. It catalyzes the reduction of anthocyanidins to flavonols, thereby affecting the accumulation of plant pigments and the color performance of flowers and fruits. The phytoene synthase gene PSY (phytoene synthase, PSY for short) is the first rate-limiting enzyme in the carotenoid biosynthesis pathway, which promotes the accumulation of carotenoids.

[0038] Meanwhile, PtMYB86 gene promotes petal expansion and flower opening, which may be that PtMYB86 gene can activate the downstream gene network in the process of petal expansion. According to the existing research, cellulose synthase gene (CesA), xyloglucan endotranshydrolase gene (XTH), aquaporin gene (PIP) and microtubule-related gene (TOR) are important functional genes involved in cell expansion, which may be involved in petal expansion and flower opening and maturation process, so the above cell expansion related genes are analyzed.

[0039] The following specific examples will be listed to explain the scheme of the application.

[0040] Unless otherwise specified in the examples, the technology or conditions are carried out according to the technology or conditions described in the literature in the art or according to the product manual. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained commercially.

[0041] The following examples use the following reagents: tryptone, yeast extract, sodium chloride, agar, cloning vector pEASYT1-Vector, Agrobacterium tumefaciens competent EHA105, and pCY-H05251 vector, which are commercially available. Among them, the pCY-H05251 vector is purchased from Shanghai Lucy Biotech Co., Ltd.

[0042] The following kits are used in the following examples: polysaccharide polyphenol plant total RNA extraction kit, HiScript III 1stStrand cDNA Synthesis Kit, FastPure Gel DNA Extraction Mini Kit, and Fast-T1 E. coli competent cells, which are purchased from Nanjing Novogene Bioinformatics Technology Co., Ltd.

[0043] The following is the composition of the medium used in the examples:

[0044] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH = 7.0.

[0045] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, pH = 7.0.

[0046] YEP liquid medium: tryptone 10 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, pH = 7.0.

[0047] YEP solid medium: tryptone 10 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, agar 15 g / L, pH = 7.0.

[0048] Resuspension formulation: 0.5 M morpholine ethanesulfonic acid MES (pH 5.6, filter sterilization), 10 mM MgCl2(high temperature sterilization), 100 mM Acetosyingone (AS, high temperature sterilization).

[0049] Example 1 Cloning of Tuberose PtMYB86 Gene

[0050] 1. Extraction of total RNA

[0051] The red flower buds and petals of the single-petal pink variety 'Dynamic' of Tuberose were selected, and the polysaccharide polyphenol plant total RNA extraction kit (Nanjing Novozyme) was used to extract total RNA. The RNA purity and integrity were detected by agarose gel electrophoresis and Nanodrop.

[0052] 2. cDNA synthesis

[0053] The extracted total RNA was used as a template, and the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozyme) was used to synthesize the first strand of cDNA.

[0054] 3. Cloning of PtMYB86 gene

[0055] The Primer5.0 software was used to design specific primers for cloning the PtMYB86 gene, and the primer sequences were as follows:

[0056] PtMYB86-F: ATGGGGAGGCACTCCTGCTGCTAC, SEQ ID NO. 3;

[0057] PtMYB86-R: CTAAATCCGTCCAAGCACTGCAG, SEQ ID NO. 4.

[0058] The high-fidelity PCR enzyme KOD FX (TOYOBO) was used to clone the CDS full-length sequence of the PtMYB86 gene, and a 50 μL reaction system was prepared for PCR amplification.

[0059] PCR amplification reaction system: 2x PCR buffer for KOD FX 25 μL; 2 mM dNTPs 10 μL; primer PtMYB86-F, primer PtMYB86-R each 1.5 μL; template cDNA 2 μL; ddH2O 10 μL.

[0060] PCR amplification reaction conditions: 94°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 61°C annealing for 30 s, 72°C extension for 5 min, 35 cycles (denaturation-extension).

[0061] 4. Sequencing verification

[0062] After amplification, the PCR product was detected by 1% agarose gel electrophoresis. The target gene band was cut off and purified by FastPure Gel DNA Extraction Mini Kit. The purified DNA fragment was ligated with cloning vector pEASYT1-Vector, and transformed into Fast-T1 E. coli competent cells, and sent to Shanghai Bioengineering Company for sequencing. Sequencing showed that the CDS region of PtMYB86 gene contained 1281 nucleotides (as shown in SEQ ID NO. 1), encoding 426 amino acids (as shown in SEQ ID NO. 2).

[0063] SEQ ID NO. 1:

[0064]

[0065] SEQ ID NO.2:

[0066] MGRHSCCYKQKLRKGLWSPEEDEKLLKHITKYGHGCWSSVPKQAGLQRCGKSCRLRWINYLRPDLKRGTFSQQEENLIIELHAVLGNRWSQIAAQLPGRTDNEIKNLWNSCLKKKLRQRGIDPNTHKPIAETESEKDKAAAAPTSGNKTSSSTPNPTVSVPSLPLIPSSTPKNSISPTKEFFLDHFVTNQESSLSSQASNSMAFFPHPQLNSSGKESVTSSQLLWLNQNTRLFDMNPEFDCQTMSSIITSVSSSILSTSMGLKPEINMPPDNPPPPRPPPPPPPPECTTLCGIQYWEVSNTSNSSRSSGNSSSGIELQSNSSFMDGGMFSWTDLIQDKESMQLNLDGEPEDLKWSEYLHGAYPISVALQNHSQPMYGDIKSENQFAIEGSSSWHQSHQQPPQQQPQRSELYSDKDFQRISAVLGRI*.

[0067] 5. Multiple sequence alignment of PtMYB86 protein

[0068] The nucleotide sequence of the PtMYB86 gene was subjected to BLAST comparison on the NCBI website, and was compared to the MYB86 or MYB61 sequences with high similarity. It was found that the similarity with the genes such as asparagus AoMYB86, pineapple AcMYB86, lady orchid PeMYB86 and date PdMYB61 was high. The amino acids encoded by the above homologous genes were subjected to multiple sequence alignment using DNAMAN and MEGA7.0 software, and the results are shown in Figure 1 It was found that the amino acid encoded by the PtMYB86 gene was also similar to the amino acid sequences of the homologous genes, and was highly conserved in many regions.

[0069] Example 2 Analysis of expression characteristics of Tuberose PtMYB86 gene in different tissues

[0070] 1. Sample preparation

[0071] Eight kinds of tissue samples of single-petal pink variety 'Dongjing' of Tuberose were collected: flower bud (stage 1), red bud (stage 2), initial opening (stage 3), full bloom (stage 4), leaf, flower stalk, bulb and root system, each sample with 3 biological replicates. Referring to Example 1, total RNA was extracted from different tissues using the polysaccharide polyphenol plant total RNA extraction kit and reverse transcribed into cDNA template for standby.

[0072] 2. qPCR detection

[0073] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene with stable expression in different organs of Tuberose was used as the internal reference gene. Real-time quantitative PCR (Real-time quantitative PCR) was used to analyze the expression difference of PtMYB86 gene in different tissues by relative quantitative ΔΔCT method.

[0074] The real-time quantitative PCR reaction system, primers and reaction program are as follows:

[0075] Internal reference primers:

[0076] PtGAPDH-116F: CACTGATGAGGATCTGGTTTCC, SEQ ID NO. 5;

[0077] PtGAPDH-116R: CGTACCAAGAGACGAGCTTAAC, SEQ ID NO. 6.

[0078] Target gene primers:

[0079] PtMYB86-117F: AGGTGGTCTCAGATTGCAGC, SEQ ID NO. 7;

[0080] PtMYB86-117R: GTGTGTGTTGGGGTCAATGC, SEQ ID NO. 8.

[0081] Reaction system: 2×SYBR qPCR Master Mix 10 μL; 0.4 μL of upstream primer (10 µM) and downstream primer (10 µM) each; 1 μL of cDNA template; RNase Free Water to 20 μL.

[0082] Amplification curve program: 95℃ 30 s, cycle 1; 95℃ 5 s, 60℃ 30 s, cycle 40 times;

[0083] Dissociation curve program: 95°C 15 s, cycle 1; 60°C 60 s, cycle 1; 95°C 0.05°C / s, cycle 1.

[0084] The results are shown in Figure 2 PtMYB86 gene was expressed in different tissues of Tuberose, such as flowers, leaves, bulbs and roots, and the expression level was the highest in flower stalks and bulbs. The expression level changed greatly during the process of flower opening, and the expression level was low in flower bud and red bud stage, and increased in early opening stage, and significantly increased in full bloom stage. It is shown that PtMYB86 gene is positively correlated with the process of flower opening.

[0085] Example 3 Construction of pCY-H05251-PtMYB86 expression vector

[0086] 1. Preparation of linearized vector

[0087] The pCY-H05251 vector was double digested with restriction enzymes KpnI and SalI, and the linearized vector was recovered.

[0088] 2. Design of homologous recombination primers

[0089] PtMYB86 gene primers with KpnI and SalI restriction site homologous arms were designed, and the target fragment was amplified by PCR, and the primer sequences were as follows:

[0090] KpnI PtMYB86-F: tagaagtaggagctcggtaccATGGGGAGGCACTCCTGCTGC, SEQ ID NO. 9;

[0091] SalI PtMYB86-R: gcccttgctcaccatgtcgacAATCCGTCCAAGCACTGCAG, SEQ ID NO. 10.

[0092] 3. Homologous recombination

[0093] The concentrations of the target gene PtMYB86 gene and the recovered vector were determined by micro nucleic acid concentration detector (NANODROP lite, Thermo Scientific), and according to the instructions of homologous recombination enzyme (ClonExpress II One Step Cloning Kit, item number: C112), the linearized vector and the target fragment were mixed at a molar ratio of 1:3, incubated at 37°C for 30 min, and the pCY-H05251-PtMYB86 plant expression vector was obtained. Figure 3 ).

[0094] 4. Verification

[0095] Take a DH5a competent E. coli (Shanghai Weidi), placed on ice, thawed naturally, 5 μL of ligation product in E. coli, light mix. On ice for 30 min, 42℃ heat shock 40 s, 2 min on ice, then add 500 μL of no-antibiotic LB liquid medium, 37℃ 200 rpm, shake culture for 30 min. The obtained bacterial liquid was coated on LB solid medium containing 50 mg / L kanamycin, and cultured at 37℃ overnight. Then pick single colony, and use primers PtMYB86-F (SEQ ID NO. 3) and PtMYB86-R (SEQ ID NO. 4) for PCR verification.

[0096] PCR amplification reaction system: DNA polymerase 12.5 μL, upstream primer, downstream primer 1 μL each, template 1 μL, ddH2O 9.5 μL;

[0097] Reaction program: 95℃ pre-denaturation 5 min; 98℃ denaturation 10 s, 61℃ annealing 10 s, 72℃ extension 10 min, 35 cycles (denaturation-extension), finally 72℃ extension 10 min, then 12℃ storage.

[0098] After gel electrophoresis of the PCR product, the identified positive clone was sent to Shanghai Bioengineering Company for sequencing, and the results showed that it was completely consistent with the target gene, indicating that the pCY-H05251-PtMYB86 expression vector was successfully obtained.

[0099] Example 4 Transient overexpression of PtMYB86 gene in Tuberose petals

[0100] 1. Cultivation of Tuberose

[0101] Tuberose bulbs were planted in pots in April, and were planted in a place with sufficient light and good ventilation. Normal cultivation and management, in May and June, flower stalks gradually grew from the basal leaf tufts, and as the flower stalks elongated, green flower buds grew at the top. In July and August, when many small flowers were formed on the flower stalks and the small flowers turned from green to bright red flower buds, they were ready for transient transformation.

[0102] 2. Preparation of Agrobacterium tumefaciens containing the target gene

[0103] (1) The pCY-H05251-PtMYB86 recombinant plasmid obtained in Example 3 was transformed into Agrobacterium tumefaciens competent EHA105, and was coated on YEP solid medium and incubated at 28℃ for 2-3 days in the dark. The transformation steps refer to the instructions for Agrobacterium tumefaciens transformation;

[0104] (2) Pick the monoclonal for PCR identification, and the correct colonies are expanded in 10 mL of YEP liquid medium containing 50 mg / L kanamycin and 25 mg / L Rif, 28°C, 180 rpm shaking culture for 12-16 h;

[0105] (3) 1 mL of bacterial solution is added to 50 mL of YEP liquid medium containing 50 mg / L kanamycin and 25 mg / L Rif for further expansion, until OD 600 0.6-0.8 is reached;

[0106] (4) The cultured bacterial solution is centrifuged at 4°C, 5000 rpm for 5 min, and under sterile conditions, the supernatant is discarded, and the bacterial pellet is resuspended with 50 mL of equal volume of transient transformation resuspension solution (pH 5.8), then shaken at low speed in a 28°C shaking bed for 3 h, and then stand for 1 h, and then used for petal infection.

[0107] 3. Petal infection

[0108] (1) Preparation for infection: sterilized 5 mL centrifuge tubes are inserted into the centrifuge tube rack in an orderly manner, 10 tubes are arranged as a group, and two groups are set, one experimental group (infection solution containing PtMYB86 target gene of Agrobacterium tumefaciens), and the other group is the control group (infection solution containing empty vector of Agrobacterium tumefaciens), then the prepared bacterial solution is injected into the centrifuge tube with a 5 mL pipette;

[0109] (2) Infection: small flowers at the red and bright bud stage are selected from 30-40 cm long and slender total inflorescences with 20-30 small flowers pulled out from the low leaf clumps of flowering gardenias in the field, the small flowers at the red bud stage are cut from the base with surgical scissors, immediately inserted into the 5 mL plastic test tube filled with the infection solution of the target gene of Agrobacterium tumefaciens, and placed in a 25°C dark condition for 2 days, while setting the flowers infected with Agrobacterium tumefaciens with empty vector as negative control, and setting 3 replicates;

[0110] 4. Phenotype observation and target gene expression analysis

[0111] After 2 days, the transiently expressed petals are observed to check the petal expansion and flower opening degree, and the flower color change, and photographed with a digital camera. qPCR is used to analyze the PtMYB86 gene expression amount in the transient overexpression experimental group and the empty vector control group.

[0112] The qPCR detection of gene expression amount results are shown in Table 1, the PtMYB86 gene expression amount in the experimental group is 3-4 times that of the empty vector control group, indicating that the gene PtMYB86 is successfully overexpressed in the petals. Figure 4 Table 1: qPCR detection of gene expression amount

[0113] Phenotype observation showed that PtMYB86 gene could significantly promote petal expansion, flower opening Figure 4 ), the flower diameter of petal of PtMYB86 gene transient overexpression reached 2.5-3.0 cm after two days, while the flower diameter of control empty vector was only 1.5-2.0 cm, almost 1.5 times, the difference was significant, at the same time, the petal color also had obvious difference, the transient overexpression of petal not only accelerated the opening of flower, but also the pink color faded, even disappeared, most of the petals had turned white, only a few places still had light color spots Figure 5 ).

[0114] 5. Anthocyanin content analysis

[0115] Take appropriate samples of the experimental group and the control group into centrifuge tubes, add 1 mL of extraction solution (methanol / water / formic acid, 70:30:1, v / v / v), high-speed vortex treatment, then ultrasonic extraction for 20 min, centrifuge at 12000 rpm for 10 min, repeat extraction twice. Combine and mix the supernatants of the two times, pass through HLB-SPE column: add 1 mL of 100% methanol solution and 1 mL of deionized water respectively, add the supernatant to the column, at the same time, add 1 mL of water solution for elution, dry the liquid, then add 1 mL of methanol solution (containing 5% formic acid) for elution, collect the eluate into a new centrifuge tube, slowly blow dry in nitrogen stream, freeze-drying treatment, add 0.2 mL of methanol solution for redissolution, dilute the sample, then perform sample detection. Use ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometer (Q Exactive HFX, Thermo, USA) and other data acquisition instruments to detect and analyze anthocyanin in the sample.

[0116] Liquid chromatography parameters:

[0117] Chromatographic column: Waters HSS T3 (100 x 2.1 mm, 1.8 μm); mobile phase: A phase is ultrapure water solution (containing 0.1% formic acid), B phase is acetonitrile solution (containing 0.1% formic acid); flow rate 0.3 mL / min; column temperature 40℃; injection volume 2 μL; elution gradient: 0 min A phase / B phase (95:5, v / v), 2 min A phase / B phase (95:5, v / v), 15 min A phase / B phase (70:30, v / v), 15.1 min A phase / B phase (5:95, v / v), 20 min A phase / B phase (5:95, v / v), 20.1 min A phase / B phase (95:5, v / v), 26 min A phase / B phase (95:5, v / v). The sample was placed in the 4℃ automatic injector during the whole analysis process. In order to avoid the influence of instrument detection signal fluctuation, random order was used for continuous analysis of samples. QC samples were uniformly inserted in the sample analysis sequence to monitor and evaluate the stability of the system and the reliability of the experimental data.

[0118] Mass spectrometry conditions:

[0119] The Q Exactive HFX high-resolution mass spectrometry system of Thermo Corporation of the United States was used to collect primary and secondary mass spectra. It was equipped with an electrospray ion source (ESI), with a sheath gas of 40 arb, an auxiliary gas of 10 arb, an ion spray voltage of +3000 V, a temperature of 350℃, and an ion transmission tube temperature of 320℃. The scanning mode was FSMS2, and the scanning mode was positive ion. The primary mass spectrum scan range was 200-700 Da.

[0120] The raw data was first pre-processed using Progenesis QI (Waters Corporation, Milford, USA) software for baseline filtering, peak identification, peak matching, retention time correction, peak alignment, etc., to obtain a data matrix containing retention time, mass-to-charge ratio and peak intensity. The secondary mass spectrum database of anthocyanin metabolites and the corresponding fragmentation rules were used to identify the peaks containing secondary mass spectrum data. The anthocyanin mass spectrum identification and quantitative results are shown in Table 1 and Figure 6 .

[0121] Table 1 Anthocyanin mass spectrum identification and quantitative results

[0122]

[0123] According to the quantitative results of the mass spectrometry of tuberose petal color, as shown in Table 1, the pigment composition of the deep red buds in the empty control was mainly composed of delphinidin and its derivatives. Among them, delphinidin-3-O-(6"-coumarin)-glucoside had the highest content (38.52%), followed by cyanidin-3-O-succinate-5-O-glucoside (13.29%) and delphinidin-3-O-glucoside (7.84%). These anthocyanins together shaped the deep red hue of the petals. Combining the data in Table 1 and Figure 6 It was found that transient overexpression of PtMYB86 significantly altered pigment composition by differentially regulating anthocyanin metabolic pathways: on the one hand, it strongly inhibited the cyanidin pathway, causing its content to drop from 7.36% to undetectable levels, and some proanthocyanidins (such as B2 and A2) and petunia derivatives also decreased; on the other hand, it generally promoted the synthesis of delphinidin pathway, with key derivatives such as delphinidin-3-O-glucoside (increasing from 7.84% to 11.90%) and delphinidin-3- The contents of O-succinate-5-O-glucoside (from 6.48% to 7.69%) and delphinidin-3-O-(6"-coumarin)-glucoside (from 38.52% to 42.33%) both increased significantly. Although the total amount of delphinidins increased, the complete absence of cyanidin led to a significant weakening of the red hue. At the same time, the reduction of accessory pigments such as proanthocyanidins may have weakened the co-coloring effect and stability of pigments, ultimately causing a decrease in flower color saturation, which macroscopically manifests as an overall lighter petal color.

[0124] Example 5: Analysis of downstream genes such as anthocyanin synthesis and petal expansion induced by transient overexpression of the PtMYB86 gene.

[0125] This embodiment analyzed the expression differences of anthocyanins and cell expansion-related genes in petals with transient overexpression of the PtMYB86 gene, using unexpressed petals as a control. The specific steps are as follows:

[0126] 1. RNA extraction

[0127] RNA was extracted from petals of plants with transient overexpression of the PtMYB86 gene and an empty vector control, according to the instructions of the Nanomagnetic Biobead Method Common Model Plant RNA Extraction Kit (Catalog No. NMR0311).

[0128] 2. RNA is reverse transcribed into cDNA

[0129] Following the instructions of Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2, prepare a 20 μL reverse transcription reaction system on ice, using the maximum amount of RNA, incubate at 37°C for 15 min, then at 85°C for 5 sec, and cool on ice to obtain cDNA.

[0130] 3. Relative quantification qPCR

[0131] The reaction system (20 μΐ) is shown in Table 2.

[0132] Table 2. Reaction system

[0133]

[0134] The reaction conditions are shown in Table 3.

[0135] Table 3. Reaction conditions

[0136]

[0137] 4. Anthocyanin gene and petal expansion gene names downstream of PtMYB86 and primers

[0138] Reference primers:

[0139] PtGAPDH-116F: CACTGATGAGGATCTGGTTTCC, SEQ ID NO. 5;

[0140] PtGAPDH-116R: CGTACCAAGAGACGAGCTTAAC, SEQ ID NO. 6.

[0141] Primers for key genes of anthocyanin and carotenoid synthesis and degradation:

[0142] 1-PtLAC-119F: CGAGCTACACGTACCGATTTAC, SEQ ID NO. 11;

[0143] 1-PtLAC-119R: GTTCTCTCTTGGGTGGATGATG, SEQ ID NO. 12;

[0144] 2-PtF3H-99F: AGCAGGGAGAGACGATGATA, SEQ ID NO. 13;

[0145] 2-PtF3H-99R: CTTGTACGGCCTCTTGGTATT, SEQ ID NO. 14;

[0146] 3-PtANR-98F: AAGACGGGAGCTTTGATGAG, SEQ ID NO. 15;

[0147] 3-PtANR-98R: GAGCTCATTCTCTGGGTCTTTAG, SEQ ID NO. 16;

[0148] 4-PtCHS-91F: GATAATCGATGCGGTCGAGATG, SEQ ID NO. 17;

[0149] 4-PtCHS-91R: GGACATGTTCCCGTACTCTTTC, SEQ ID NO. 18;

[0150] 5-PtANS-98F: GGATGGACTACTTCTTTCACCTC, SEQ ID NO. 19;

[0151] 5-PtANS-98R: CTCCTGCATCACCTCAATGTAT, SEQ ID NO. 20;

[0152] 6-PtPSY-97F: GACACAAGGAAGTGGAGGTATC, SEQ ID NO. 21;

[0153] 6-PtPSY-97R: CCATCACAGGTACACTCATAAGG, SEQ ID NO. 22;

[0154] Petals expansion downstream genes primers:

[0155] 7-PtPIP-128F: AGTTCACCGCCAAAGACTAC, SEQ ID NO. 23;

[0156] 7-PtPIP-128R: GATGTAGAGGAAGAGGAGGGT, SEQ ID NO. 24;

[0157] 8-PtCESA-114F: GCTCAGAAGGTTCCAGAAGAAG, SEQ ID NO. 25;

[0158] 8-PtCESA-114R: CCCTCCACTTTGACCAAGAAA, SEQ ID NO. 26;

[0159] 9-PtXTH-104F: GAGTTCTTGGGCAGTAGAGAAG, SEQ ID NO. 27;

[0160] 9-PtXTH-104R: GGGTCAAACCACAAGCTAATTC, SEQ ID NO. 28;

[0161] 10-PtTOR1-114F: CAGCAGAAGAGCTTGGGATAA, SEQ ID NO. 29;

[0162] 10-PtTOR1-114R:ACCCGAATAGCTTCCAAAGTAG, SEQ ID NO.30;

[0163] 11-PtTOR2-105F:AGAAAGAGGCACTAAGCAGAAAG, SEQ ID NO.31;

[0164] 11-PtTOR2-105R:CTGTTGTCATCCCGATTGTAGT, SEQ ID NO. 32.

[0165] 5. Expression analysis of anthocyanin-related genes downstream of PtMYB86

[0166] The expression of downstream anthocyanin-related genes of PtMYB86 is as follows: Figure 7 As shown, from Figure 7 It can be seen that after transient overexpression of the PtMYB86 gene, the expression level of the laccase gene LAC significantly increased, reaching eight times that of the empty vector. The expression levels of key genes for anthocyanin synthesis and metabolism, such as F3H, ANR, CHS, and ANS, significantly decreased. In particular, the expression levels of ANR and ANS, the two anthocyanin reductase and synthase genes, decreased significantly, reaching only one-sixth and one-third of those in the empty vector, respectively. Meanwhile, the expression level of PSY, the key gene for yellow carotenoids in petals, remained basically unchanged. This indicates that the transient expression of the PtMYB86 gene caused a sharp increase in the expression of the laccase LAC gene and a significant decrease in the expression levels of key anthocyanin synthesis enzyme genes, resulting in changes in the composition and degradation of anthocyanins. This is the key factor that enables the PtMYB86 gene to accelerate petal fading.

[0167] 6. Expression analysis of downstream petal expansion-related genes of PtMYB86

[0168] The expression of PtMYB86 downstream cell expansion-related genes is as follows: Figure 8 As shown, from Figure 8It can be seen that after the transient overexpression of PtMYB86 gene, the expression of most genes related to cell expansion, such as cellulose synthase (PtCesA), xyloglucan endotranshydrolase gene (PtXTH) and microtubule-related genes (PtTOR, TOR2), is increased, especially the xyloglucan endotranshydrolase gene (PtXTH) is sharply increased, which is almost six times of the empty vector, the microtubule-related genes (PtTOR1 and TOR2) are also significantly increased, which is twice of the empty vector, the cellulose synthase (PtCesA) is 1.2 times of the empty vector, which is also significantly higher than the control; only the aquaporin gene (PtPIP) decreases, which may be because the transient overexpression of the target gene promotes the early maturation and aging of the petals and the production of ethylene, thereby inhibiting the expression of the aquaporin gene.

[0169] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A Tuberose PtMYB86 gene, characterized in that, The nucleotide sequence of the Tuberose PtMYB86 gene is shown as SEQ ID NO.

1.

2. A protein encoded by the vanilla PtMYB86 gene of claim 1, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

2.

3. A recombinant vector comprising the Tuberose PtMYB86 gene of claim 1.

4. A recombinant microorganism comprising the recombinant vector of claim 3.

5. The use of the vanilla PtMYB86 gene of claim 1, the protein of claim 2, the recombinant vector of claim 3 or the recombinant microorganism of claim 4 in regulating the opening process of vanilla flowers, characterized in that, Overexpression of the Tuberose PtMYB86 gene promotes the opening of Tuberose flowers.

6. Use according to claim 5, characterized in that, Promoting the opening of Tuberose flowers includes promoting the expansion of Tuberose petals.

7. The use of the vanilla PtMYB86 gene of claim 1, the protein of claim 2, the recombinant vector of claim 3 or the recombinant microorganism of claim 4 in regulating the content of anthocyanins in vanilla flowers, characterized in that, Overexpression of the Tuberose PtMYB86 gene reduces the content of anthocyanins, which include proanthocyanidins and anthocyanins.

8. A method for promoting the opening of flowers of Tuberose, characterized by, The method comprises the step of increasing the expression level of the Tuberose PtMYB86 gene in Tuberose or its flowers, the nucleotide sequence of the Tuberose PtMYB86 gene is shown as SEQ ID NO.

1.

9. The method of claim 8, wherein, Promoting the opening of Tuberose flowers includes promoting the expansion of Tuberose petals.

10. A method of promoting anthocyanin degradation in a Tuberose flower, characterized by, The method comprises the step of increasing the expression level of the Tuberose PtMYB86 gene in Tuberose or its flowers, the nucleotide sequence of the Tuberose PtMYB86 gene is shown as SEQ ID NO. 1; the anthocyanins include proanthocyanidins and anthocyanins.

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