Application of a tyrosine decarboxylase gene in regulating plant growth

Abstract

The invention belongs to the field of biotechnology, and specifically relates to an application of a tyrosine decarboxylase gene in regulating plant growth. The tyrosine decarboxylase gene MdTyDC promotes leaf senescence, plant flowering, and fruit ripening through overexpression, laying a theoretical foundation for improving fruit quality and preserving freshness.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a tyrosine decarboxylase gene in regulating plant growth. Background Art

[0002] Flowers and fruits are important links in plant reproduction and population reproduction, and are also key factors in determining crop yields. Flowers and fruits are the main product organs of plants with seeds and fruits as their main products, which constitute an important basis for crop yields and are also the key to plant reproductive growth and population reproduction. Flowering, as an important reproductive trait, is a key factor in determining the success or failure of plant reproduction. It requires precise integration of endogenous and exogenous information to start the entire flowering process. The flowering period of plants is determined by environmental and genetic factors, and there are large differences between and within different species. In recent years, people have conducted in-depth research on the flowering mechanism of plants from multiple perspectives such as physiology, biochemistry, metabolism, ecology, genetics and evolution. In particular, the development of molecular biology technology has enabled people to understand the regulatory mechanism of plant flowering from multiple levels such as genetic composition, expression regulation, and signal transduction, and provided new ideas for regulating the flowering time of plants through methods such as genetic engineering. The flowering mechanism of plants is complex, and conventional breeding methods are difficult to achieve effective control of flowering time. The use of genetic engineering and other technologies has provided a new idea for plant flowering breeding. However, how to effectively control the cloning of flowering genes is an important issue.

[0003] Fruit ripening is an important stage in the growth and development of plants. For plants, this is a necessary process for them to complete the alternation of generations and reproduce. For humans, fruits of various crops have become an important source of food for humans, providing people with sufficient energy and rich nutrients. Fruit ripening refers to a series of physiological and biochemical reactions that occur inside the fruit after the fruit has completed its growth and expansion in the late stage of development, including typical characteristics such as peel chlorosis, chlorophyll degradation, anthocyanin and carotenoid synthesis, aroma synthesis and fruit softening. This involves a series of complex metabolic processes and is accompanied by the production of various secondary metabolites. Studies have shown that the rational use of plant growth regulators in fruit production plays an important role in improving fruit quality. Ethylene, as an important plant hormone, is involved in various developmental processes of plants, including the time of fruit ripening, nutritional senescence, and responses to different biological and abiotic stresses. The ripening of climacteric fruits (such as apples) is accompanied by an increase in respiration rate and subsequent ethylene bursts, which are required to trigger multiple processes related to ripening, such as the color, texture, flavor and aroma of the flesh. Ethylene signal transduction begins with the binding of ethylene to ethylene receptors and ends with a transcriptional cascade involving the EIN3 / EIL (EIN3-like protein) and ERF (ethylene response factor) families.

[0004] Senescence, the final stage of leaf development, involves the degradation of cellular components and their remobilization to the growth organ. The reduction in chlorophyll concentration is considered to be one of the most significant features of the senescence process. For apple (Malus domestica), the timing of leaf senescence is crucial for fruit yield and quality, because premature senescence leads to reduced fruit yield, while delayed leaf senescence disrupts nutrient redistribution, which also has an adverse effect on fruit quality formation. Leaf senescence is mainly regulated by the levels of environmental and endogenous signals and their interactions. Environmental factors include stress factors; endogenous signals include plant hormones, senescence, and reproductive development. Some plant hormones such as abscisic acid (ABA), ethylene (ET), jasmonic acid (JA), methyl jasmonate (MeJA), and salicylic acid (SA) have been shown to induce senescence. Many senescence-associated genes (SAGs) have been identified and highly expressed during senescence. For example, SAG12 has been widely used as a molecular marker for MeJA- and ABA-induced leaf senescence. SEN1 is also known as a marker gene for age-triggered and darkness-induced senescence. The pyrophosphate a-oxygenase gene (PAO) is a key chlorophyll degradation gene that is highly upregulated during senescence and synchronized with chlorophyll degradation. Many studies have shown that ethylene is a positive regulator of senescence or an inducer of upregulation of certain SAGs. In addition, ethylene is involved in the regulation of chlorophyll degradation during leaf senescence and is considered a promoter of the senescence process.

[0005] Tyrosine decarboxylase (TyDC) is a common enzyme ubiquitous in plants and mediates many secondary reactions. Current studies have shown that TyDC is regulated in many biotic or abiotic stresses. Overexpression of MdTyDC promoted arbuscular mycorrhizal symbiosis by increasing dopamine content, thereby improving salt tolerance in apple. In addition, overexpression of MdTyDC enhanced salt tolerance in apple plants by improving photosynthetic capacity, scavenging ROS, and mediating ion homeostasis. Arabidopsis AtTyDC transcription responded greatly to wounding and drought stress, suggesting that AtTyDC is involved in these stress stimuli.

[0006] Apple (Malus domestica Borkh.) is widely planted in the world, and China, as the world's major apple producer and consumer, ranks first in the world in terms of cultivated area and output. Its market price and unit benefit depend on the fruit quality of apples, so improving fruit quality has become a major problem facing the industry. However, due to the complex genetic background of apples and the long breeding cycle, it is difficult to obtain varieties that combine all the best qualities through traditional hybridization methods. Therefore, exploring the mechanism of leaf senescence and fruit ripening as soon as possible will help breeders improve fruit quality and is of great significance to accelerate the development of my country's apple industry. At present, the expression of MdTyDC involves many abiotic and biotic stresses and plant development, but little is known about its role in plant senescence, promoting plant flowering and fruit ripening. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an application of a tyrosine decarboxylase gene in regulating plant growth.

[0008] The invention discloses an application of a tyrosine decarboxylase gene in regulating plant growth, characterized in that the gene is MdTyDC and the gene sequence number is MDP0000850540.

[0009] Preferably, the regulating plant growth is promoting plant leaf senescence by overexpressing MdTyDC.

[0010] Preferably, plant leaf senescence is promoted jointly by overexpression of MdTyDC and ACC treatment.

[0011] Preferably, the regulating plant growth is promoting plant flowering by overexpressing MdTyDC.

[0012] Preferably, the regulating plant growth is promoting plant fruit ripening by overexpressing MdTyDC.

[0013] Preferably, overexpression of MdTyDC shortens the time from pollination to fruit coloring to promote plant fruit ripening.

[0014] Preferably, the plant is apple, tomato or Arabidopsis thaliana.

[0015] The method for overexpressing MdTyDC comprises the following steps:

[0016] The cDNA of plant leaves containing the MdTyDC gene was used as a template for PCR amplification to obtain the amplified product;

[0017] The amplified product was connected to the overexpression vector pGWB411 to obtain the overexpression vector;

[0018] The overexpression vector is used to transform Agrobacterium to upregulate the expression level of the MdTyDC gene.

[0019] A method for regulating plant growth comprises introducing the MdTyDC gene into a plant to be regulated to obtain a transgenic plant, overexpressing the MdTyDC gene, and regulating plant growth.

[0020] Preferably, the plant to be regulated is a plant that needs to promote flowering and / or promote fruit ripening.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) When the transgenic apple and Arabidopsis thaliana cloned with the tyrosine decarboxylase gene MdTyDC were treated with ACC under dark conditions, overexpression of MdTyDC promoted leaf senescence by upregulating SAG. There is a positive correlation between the expression of MdTyDC and leaf senescence, which can be used as a marker to indicate plant senescence and provide an important reference for scientifically evaluating the degree of plant senescence.

[0023] (2) Compared with the wild type, the transgenic tomatoes overexpressing the tyrosine decarboxylase gene MdTyDC in the present invention shorten the time from pollination to fruiting, and have a higher carotenoid content and expression of carotenoid biosynthesis genes, which has a promoting effect on plant flowering and fruit ripening, and provides a safe and effective method for regulating fruit growth and development and improving fruit quality.

[0024] (3) In the present invention, the overexpression of the tyrosine decarboxylase gene MdTyDC induces the transduction of ethylene biosynthesis and signal genes, thereby shortening the ripening time of the fruit, providing an important theoretical basis for accelerating the development process of the fruit.

[0025] (4) The present invention obtains MdTyDC overexpression transgenic materials through transgenic technology, preliminarily elucidates the function and application of the tyrosine decarboxylase gene MdTyDC in regulating aging, promoting plant flowering and fruit ripening, and laying a theoretical foundation for improving fruit quality and preserving freshness. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the identification result of MdTyDC overexpressing transgenic plants; A: the identification result of DNA of resistant buds of apple plants and the quantitative result of resistant buds of apple plants, B: the identification result of DNA of resistant buds of Arabidopsis and tomato plants; the values ​​are the mean ± SD of 3 replicates; one-way ANOVA (Tukey's multiple range test) was used, * indicates that P < 0.05 is statistically significant; **, P < 0.01;

[0027] Figure 2is the qRT-PCR analysis of MdTyDC expression in apple leaves; A: leaf phenotypes at non-senescence (NS), early senescence (ES) and late senescence (LS) stages, B: relative expression of MdTyDC, C: MdTyDC response to water, 30 μM abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC), methyl jasmonate (MeJA) and salicylic acid (SA) treatment for 8 h, D: expression of MdTyDC after treatment with 30 μM AC for different time periods as control; values ​​are the mean ± SD of 3 replicates; one-way ANOVA (Tukey's multiple range test) was used, * indicates, P < 0.05 indicates statistically significant difference; **, P < 0.01;

[0028] Figure 3 MdTyDC overexpression promotes apple leaf senescence; A: Phenotypes of wild type (WT) and transgenic apple leaves after 0 and 14 days of darkness treatment, B: Total chlorophyll content, C: Phenotypes of wild type (WT) and transgenic apple leaves after 0 and 14 days of ACC treatment, D: Total chlorophyll content; Values ​​are the mean ± SD of 3 replicates; One-way ANOVA (Tukey's multiple range test) was used, * indicates that P < 0.05 is statistically significant; **, P < 0.01;

[0029] Figure 4 The relative expression of senescence-related genes MdSAG12, MdSAG18, MdSAG20, MdSAG29, MdPAO, and MdSEN4 in wild-type and transgenic apple leaves was detected by real-time fluorescence quantitative PCR (qRT-PCR); A: expression of SAG in apple leaves treated with dark for 14 days, B: expression of SAG in apple leaves treated with ACC for 14 days; values ​​are the mean ± SD of 3 replicates; one-way analysis of variance (Tukey's multiple range test) was used, * indicates that P < 0.05 is statistically significant; **, P < 0.01;

[0030] Figure 5 Phenotypic observation of transgenic Arabidopsis and tomato; A: 4-week-old Arabidopsis leaf phenotype, B and C: 4-week-old Arabidopsis leaf length and width, D: Arabidopsis flowering days record, E: 40-day-old tomato leaf phenotype, F: 40-day-old tomato stem height, G: tomato flowering days record; Values ​​are the mean ± SD of 3 replicates; One-way ANOVA (Tukey's multiple range test) was used, * indicates, P < 0.05 indicates statistically significant difference; **, P < 0.01;

[0031] Figure 6MdTyDC overexpression promotes Arabidopsis leaf senescence; A: Phenotypic observation of 4-week-old Arabidopsis detached leaves under darkness and ACC treatment at 0 and 4 days, B: Total chlorophyll content of 4-week-old Arabidopsis detached leaves under darkness and ACC treatment at 0 and 4 days, C: Phenotypic observation of 4-week-old Arabidopsis plants under darkness and ACC treatment at 0 and 7 days, D: Total chlorophyll content of 4-week-old Arabidopsis plants under darkness and ACC treatment at 0 and 7 days; Values ​​are the mean ± SD of 3 replicates; One-way ANOVA (Tukey's multiple range test) was used, * indicates that P < 0.05 is statistically significant; **, P < 0.01;

[0032] Figure 7 is the expression of SAG in 4-week-old Arabidopsis plants under dark and ACC treatment for 0 and 7 days; AD: expression levels of AtSAG12, AtSAG29, AtPAO and AtSEN4; values ​​are the mean ± SD of 3 replicates; one-way ANOVA (Tukey's multiple range test) was used, * indicates that P < 0.05 is statistically significant; **, P < 0.01;

[0033] Figure 8 Phenotypic analysis of wild type and transgenic tomatoes; A: Phenotypes of mature fruits of wild type and MdTyDC transgenic plants, B: Time record from pollination to maturity, C: Determination of total carotenoid content in WT and MdTyDC transgenic plants at stages 4-6, DF: Expression of carotenoid biosynthesis genes SlPSY1, SlPDS and SlZDS; Values ​​are the mean ± SD of 3 replicates; One-way ANOVA (Tukey's multiple range test) was used, * indicates that P < 0.05 is statistically significant; **, P < 0.01;

[0034] Fig. 9 The expression levels of ethylene biosynthesis and signaling genes (SlACS2, SlACO1, SlACO3, SlRIN, SlE4, SlE8, SlERF1 and SlERF5) in WT and MdTyDC transgenic fruits at stages 4-6; the values ​​are the mean ± SD of 3 replicates; one-way ANOVA (Tukey's multiple range test) was used, * indicates a statistically significant difference with P < 0.05; **, P < 0.01. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0036] Tyrosine decarboxylase (TyDC) is a 5'-pyridoxal phosphate (PLP)-dependent decarboxylase that can catalyze the decarboxylation of L-tyrosine to form tyramine. It is a key enzyme in the dopamine anabolic pathway in plants, a common enzyme ubiquitous in plants, and mediates many secondary reactions. In previous studies, the expression of MdTyDC was involved in many abiotic and biotic stresses and plant development. However, little is known about its role in plant aging. Based on this, the present invention provides an application of an apple tyrosine decarboxylase gene MdTyDC in regulating aging, promoting plant flowering and fruit ripening.

[0037] Example 1

[0038] Related treatments for apple, Arabidopsis, and tomato plants

[0039] 1. Genetic transformation of apple

[0040] (1) The MdTyDC gene sequence was retrieved using the apple genome database. The MdTyDC gene sequence number is MDP0000850540.

[0041] The primers for MdTyDC gene detection are:

[0042] Forward primer: 5'-ATGCCTAGCGCTCTGGACCCTG-3', recorded as SEQ ID NO.51;

[0043] Reverse primer: 5'-TTATATATTATTTTTCATGAGGATGGCATCTG-3', recorded as SEQ IDNO.52.

[0044] PCR amplification was performed using cDNA from the leaves of 'Golden Crown' apple as a template. The target band was detected by gel electrophoresis, and the correct target band was recovered. After adding the A tail, the product was connected to pMD19T-simple and transformed into Escherichia coli (DH5α competent state). After PCR detection of single colonies, the single clones with the correct bands shown by gel electrophoresis were shaken and sequenced. The correctly sequenced plasmid was connected to the overexpression vector pGWB411 based on the Gateway system to construct the overexpression vector.

[0045] pGWB411 overexpression primers Sequencing primers are:

[0046] attB1 Forward primer: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCCTAGCGCTCTGGACCCTG-3', recorded as SEQ ID NO.53;

[0047] attB2 Reverse primer: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTATATTATTTTTCATGAGGATGGCA-3', recorded as SEQ ID NO.54.

[0048] (2) The successfully constructed overexpression vector was transformed into the EHA105 Agrobacterium strain, and the single Agrobacterium colony that was positive in PCR detection was used for the subsequent apple leaf transformation experiment. The transformation method is as follows:

[0049] Resuspend and activate Agrobacterium in M20 and dilute OD 600 To 0.6-0.8 for later use. Take a healthy leaf of 'GL-3' grown in tissue culture for 30 days, place it in M20 resuspension, make 3-4 wounds with a blade (cut the veins without damaging the edges), and soak for 8-10 minutes. Then dry the bacterial solution on sterile filter paper, place the leaf with the back facing up on a co-culture plate and culture it in the dark using co-culture medium. After 3 days, rinse the leaves with sterile cephalosporin water 3 times, 8 minutes each time, dry the water and transfer to a delayed plate and culture it on a delayed screening medium for 2 days. Transfer the leaves to kanamycin screening medium and culture them in the dark for 3-4 weeks, and then culture them in the light after resistant buds appear. After the resistant buds grow up, move them to a subculture medium containing kanamycin and continue screening and culture for 20-30 days. The formula of the M20 resuspension and culture medium used is as follows:

[0050] M20 resuspension: 4.43 g MS powder, 20 g sucrose, 20 mM sodium citrate, 0.1 mM acetosyringone + 1 mM betaine, dissolved in 1 L distilled water, pH 5.8;

[0051] Co-culture medium: MS 4.43 g, sucrose 30 g, agar 7 g, 2.0 mg / LTDZ, 0.5 mg / L NAA, 1 M NaOH 380 μL dissolved in 1 L distilled water, autoclaved at 121 °C for 20 min, cooled to room temperature, and then added with 0.1 mM acetosyringone + 1 mM betaine in an ultra-clean workbench;

[0052] Delayed screening medium: MS 4.43 g, sucrose 30 g, agar 7 g, 2.0 mg / L TDZ, 0.5 mg / L NAA, 1 M NaOH 380 μL dissolved in 1 L distilled water, autoclaved at 121 °C for 20 min, cooled to room temperature, and added 250 mg / L cephalosporin in a clean bench;

[0053] Kanamycin screening medium: MS 4.43g, sucrose 30g, agar 7g, 2.0mg / LTDZ, 0.5mg / LNAA, 1MNaOH 380μL dissolved in 1L distilled water, autoclaved at 121℃ for 20min, cooled to room temperature, and then added with 25mg / L kanamycin and 250mg / L cephalosporin in a clean bench;

[0054] Subculture screening medium: MS 4.43g, sucrose 30g, agar 8g, 0.3mg / L 6-BA+0.2mg / LIAA, 25mg / L kanamycin, 1M NaOH 380μL dissolved in 1L distilled water.

[0055] (3) Identification of transgenic apple plants overexpressing MdTyDC

[0056] The resistant buds after screening were taken and identified at the DNA and RNA levels.

[0057] 2. Arabidopsis transformation

[0058] The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 and wild-type Arabidopsis thaliana (Col-0) was infected using the "flower dipping method" with a resuspension solution of 5% sucrose solution + 0.02% surfactant Silwet L-77. T1 seeds were harvested and surface sterilized and sown to a Kan (50 mg L -1 ) on MS medium, vernalized at 4℃ for 3 days, moved to the tissue culture room, grown for 2 weeks, and transplanted the seedlings that grew normally. After DNA and RNA level detection, the positive seedlings T2 generation seeds were collected. The T2 generation seeds were screened in the same way, and the homozygous T3 generation seeds were collected for subsequent experiments.

[0059] 3. Tomato Transformation

[0060] Tomato (cvAilsa Craig) transformation was performed at Wuhan Double Helix Biotechnology Co., Ltd. (https: / / www.bioon.com.cn / company / index / 63842b02387d).

[0061] In the present invention, we obtained three transgenic apple lines overexpressing MdTyDC: OE-2, OE-5 and OE-7; three transgenic Arabidopsis lines overexpressing MdTyDC: OE-1, OE-3 and OE-4; and two transgenic tomato lines overexpressing MdTyDC: OE-10 and OE-11.

[0062] Figure 1 The results of the identification of the transgenic plants overexpressing MdTyDC in Example 1 of the present invention; A is the DNA result of the resistant buds of apple plants and the quantitative result of the resistant buds of apple plants. The test results show that the expression of the MdTyDC gene is upregulated by 6 to 9 times compared with WT. B is the DNA result of the resistant buds of Arabidopsis and tomato plants.

[0063] 4. Related Processing

[0064] 1. Chlorophyll content determination and qRT-PCR determination

[0065] Six-month-old apple plants and four-week-old Arabidopsis detached leaves in the same position were placed in the dark for senescence phenotype observation (dark induction). In order to make darkness and ACC (1-aminocyclopropane-1-carboxylic acid) coexist, the detached leaves of apple and Arabidopsis were placed in a plate with 50μM ACC solution under dark conditions (ACC treatment). When the senescence phenotype appeared, all leaves were placed at 24°C for chlorophyll content determination and qRT-PCR determination.

[0066] For ACC treatment of Arabidopsis seedlings, 4-week-old Arabidopsis plants were sprayed with 50 μM ACC solution and placed under weak light conditions (photon flux density: 20 μmol·M -2 ·s -1 After phenotypic recording, leaves from the same location were used for chlorophyll content and qRT-PCR determination.

[0067] 2. Phenotypic observation of MdTyDC overexpression in Arabidopsis and tomato.

[0068] Phenotypic observation and flowering time recording were performed using Arabidopsis and tomato overexpressing MdTyDC. Leaf length and width were measured using four-week-old Arabidopsis plants, and stem height was measured using one-month-old tomato plants. Flowering time was calculated every 6 hours when the petals of Arabidopsis and tomato were fully open.

[0069] 3. Fruit maturity analysis.

[0070] T3 seeds of tomato lines overexpressing MdTyDC (OE) and wild-type (WT) were sown to measure differences during fruit ripening. The experiment was repeated three times to determine pollination and ripening times. Six stages were defined during fruit ripening, with stage 1 being fruits at the immature stage 18 days after pollination, stage 2 being fruits at the ripe stage 28 days, stage 3 being the fruit coloring period of the OE line at 38 days, stage 4 being the fruit coloring period of the WT line at 43 days, and stage 6 being stage 4 + 10 days of the OE line. Fruits from stage 4 to stage 6 were selected for total carotenoid concentration and qRT-PCR measurements.

[0071] Total chlorophyll content was determined by spectrophotometry: 8 mL of 80% acetone was added to 0.1 g of fresh leaves for at least 24 h to extract the pigment, during which the mixture was shaken 3-4 times until the leaves turned white. The optical density was measured at 663, 645 and 470 nm using a UV 2250 spectrophotometer (Shimadzu, Kyoto, Japan). Calculation formula:

[0072] Ca (mg / g fresh weight) = (12.7D 663 -2.69D 645 )×V / 1000W

[0073] C b (mg / g fresh weight) = (22.9D 645 -4.68D 663 )×V / 1000W

[0074] C 总 (mg / g fresh weight) = (20.0D 645 +8.02D 663 )×V / 1000W

[0075] Cx·c (mg / g fresh weight) = (1000D 470 -3.27Ca-104C b ) / 229×V / 1000W

[0076] Where: Ca and Cb are the concentrations of chlorophyll a and b respectively; C 总 is the total chlorophyll concentration; Cx·c is the total carotenoid concentration; D 663 , D 645 and D 470 are the optical densities of chloroplast pigment extracts at wavelengths of 663 nm, 645 nm and 470 nm, respectively; v is the volume of the extract (ml), W is the weight of the leaf extract (g), mg / g fresh weight = mg / gFW

[0077] qRT-PCR The plant RNA extraction kit (Vicband, Hong Kong, China) was used as described. MDH, AtActin2, and SlCAC were used as reference genes for apple, Arabidopsis, and tomato to calculate the ΔCt value (Perini eral. 2014). The sequences of the primers are shown in Table 1.

[0078] All data were analyzed based on Tukey’s multiple range test to determine the differences between values ​​using SPSS 19.0 software (IBM Corp., Armonk, NY, USA), and P values ​​< 0.05 were considered significant.

[0079] Table 1 Primers used for real-time fluorescence quantitative RT-PCR

[0080]

[0081]

[0082] Example 2

[0083] Analysis of the expression pattern of MdTyDC during apple senescence

[0084] The color change of apple leaves during senescence is significant, from the non-senescent (NS) stage to the early senescent (ES) stage and then to the late senescent (LS) stage ( Figure 2 A). The expression of MdTyDC is highly induced by the aging process. From the NS stage to the ES stage, the expression of MdTyDC increases significantly by about 5 times, and then from the ES stage to the LS stage, the expression of MdTyDC increases by about 7 times ( Figure 2 B), the results are consistent with the discoloration of apple leaves.

[0085] In addition, since plant hormones such as ABA, ethylene, jasmonic acid (MeJA) and salicylic acid (SA) are involved in the senescence process and accelerate leaf senescence, we detected the expression of MdTyDC under various plant hormone treatments, among which 1-aminocyclopropyl-1-carboxylic acid (ACC) is a direct precursor of ethylene, and changes in ACC content are usually used to evaluate the response of plants to ethylene. The results showed that ABA, ACC, MeJA and SA treatments increased the expression level of MdTyDC, and the expression induced by ACC was significantly increased compared with other treatments ( Figure 2 C). In ACC treatment, the expression of MdTyDC increased significantly from 1 to 12 hours and reached a peak at 12 hours ( Figure 2 D) These data indicate that MdTyDCs are responsive to phytohormone induction and are highly induced by ACC.

[0086] Example 3

[0087] Overexpression of MdTyDC promotes leaf senescence in apple

[0088] (1) Overexpression of MdTyDC promotes apple leaf senescence and accelerates ACC-induced leaf senescence

[0089] Three apple lines (OE-2, 5, and 7) overexpressing MdTyDC were used to investigate its role in leaf senescence. As expected, after 14 days of dark induction, apple leaves in the MdTyDC overexpressing lines showed obvious senescence symptoms compared to WT ( Figure 3 A). The color change in the OE line was enhanced, which was consistent with the sharp decrease in chlorophyll content in the OE line compared with that in the WT ( Figure 3 B). In addition, the color change of OE treated with ACC was much more significant than that of WT ( Figure 3 C). Similarly, the chlorophyll content in the OE line was significantly lower than that in the WT ( Figure 3 D) These results indicate that darkness induces profound senescence in the OE line, and ACC treatment accelerates and exacerbates this process.

[0090] (2) MdTyDC overexpression leads to higher expression of SAG genes

[0091] The expression of some senescence-related genes was used as markers in the senescence process. Therefore, we examined the gene expression in apple leaves under all dark and ACC treatments by qRT-PCR analysis. The expression of senescence-related genes, such as MdSAG12, MdSAG18, MdSSAG20, MdSAG29, MdPAO, and MdSEN4, was significantly increased in the OE line compared with the WT after 10 days of dark induction ( Figure 4 A). The changes of OE and WT in ACC treatment were similar ( Figure 4 B). It is noteworthy that the expression of some genes in ACC treatment was much higher than that in darkness alone ( Figure 4 ). For example, under darkness induction, the expression of MdPAO in the OE line increased 3-5 times compared with the WT, while under ACC treatment, the expression in the OE line increased 25-35 times compared with the WT. The same trend was also found in the expression of MdSAG12, MdSAG18, and MdSEN4. These results suggest that overexpression of MdTyDC promotes leaf senescence by upregulating senescence-related genes. In addition, ACC treatment significantly increased the expression of senescence-related genes in the OE line.

[0092] Example 4

[0093] Overexpression of MdTyDC promotes growth and flowering in Arabidopsis and tomato

[0094] In this study, 4-week-old Arabidopsis plants and 1.5-month-old tomato plants were used for phenotypic observations. In Arabidopsis, overexpression of MdTyDC altered leaf shape, with higher leaf length and width ( Figure 5 AC). Moreover, the MdTyDC overexpression line shortened the flowering time of Arabidopsis by at least 6 days ( Figure 5 D). For tomato, overexpression of MdTyDC significantly promoted growth, with plant height being higher than that of WT ( Figure 5 E, F). Similarly, tomato lines overexpressing MdTyDC flowered 3 days earlier than WT ( Figure 5 G) These results suggest that MdTyDC may play a role in the regulation of flowering in plants.

[0095] Example 5

[0096] Overexpression of MdTyDC promotes leaf senescence in Arabidopsis and tomato

[0097] To further characterize the role of MdTyDC in ACC-induced leaf senescence, we used three Arabidopsis plants overexpressing MdTyDC (OE-1, 3, and 4). First, detached leaves were examined under darkness induction. WT leaves showed a senescent phenotype, with yellow and necrotic spots as expected. However, detached leaves treated with ACC or overexpressing MdTyDC enhanced the senescent phenotype ( Figure 6 A). After ACC treatment, detached leaves overexpressing MdTyDC showed a more obvious senescence phenotype. In addition, the decrease in chlorophyll was similar to the trend of senescence process, and WT and MdTyDC overexpressing detached leaves treated with ACC showed similar lower chlorophyll content. In addition, ACC-treated leaves overexpressing MdTyDC showed a sharp decrease in chlorophyll content ( Figure 6 B).

[0098] To further explore the interaction between MdTyDC and ACC-induced senescence, four-week-old Arabidopsis thaliana that had been naturally aged were subjected to darkness and ACC treatment. After 4 days of darkness-induced treatment or 5 days of ACC treatment, transgenic and wild-type leaves treated with ACC showed similar senescence phenotypes, and the transgenic leaves had a more profound senescence phenotype with the presence of ACC treatment ( Figure 6 C). The changes in chlorophyll content are consistent with the above senescence phenotype ( Figure 6 D). Consistent with the earlier observations, the chlorophyll content was ranked from high to low as follows: WT leaves under dark induction, WT leaves treated with ACC, OE lines under dark induction, and OE lines treated with ACC. These results suggest that MdTyDC and ACC enhance each other in inducing leaf senescence.

[0099] (2) Overexpression of MdTyDC in Arabidopsis induces expression of SAG genes

[0100] We also examined the expression of senescence-related genes during natural senescence in 4-week-old Arabidopsis. The results showed that AtSAG12, AtSAG29, AtPAO, and AtSEN4 were highly induced in the overexpression lines compared with the wild type under darkness induction and ACC treatment ( Figure 7 Among them, the expression of AtSAG12, AtSAG29 and AtPAO increased more significantly ( Figure 7 AC).

[0101] Example 6

[0102] MdTyDC overexpression promotes fruit ripening

[0103] (1) MdTyDC overexpression promotes fruit ripening

[0104] MdTyDC affects flowering time in Arabidopsis and tomato, which prompted us to explore whether MdTyDC affects fruit ripening. Next, we obtained two independent tomato OE lines (OE-10 and 11) to further confirm the role of MdTyDC in fruit ripening. Stage 3, when the fruit color changes from green to yellow (coloring period), was 37 days in the OE line. Compared with the WT, the time from pollination to stage 3 was shortened in the OE line, two and three days earlier ( Figure 8 A, B). At the maturity stage of 4 to 5, the carotenoid content in the OE line was higher than that in the WT ( Figure 8 C). At stage 6, there was no significant difference between WT and OE lines, which may be due to the same degree of fruit maturity. To further confirm that MdTyDC affects carotenoid accumulation and fruit ripening, the relative expression of carotenoid biosynthesis-related genes was detected by qRT-PCR. The results showed that at stages 4 to 6, the expression of SlPSY1 and SlPDS was significantly increased compared with WT ( Figure 8 D, E). Although only OE-10 had higher MdZDS expression at stage 5, the expression levels at stages 4 and 6 were higher than those of WT during maturity ( Figure 8 F). These results suggest that overexpression of MdTyDC promotes fruit ripening by shortening the time from pollination to coloration stage and promoting carotenoid accumulation and expression of related genes.

[0105] (2) Overexpression of MdTyDC in tomato increases gene expression of ethylene biosynthesis and ethylene response genes

[0106] The hormone ethylene plays an active role in the ripening of climacteric fruits. The release of ethylene is usually accompanied by the upregulation of the expression of ethylene biosynthesis and ethylene response genes. Compared with WT, the expression of SlACS2, SlACO3, SlRIN, SlERF1 and SlERF5 was higher in stages 4 to 6 ( Fig. 9 For S1ACO1, S1E4, and S1E8, higher expression than in WT was detected in stages 4 and 5, however, at stage 6, there was no significant difference between WT and OE lines ( Fig. 9 A, B). Our results showed that overexpression of MdTyDC promoted fruit ripening by upregulating ethylene biosynthesis and ethylene-responsive genes.

[0107] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0108] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An application of a tyrosine decarboxylase gene in regulating plant growth, characterized in that: The gene is MdTyDC, the gene sequence number is MDP0000850540, and the plant is tomato or Arabidopsis; The plant growth refers to the senescence of Arabidopsis and tomato leaves, the flowering of Arabidopsis and tomato, or the ripening of tomato fruits; The regulating plant growth is to promote Arabidopsis and tomato leaf senescence by overexpressing MdTyDC; The regulating plant growth is to promote flowering of Arabidopsis and tomato by overexpressing MdTyDC; The regulating plant growth is promoting tomato fruit ripening by overexpressing MdTyDC.

2. The use according to claim 1, characterized in that: Overexpression of MdTyDC and ACC treatment jointly promoted leaf senescence in Arabidopsis and tomato.

3. The use according to claim 1, characterized in that: Overexpression of MdTyDC shortens the time from pollination to fruit coloring and promotes tomato fruit ripening.

4. The use according to claim 1, characterized in that: The method for overexpressing MdTyDC comprises the following steps: The cDNA of plant leaves containing the MdTyDC gene was used as a template for PCR amplification to obtain the amplified product; The amplified product was connected to the overexpression vector pGWB411 to obtain the overexpression vector; The overexpression vector is used to transform Agrobacterium to upregulate the expression level of the MdTyDC gene.

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