CN117051012A - PAP5 gene for cultivating color-leaf poplar and application thereof - Google Patents

Abstract

The invention provides a PAP5 gene for cultivating colored-leaf poplar and application thereof, belonging to the technical field of plant genetic engineering. The invention can obviously change the leaf color of the 741 poplar by over-expressing the PAP5 gene of the full red poplar (middle forest 2025 poplar bud variant) in the poplar, and cultivate the transgenic 741 poplar with red leaf color of the full plant; compared with the control plants, the PAP5 gene over-expression plants accumulate a large amount of pigment in the epidermal tissues of stems, leaves, veins and petioles. The invention discovers that the full red poplar PAP5 gene is excessively expressed in poplar, can cultivate a new poplar germplasm material with obviously changed leaf color, and lays a foundation for cultivating color leaves Yang Xinpin. The achievement of the invention greatly promotes the cultivation of tall and fast-growing color tree, and has important function for promoting the research and development and utilization of color tree in China.

Description

PAP5 gene for cultivating color-leaf poplar and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a PAP5 gene for cultivating colored-leaf poplar and application thereof.

Background

The colorful plants have bright colors and great application value in enriching the colors of the light flowers and the seasons of the city, improving the ecological environment of the city and improving the landscape value of the city. The main cultivated colored-leaf tree species in China at present mainly comprise low-level arbor or shrubs (such as purple leaf plums, golden leaf glossy privet fruits and the like) and seasonal color-changing leaf tree species (such as maples and the like), and the tall arbor which shows a stable colored-leaf phenotype in the whole growing period is lacked. Poplar is an important afforestation tree species in China, and is widely applied to landscape design and landscaping. The cultivation of the color leaf poplar variety with bright color and stable color character plays an important role in promoting the quality improvement of the color leaf tree germplasm and the development and utilization of the color leaf tree. The breeding work of the color leaf poplar variety is started from the beginning of the century in China, the variety breeding is mainly carried out through screening of natural bud variants, and new varieties such as Quan Gongyang, jin Gongyang and the like are sequentially examined and approved. However, these varieties of colored-leaf poplar require maintenance of color traits by stumping or topping each year in production, and at present, no colored leaf Yang Pinchong capable of maintaining stable colored-leaf traits throughout the growing period is seen on the market.

Disclosure of Invention

The invention provides a PAP5 gene for cultivating colored-leaf poplar and application thereof, wherein the leaf color of poplar can be obviously changed by over-expressing Quan Gongyang (middle forest 2025 poplar bud variant) PAP5 gene in 741 poplar, transgenic 741 poplar with red leaf color of whole plant is cultivated, and the red intensity of upper tender leaves is higher than that of middle lower leaves.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a PAP5 gene for cultivating color-leaf poplar, wherein the nucleotide sequence of the PAP5 gene is shown as SEQ ID NO. 1.

The invention provides application of the PAP5 gene in breeding poplar with a colored leaf phenotype.

The invention provides application of the PAP5 gene in improving the anthocyanin-3-O-rutinoside content of poplar.

The invention provides a protein encoded by the PAP5 gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention provides a recombinant vector, which is obtained by inserting the PAP5 gene into a plant binary expression vector delta pCAMBIA1302.

The invention provides a method for obtaining poplar with a colored leaf phenotype, which comprises the following steps: the recombinant vector is transformed into poplar by agrobacterium-mediated method to obtain poplar with color leaf phenotype.

Preferably, the agrobacterium comprises agrobacterium EHA105.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, an agrobacterium-mediated genetic transformation method is utilized to transfer the anthocyanin biosynthesis key regulatory gene PAP5 into the poplar fine variety 741 poplar, so that the leaf color of the poplar can be obviously changed, and the transgenic 741 poplar with red leaf color of the whole plant is cultivated; compared with a control plant, a large amount of pigment is accumulated in the epidermal tissue of the stem, leaf vein and leaf stalk of the PAP5 gene over-expression plant, and the content of anthocyanin Cyanidin-3-O-rutinoside in the body is obviously improved. The transgenic 741 poplar has stable color character and does not change along with the growth and development of plants.

The invention discovers that the full red poplar PAP5 gene is over-expressed in poplar, a new poplar germplasm material with obviously changed leaf color can be cultivated, and stable color leaf character is maintained in the whole growth period. The 741 poplar leaves over-expressing the PAP5 gene of the invention are red, wherein the red intensity of the upper tender leaves is higher than that of the middle and lower leaves, the color is gorgeous, a foundation is laid for cultivating the Yang Xinpin species of the color leaves, and meanwhile, the cultivation of tall and fast-growing color leaf trees is greatly promoted, thus having important function for promoting the research, development and utilization of the color leaf trees in China.

Drawings

FIG. 1PAP5 overexpression vector construction flow chart.

FIG. 2 shows a gel electrophoresis pattern of a PAP5 gene-transferred positive plant (M is DL2000Marker;1,2,3 indicates amplification products using genomic DNAs of candidate transgenic lines L2, L14 and L15 as templates, respectively, with a size of 1852bp; and WT indicates amplification products using poplar DNA of a recipient plant as a template).

FIG. 3 results of RT-qPCR detection of the relative expression levels of PAP5 in control and transgenic plants.

FIG. 4PAP5 over-expression plant phenotype results.

FIG. 5 anatomical analysis of PAP5 over-expression plants (A and E are cross-sections of control and PAP5 transgenic plant stems, respectively; B and F are cross-sections of control and PAP5 transgenic plant petioles, respectively; C and G are cross-sections of control and PAP5 transgenic plant veins, respectively; D and H are cross-sections of control and PAP5 transgenic plant leaves, respectively; scale: 200 μm).

FIG. 6 UPLC-MS/MS analysis results of PAP5 transgenic plant extracts (A is UPLC chromatogram of control and transgenic plant leaf extracts; B is mass spectrum of peak a1 and cyanidin-3-O-rutinoside standard).

Detailed Description

The invention provides a PAP5 gene for cultivating a color leaf Yang Shuxin variety, and the nucleotide sequence of the PAP5 gene is shown as SEQ ID NO. 1. The PAP5 gene is derived from all red poplar, quan Gongyang is a bud variant of the woody 2025 poplar in the black Yang Pinchong. The amino acid sequence of the protein encoded by the PAP5 gene is MVSSSGIRKGAWTREEDILLRDCVEKYGEGRWHQVSS KAGLNRCRKSCRLRWLSYLKPGIKRGQYSEDEEDLIIKLHRLLGNRWSLIAGRLPGRTANDLKNYWNTNLSKKVVSGTREAQTKPEPKAITKANIIKPRPHKFKSLCWLGGKGIPFFNGGFQYGYDLCKPWSTSALSPSDIIEVESMRWESLLDDKEISVSSNTGCLRSGSESDQEPIKSLFAEDSAPEGMRIGDVFCEQGQHCWSGNSFDAADLWNLVNT (SEQ ID NO: 2).

In the present invention, the PAP5 gene is amplified by a forward primer (5 '-3'): ATGCACGTCGCTGTAGTTAGA (SEQ ID NO: 3) and reverse primer (5 '-3'): GCCTGAGACGTGGAACTTATT (SEQ ID NO: 4) by PCR amplification. The PCR amplification system (25. Mu.L) of the present invention: gold medal Mix (Green) ^a 22. Mu.L, 1.0. Mu.L of forward primer (2.0 mM), 1.0. Mu.L of reverse primer (2.0 mM) and 1. Mu.L of template (cDNA). The PCR amplification procedure of the present invention: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10sec, annealing at 55-58℃for 10sec, extension at 72℃for 1-2min,35 cycles; extending at 72 ℃ for 5min; and (5) storing at 16 ℃.

The invention provides application of the PAP5 gene in breeding poplar with colored leaf phenotype. The over-expression of the PAP5 gene in poplar can cause the poplar leaf to turn red.

The invention provides application of the PAP5 gene in improving the anthocyanin-3-O-rutinoside content of poplar. The over-expression of the PAP5 gene can cause the increase of anthocyanin Cyanidin-3-O-rutinoside content in poplar leaves.

The invention provides a recombinant vector, which is obtained by inserting the PAP5 gene into a plant binary expression vector delta pCAMBIA1302. The plant binary expression vector delta pCAMBIA1302 is obtained by modifying a commercial vector pCAMBIA1302. The nucleotide sequence of the plant binary expression vector delta pCAMBIA1302 is shown as SEQ ID NO. 13.

The invention provides a method for obtaining poplar with a colored leaf phenotype, which comprises the following steps: the recombinant vector is transformed into poplar by agrobacterium-mediated method to obtain poplar with red leaf phenotype. The agrobacterium of the invention includes agrobacterium EHA105.

In the present invention, all reagents are commercially available products well known to those skilled in the art unless specified otherwise.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 cloning of the full-Length coding sequence of poplar PAP5

1. Total RNA extraction

The total RNA of poplar was extracted by RNAprep Pure Plant Kit (Tiangen, beijing) and the specific steps were carried out according to the instructions.

2. Reverse transcription reaction system and program

Total RNA was reverse transcribed into cDNA using RNAPCR Kit (AMV) Ver 3.0 (TaKaRa). The reaction system is as follows: 5 XBuffer 2.0. Mu.L; dNTP mix (10 mM) 1.0. Mu.L; AMV Reverse Transcriptase (5U/. Mu.l) 0.5. Mu.l; oligo (dT) 18 (2.5 pmol/. Mu.l) 0.5. Mu.l; RNase Inhibitor (40U/. Mu.l) 0.25. Mu.L; total RNA 5.75. Mu.L. The reaction procedure is: 30 ℃ for 10min;45 ℃ for 30min;99 ℃ for 5min;15 ℃ for 5min.

PCR amplification of DNA fragments

Amplification system (25 μl): gold medal Mix (Green) ^a : 22. Mu.L, forward Primer (2.0 mM): 1.0 μl, reverse Primer (2.0 mM): 1.0 μl, template (genomic DNA or cDNA): 1 μl; amplification procedure: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10sec, annealing at 55-58℃for 10sec, extension at 72℃for 1-2min,35 cycles; extending at 72 ℃ for 5min; and (5) storing at 16 ℃.

4. Leaf tissue of Quan Gongyang seedlings was collected and ground into powder with liquid nitrogen. Total RNA was extracted using RNAprep Pure Plant Kit (Tiangen, beijing) as described in step 1 of this example. Total RNA was reverse transcribed to cDNA using RNAPCR Kit (AMV) Ver 3.0 (TaKaRa), the reaction system and procedure were as described in step 2 of this example. Specific primers were designed using the populus tomentosa reference genomic sequence (table 1), and the full-length coding sequence of Quan Gongyang PAP5 was PCR amplified using cDNA as template: ATGGTGAGTTCATCAGGAATAAGGAAAGGTGCATGGACCAGAGAGGA AGATATACTTCTAAGGGATTGCGTTGAGAAATATGGTGAAGGAAGATGGCATCAAGTTTCTTCCAAAGCAGGCTTGAATAGATGCAGGAAAAGCTGCAGGTTGAGGTGGTTGAGTTATCTCAAGCCAGGTATCAAGAGAGGACAGTATTCTGAGGACGAAGAAGACTTGATTATCAAGCTACACAGGTTGCTTGGCAATAGGTGGTCATTGATTGCTGGTAGACTTCCAGGAAGAACAGCAAATGATTTAAAGAATTATTGGAACACAAACCTGAGTAAGAAGGTGGTTTCTGGCACCAGAGAAGCTCAAACAAAACCAGAACCAAAAGCAATAACAAAAGCCAACATAATAAAGCCTCGACCTCATAAATTCAAAAGTTTATGCTGGTTAGGAGGAAAAGGTATTCCATTTTTCAATGGTGGTTTTCAATATGGGTACGATCTTTGTAAGCCATGGTCTACATCAGCATTGTCCCCTTCCGATATTATTGAAGTTGAAAGTATGCGGTGGGAAAGCTTGTTAGATGACAAAGAAATTAGTGTATCGAGCAACACCGGATGTCTACGTTCGGGGTCAGAATCTGACCAAGAGCCCATCAAAAGTCTTTTTGCAGAGGACAGCGCTCCAGAAGGGATGAGAATTGGAGACGTGTTCTGTGAACAAGGACAGCATTGTTGGAGTGGCAATTCTTTTGATGCAGCAGACCTTTGGAATTTAGTCAATACTTGA (SEQ ID NO: 1). The PCR amplification system and procedure were as described in step 3 of this example. The PCR amplified product is subjected to agarose gel electrophoresis, and the amplified product with the expected size is subjected to tapping purification and then is connected with a pEASY-Blunt carrier for sequencing. The full-length PAP5 coding sequence was determined by sequence analysis.

TABLE 1 cloning primer information for PAP5 coding sequence

EXAMPLE 235S PAP5 overexpression vector construction

The binary vector delta pCAMBIA1302 is taken as a framework to construct a 35S/PAP 5 overexpression vector. Plant binary expression vector Δpcambia1302 was engineered from the commercial vector pCAMBIA1302. The nucleotide sequence of the plant binary expression vector delta pCAMBIA1302 is shown as SEQ ID NO. 13.

The full-length coding sequence of PAP5 is cloned into a delta pCAMBIA1302 vector by adopting a homologous recombination method, and the specific steps comprise: (1) The ΔpCAMBIA1302 vector was digested with the restriction enzymes BamHI and SalI, available from NEB company, and the digestion system and procedure were performed as described. (2) Specific primers containing homology arms were designed (Table 2) and the PAP5 coding sequence was amplified using the full-length PAP5 coding sequence cloned in example 1 as a template. (3) The amplified PAP5 coding sequence was ligated into the ΔpCAMBIA1302 vector using homologous recombinase 2X Assembly Master Mix (Zhongmeitai and Beijing) to obtain a 35S: PAP5 overexpression vector (FIG. 1). The method steps of homologous recombination are carried out by reference to the specification.

TABLE 2 construction of PAP5 overexpression vector primer information

EXAMPLE 3 genetic transformation of poplar

1. Preparation of hormone and antibiotic

Zein (ZT) formulation: dissolving with dilute hydrochloric acid, adjusting pH to 5.80-5.85, filtering, sterilizing, and preserving at 4deg.C.

Acetosyringone (AS) formulation: dimethyl sulfoxide (DMSO) was dissolved and ready to use.

Rifampin (Rif) formulation: dimethyl sulfoxide (DMSO) was dissolved and ready to use.

NAA: dissolving in ethanol, filtering, sterilizing, and storing at 4deg.C.

IBA: dissolving in ethanol, filtering, sterilizing, and storing at 4deg.C.

Cephalosporin (Cef): water-soluble bacteria, filtering, sterilizing, and preserving at-20deg.C.

2. Culture medium formula

WPM resuspension: WPM powder 2.41 g/L+sucrose 30g/L+AS 100. Mu. Mol/L.

CM1: WPM powder 2.41 g/L+sucrose 30g/L+AS 100. Mu. Mol/L+NAA 1 mg/L+ZT2 mg/L+agar powder 6g/L.

CM2: WPM powder 2.41 g/L+sucrose 30g/L+NAA 1mg/L+ZT 2mg/L+Cef400mg/L+Hyg 10 mg/L+agar powder 6g/L.

CM3: WPM powder 2.41 g/L+sucrose 30g/L+NAA 0.1 mg/L+ZT2 mg/L+Cef400mg/L+Hyg 10 mg/L+agar powder 6g/L.

CM4: WPM powder 2.41 g/L+sucrose 30g/L+NAA 0.1 mg/L+Cef400mg/L+Hyg 10 mg/L+agar powder 6g/L.

After each medium was weighed, it was autoclaved at 121℃for 15 minutes. And adding ZT, hyg and Cef when the culture medium is cooled to below 60 ℃.

3. Genetic transformation process

1) Agrobacterium activation: agrobacterium EHA105 carrying the 35S: PAP5 recombinant vector was inoculated into liquid YEB containing 50mg/Lkan and 50mg/L Rif, and cultured with shaking at 28 ℃; after the bacterial liquid grows to OD ₆₀₀ At 0.8, 100-200 μl of bacterial liquid is inoculated into fresh YEB culture medium, and shake-cultured at 28deg.C to OD ₆₀₀ =0.8-1.0; centrifugally collecting thalli, re-suspending thalli by using WPM heavy suspension, and then shake culturing for 1-2h at 28 ℃ until OD ₆₀₀ About 0.6, placed on ice as an aggressive dye solution for conversion.

2) Infection with agrobacterium: cutting upper leaf and stem of tissue culture seedling growing for about 4 weeks into pieces of 0.5X0.5 cm ² And stem segments with the length of 0.5cm are placed in the infection liquid to be suspended for infection for 10-15 minutes.

3) Co-cultivation: the infected explant is placed on filter paper to absorb bacterial liquid, then laid on CM1 culture medium and cultured for 2 days in dark environment at 25 ℃.

4) Selection and culture: the explants were transferred to CM2 medium and cultured in dark at 25℃for 3-4 weeks, and white or pale yellow callus appeared at leaf wounds. Fresh medium was changed every 10-14 days during this period.

5) Adventitious bud induction: the explants with the callus were transferred to CM3 medium and incubated at 25℃for about 3 weeks, with adventitious buds appearing on the callus. Fresh medium was changed every 10-14 days during this period.

6) Adventitious root induction: when adventitious buds grow to 1-2 CM, they are excised and transferred to CM4 medium, and adventitious roots begin to appear about one week.

7) Transplanting: when the seedlings grow to 8-10CM on the CM4 culture medium, transplanting the seedlings into soil for culture.

Example 4 identification of transgenic Positive plants

PCR amplification to detect whether the target Gene is integrated into the recipient plant genome

1) DNA extraction: genomic DNA was used as Plant Genomic DNAKit (Tiangen, beijing) and the detailed procedures were as described in the specification.

2) PCR amplification of DNA fragment: amplification system (25 μl): gold medal Mix (Green) ^a : 22. Mu.L, forward Primer (2.0 mM): 1.0 μl, reverse Primer (2.0 mM): 1.0. Mu.L, template (genomic DNA): 1 μl; amplification procedure: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10sec, annealing at 55-58℃for 10sec, extension at 72℃for 1-2min,35 cycles; extending at 72 ℃ for 5min; and (5) storing at 16 ℃.

3) The genomic DNA of the candidate positive plants was extracted by the procedure of step 1) of this example. Specific primers (Table 3) were designed on both sides of the adjacent multiple cloning sites based on the plant binary expression vector sequences, and PCR amplification was performed using genomic DNA of the candidate plants as templates, according to the procedure of step 2) of this example. The PCR amplified fragment contains the target gene, the agarose gel electrophoresis result of the PCR amplified product is shown in figure 2, the PCR amplified product of the candidate transgenic strain has obvious band, the band size is consistent with that of the PCR product of positive control (plasmid), and the PCR product of negative control (water and WT) has no target band, which indicates that the target gene is successfully integrated into the genome of the receptor plant.

TABLE 3 primer information for detection of PAP5 integration into the recipient plant genome

RT-qPCR detection of PAP5 expression levels in transgenic plants

1) RT-qPCR reaction system and program

First, RNA was reverse transcribed into cDNA using PrimeScriptTM RTreagentKit (Perfect Real Time) (TakaRa), and the reaction system was: 5X PrimeScript Buffer (forReal Time) 2. Mu.L; primeScript RT Enzyme Mix I0.5.5. Mu.L; oligo dT Primer (50. Mu.M) 0.5. Mu.L; random 6mers (100. Mu.M) 0.5. Mu.L; 400ng of total RNA; supplementing RNase Free ddH ₂ O to 10. Mu.L. The reaction procedure is: 15min at 37 ℃; 5sec at 85 ℃.

Using cDNA as template, usingPremix Ex Taq ^TM II (Tli RNaseH Plus), carrying out RT-qPCR reaction, wherein the reaction system is as follows: SYBR Premix Ex Taq II (Tli RNaseH Plus) (2×): 10. Mu.L; PCR forward primer (10. Mu.M): 0.8. Mu.L; PCR reverse primer (10. Mu.M): 0.8. Mu.L; cDNA template [ ]<100 ng): 2. Mu.L; sterilizing water: 6.4. Mu.L. The reaction procedure is: pre-denaturation: 95 ℃ for 30sec; PCR reaction (40 cycles): 95℃for 5sec and 60℃for 30sec; dissolution profile analysis: 95℃for 5sec and 60℃for 60sec.

2) Total RNA from leaves of transgenic poplar and control plants (plants transformed with empty vector in the same manner as in example 3) in the growth period of 2 months was extracted in the same manner as in example 1, step 1, and cDNA was synthesized by reverse transcription in a single strand, as in example 2, step 1). The relative expression level of PAP5 in transgenic plants was detected by RT-qPCR using cDNA as template and specific primers (Table 4) were designed. The RT-qPCR reaction system and procedure are as described in step 2, step 1) of this example. Populus sectional (GenBank No. XM_ 002316253) was used as an internal control and three biological replicates were performed for each transgenic plant. RT-qPCR assay showed that PAP5 was expressed in higher amounts in leaves of transgenic plants compared to control plants (FIG. 3).

TABLE 4 RT-qPCR detection primer information for PAP5 overexpressing plants

Example 5 phenotypic analysis of PAP5 overexpressing plants

The obtained transgenic plants and control plants (plants transformed with empty vectors) are cultured together in a greenhouse, and under the normal culture environment, the whole transgenic plants are red, wherein the red intensity of the upper tender leaves is higher than that of the middle lower leaves, and the transgenic plants are bright in color, compared with the control plants which are green (figure 4).

The stem, leaf, petiole tissues of the control and transgenic plants were further dissected. The results showed that there was a large number of red pigment cells distributed in the epidermis tissue of the stems, veins, petioles and upper and lower epidermis of the leaves of the transgenic plants, whereas the control plants observed only a small number of red pigment cells in the epidermis tissue of the petioles (fig. 5). Anatomical studies indicate that the distribution of a large number of pigment cells in the epidermal tissue imparts a red phenotype to the transgenic plants.

Example 6 analysis of anthocyanin Components and content in PAP5 over-expressed plants

Leaf tissue of control (wild type plant) and transgenic poplar grown for 3-4 months was used to detect anthocyanin components and content by UPLC-MS/MS. The UPLC-MS/MS detection flow is as follows:

1. anthocyanin extraction

Poplar leaves were collected and ground to a powder in liquid nitrogen, and 0.5g of the powder was weighed and resuspended in 1ml of extract (2% methanol formate). The resuspension was incubated for 5 minutes at room temperature and then sonicated for 20 minutes. Centrifuge at 1000rpm for 3 minutes at room temperature and collect the supernatant. Adding petroleum ether with volume of 2 times, vortex oscillating, and extracting nonpolar pigment such as chlorophyll. Repeating the above petroleum ether extraction steps until the petroleum ether at the upper layer is colorless. Supernatant was centrifuged at 12000rpm for 10 minutes, and the supernatant was collected for UPLC-MS/MS analysis.

UPLC-MS/MS detection

Using ACQUITY Ultra Performance Liquid Chromatograph (UPLC I-CLASS, waters) and Xevo ^TM The UPLC-MS/MS analysis was performed by a TQ-MS triple quadrupole mass spectrometer (Waters, milford, USA) system. The chromatographic column is ACQUITYHSS T3 chromatographic column (2.1X100 mm,1.8 μm inner diameter), column temperature set to 35℃and flow rate 0.35mL min ^-1 . The mobile phases were A (water/formic acid; 99.5:0.5; v/v) and B (pure acetonitrile). The gradient elution procedure was demonstrated as a concentration change of solvent B over elution time as follows: 5% b 0min; 10% b 7 min; 30% b 15min; 60% b for 20 min; 90% b 21 min; 5% b 23 min; 5% B25 min. MS detection adopts positive ion mode, capillary voltage is 3.83kV, taper hole voltage is 50V, desolvation gas temperature is 500 ℃, ion source temperature is 150 ℃, desolvation gas flow rate is 995Lh ^-1 The collision gas flow rate is 0.14mL min ^-1 The collision energy was 18eV and the scan range was 100-1200 (m/z). Cyanidin-3-O-rutinoside (Shanghai Mike Lin Shenghua Co., shanghai, china) was used as a standard to quantify the amount of Cyanidin-3-O-rutinoside in control and transgenic poplar.

The results of the UPLC-MS/MS analysis showed that the transgenic plant leaf extract exhibited two major signal peaks at 525nm, with the maximum peak (a 1) occurring around 9.12min, whereas the control plant leaf extract did not detect the visible signal peak (FIG. 6A). MS/MS analysis of the a1 Compound, the MS/MS mass spectrum of the a1 peak shows an m/z 595[ M ]] ⁺ Molecular ion of (C) and an m/z287[ Y0 ]] ⁺ Is shown in FIG. 6B. Based on mass spectral information, a1 was inferred to be cyanidin-3-O-rutinoside. This was confirmed by UPLC-MS/MS analysis of the Cyanidin-3-O-rutinoside standard (FIG. 6B). The present study further quantified the content of cyanidin-3-O-rutinoside in leaves of the overexpressed plants, and the anthocyanin content in the PAP5 gene overexpressed plants was 889-1295 mu g g ^-1 FW. These results demonstrate that over-expression of PAP5 significantly promotes the biosynthesis of anthocyanin Cyanidin-3-O-rutinoside in transgenic poplar.

In conclusion, through the transgenic technology, the full red poplar PAP5 gene is transferred into a poplar fine variety 741 poplar, and is stably expressed in high quantity in 741 Yang Tina, the obtained transgenic 741 Yang Shese presents red, wherein the red intensity of young leaves at the upper part of a plant is higher than that of young leaves at the middle and lower parts of the plant, and the content of anthocyanin 3-O-rutinoside in the body is obviously improved. The transgene 741 Yang Seze obtained by the invention is gorgeous, has better ornamental value and is an excellent new germplasm of the populus with colored leaves.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (7)

1. The PAP5 gene for cultivating color leaf poplar is characterized in that the nucleotide sequence of the PAP5 gene is shown as SEQ ID NO. 1.

2. Use of the PAP5 gene according to claim 1 for breeding poplar with a colored-leaf phenotype.

3. Use of the PAP5 gene according to claim 1 for increasing the anthocyanin-3-O-rutinoside content in poplar.

4. The protein encoded by the PAP5 gene according to claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.

5. A recombinant vector obtained by inserting the PAP5 gene according to claim 1 into a plant binary expression vector ΔpCAMBIA1302.

6. A method for obtaining poplar with a colored leaf phenotype, comprising the steps of: transforming the recombinant vector of claim 5 into poplar by agrobacterium-mediated transformation to obtain poplar with color leaf phenotype.

7. The method of claim 6, wherein the agrobacterium comprises agrobacterium EHA105.