LU102583B1 - Gene smfls for peel coloring and use of the same - Google Patents

Abstract

The invention relates to a key gene SmFLS for peel coloring cloned from eggplant and its use in regulating the peel coloring of eggplant. According to the eggplant genome data, the full-length primer was designed and the key gene SmFLS was cloned. It's nucleic acid sequence is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:2. Using Agrobacterium-mediated method, the SmFLS was transferred into eggplants. The results showed that the flavonol content of the transgenic plants was significantly higher than that of the non-transformed plants, resulting in a lighter peel color. The invention is helpful to further understand the coloring mechanism of eggplant peels, so that biotechnological means can be used to regulate the peel coloring of eggplant and breed new eggplant varieties with uniform coloring.

Description

BL-5204 GENE SMFLS FOR PEEL COLORING AND USE OF THE SAME LU102583 Technical Field of the Invention

[0001] The invention relates to a key gene SmFLS for peel coloring cloned from eggplant and its use in regulating the peel coloring of eggplant.

Background of the Invention

[0002] Eggplant (Solanum melongena L.) is originated in India, but China is the largest eggplant growing country in the world. Eggplant is cultivated in large areas in both the north and the south of China. But the shape and color of the fruits vary greatly: in the northeast, the green egg eggplant is mainly cultivated, while the slender purple eggplant in the north China, the green long eggplant in the Central Plains, purple eggplant in southwest China and purple red eggplant is mainly cultivated in South China and Central China. Therefore, peel color has become one of the important breeding goals of eggplant.

[0003] Flavonol synthase (FLS) is one of the key enzymes for the synthesis of flavonol. In the biosynthetic pathway of plant flavonoids, using dihydroflavonol as a substrate, leucocyanidins and flavonols were synthesized respectively under the catalysis of dihydroflavonol 4-reductase (DFR) and FLS. FLS can catalyze dihydromyricetin (DHM), dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to produce myricetin, kaempferol and quercetin respectively (Winkel- shirley, 2001). The competition relationship between FLS and DFR affects the accumulation of anthocyanin and flavonol. When FLS is overexpressed, the flavonol content increases, and the flower or fruit color will become lighter. Therefore, studying the function of FLS not only affects the biosynthesis of plant flavonoids, but also has an important impact on the formation of plant flower color and fruit color. [

[0004] Overexpression of McDFR or silencing of McFLS increased the anthocyanin content. On the contrary, overexpression of McFLS or silencing of McDFR increased flavonol content in Begonia leaves and apple peels (Tian et al., 2015). In transgenic lines, when the expression level of DFR and ANS were up-regulated by 1.95 and 1.56 times, respectively, the expression level of FLS was lower (Yao et al., 2017).

[0005] Therefore, studying the effect of FLS on peel coloring is important to breed new eggplant varieties with uniform coloration. Summary of the Invention

[0006] In view of the above problems, the purpose of the present invention is to clone the key -1-

BL-5204 gene SmFLS for peel coloring through a pair of full-length primers designed from the eggplant LU102583 genome data. The invention is helpful to further understand the peel coloring mechanism of eggplant, so that biotechnological means can be used to regulate the peel coloring of eggplant and breed new eggplant varieties with uniform coloring.

[0007] According to one aspect of the present invention, a gene SmFLS for peel coloring cloned from eggplant is provided, which encodes the amino acid sequence shown in SEQ ID NO: 2.

[0008] The nucleic acid sequence of the gene SmFLS for peel coloring is shown in SEQ ID NO:1.

[0009] Preferably, the maximum open reading frame (ORF) of the SmFLS gene is 1014 bp.

[0010] The gene SmFLS for peel coloring encodes 337 amino acids and has two conserved domains, H-X-D and R-X-S, which belongs to the family of 2-oxoglutarate-dependent dioxygenases.

[0011] According to another aspect of the present invention, the use of the key gene SmFLS in regulating the peel coloring of eggplant is also provided. Wherein, the key gene SmFLS for peel coloring is forwardly connected with the plant expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transferred into the eggplant by the Agrobacterium-mediated method. The flavonol content in the peel of the transgenic eggplants increases.

[0012] Specifically, the SmFLS gene is forwardly connected to the vector pBI121 to obtain the recombinant plasmid pBI121-SmFLS, and the recombinant plasmid pBI121-SmFLS is transferred into eggplant by the Agrobacterium-mediated method, the flavonol content in the transgenic eggplants is significantly increased.

[0013] According to another aspect of the present invention, a vector containing the key gene SmFLS for peel coloring is provided.

[0014] According to another aspect of the present invention, a host cell containing the above- mentioned key gene SmFLS for peel coloring is provided.

[0015] The key gene SmFLS for peel coloring obtained by the present invention can regulate the peel coloring of eggplants by means of biotechnology and breed new eggplant varieties with uniform coloring. Brief Description of the Drawings

[0016] . Figure 1 shows an alignment of amino acid sequences encoded by FLS homologous genes. 2-

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[0017] Figure 2 shows the results of PCR detection of SmFLS transgenic plants. LU102583

[0018] Figure 3 shows the determination results of flavonol content in SmFLS transgenic plants. Detailed Description of the Embodiments

[0019] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0020] The FLS homologous gene provided by the present invention is cloned from eggplant peel and named as SmFLS gene. The ORF sequence is shown in SEQ ID NO:1 (sequence 1).

[0021] The protein encoded by the SmFLS gene is named SmFLS protein, and its amino acid sequence is shown in SEQ ID NO: 2 (sequence 2).

[0022] Total RNA was isolated from peels using a plant RNA extraction kit 2.0 (TIANDZ, Inc. Beijing, China, with DNase).

[0023] 1. Cloning and sequence analysis of SmFLS gene

[0024] The cloning and sequence analysis of the above SmFLS gene include the following steps:

[0025] 1.1 RNA extraction

[0026] Total RNA was isolated from peels using a plant RNA extraction kit 2.0 (TIANDZ, Inc. Beijing, China, with DNase).

[0027] 1.2 Reverse transcription

[0028] The first Strand Reverse Transcription Kit from Dalian Takara Bioengineering Company (TAKARA) is used to obtain the first strand of cDNA, used as a template for subsequent PCR amplification.

[0029] 1.3 Designing primer

[0030] According to the eggplant genome data (http://eggplant.kazusa.or.jp/), a pair of primers for SmFLS full-length amplification is designed. FLS-F: 5’ - ATGAAAACAGTTCAAGGTCAGA -3’ FLS-R: 5’ - TCACTGAGGAAGTTTGTTAAGC -3”

[0031] The reaction system is: 12.5 pl of PCR buffer , 8.4 ul of sterile deionized water, 0.1 pl of 3-

BL-5204 Taq enzyme, 1 pl (10 pM) each of forward and reverse primers, 2 pl of cDNA template. PCR LU102583 amplification conditions: denaturation at 94 °C for 3 min; denaturation at 94 °C for 0.5 min, annealing at 58 °C for 0.5 min, extension at 72 °C for 2 min, 32 cycles; extension at 72 °C for 10 min.

[0032] 1.4 DNA fragment recovery

[0033] The target DNA fragment obtained from PCR amplification is recovered with TAKARA's Agarose Gel DNA Purification Kit Ver 2.0.

[0034] The target DNA fragment was ligated with pMD19-T vector at 16 °C for 2 to 4 hours and the ligation product was transformed into JM109 competent cells. The transformants were screened by blue and white spots, and the white spots were selected for further PCR detection. The positive transformants were picked and sent to the company for sequencing. The full length of SmFLS is 1080 bp, and its ORF is 1014 bp (SEQ ID NO: 1), encoding 337 amino acids (SEQ ID NO: 2).

[0035] 1.5 Sequence analysis

[0036] Searching for homologous sequences from NCBI for comparison (Fig. 1), the results show that SmFLS has the highest homology of 90% with the FLS gene in potato and a homology of 89% in peppers. The homologies with FLSs in tomato, tobacco and petunia are over 85%.

[0037] SmFLS belongs to the family of 2-ketoglutarate-dependent dioxygenases, with a typical 20G-Fell_Oxy oxygenase active region (214 ~ 310 aa), one DIOX_N functional domain (55 ~165 aa), and two conserved domains, H-X-D and R-X-S (Fig. 1). These structures are related to its protein function.

[0038] 2. Construction of plant expression vector and genetic transformation

[0039] A plant expression vector containing SmFLS was constructed and transferred into eggplant by Agrobacterium-mediated method to further verify the function of SmFLS gene.

[0040] 2.1 Amplification of coding region with restriction site

[0041] Designing primers with restriction sites: FLSmq-F: CGGGATCCGCACAACAGCCCTGATGGAG BamHI FLSmq-R: CGAGCTCGACGATTTCACTGAGGAAGTTTGTT Sacl _4-

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[0042] PCR reaction system: 25 ul of total reaction volume containing 2 ul of template first strand102583 cDNA, 1 pl each of upstream and downstream primers (10 pmol/L), 0.1 ul (5 U/ul) of Tag DNA polymerase, 12.5 ul of 2x Mix buffer, making up the volume with sterile redistilled water.

[0043] The PCR reaction conditions were: denaturation at 94 °C for 3 min; denaturation at 94 °C for

0.5 min, annealing at 58 °C for 0.5 min, extension at 72 °C for 2 min, 35 cycles; extension at 72 °C for 10 min. The amplified FLSmq was connected with pMD19-T, named pMD19-SmFLS, and transformed into JM109 competent cells. The transformants were screened with blue and white spots, and the white spots were selected for further PCR detection. The positive transformants were picked and sent to the company for sequencing. The sequencing results confirmed it is the SmFLS gene including the coding region and restriction site.

[0044] 2.2 Construction of plant expression vector

[0045] The target DNA fragment was cut from the recombinant plasmid pMD19-SmFLS with restriction enzymes BamHI and SacI, and the pBI121 vector was digested with BamHI and SacI too. After separation by agarose gel electrophoresis, the SmFLS and the large fragment of the pBI121 vector were recovered separately. Using T4 DNA ligase to ligate SmFLS into pBII2! vector, the ligation product was transformed into JM109 competent cells. After PCR identification, the plant expression vector pBI121-SmFLS was obtained.

[0046] 2.3 Introducing plant expression vector into Agrobacterium

[0047] After thawing the competent cells of Agrobacterium GV3101 on ice, 1 ug of pBII2I-SmFLS plasmid DNA was added, mixing well and ice bathing for 30 min. The mixture was placed in liquid nitrogen for 1 min, then quickly placed in water bath at 37 °C for 5 min. After the mixture thawing,

0.8 ml of fresh LB liquid medium (without antibiotics) was added, shaking culture at 28 °C for 2 to 4 hours. 200 ul of bacterial solution was smeared on the LB solid medium containing 30 mg/L kanamycin (Kan) and 400 mg/L timentin (Tim), which was cultured at 28 °C for 2 to 3 days. The colony was detected by PCR.

[0048] 2.4 The target gene is transferred into eggplant

[0049] The single positive Agrobacterium clones were picked and cultured in 10 ml of LB liquid medium containing Tim (400 mg/L) and Kan (30 mg/L) at 28 °C and shaking with the speed of 200 rpm until the optical density (OD600) of the bacterial liquid is between 1.0 and 1.5.

[0050] 100 ml of Agrobacterium solution with pBI121-SmFLS was prepared and centrifuged at -5-

BL-5204 5000 rpm for 10 min at room temperature. The supernatant was discarded and the precipitate was 102583 suspended in LB liquid medium containing Tim (400 mg/L) and Kan (30 mg/ In L). When the ODsoo of the resuspended bacterial solution is about 0.6, it can be used for transformation. Pouring the Agrobacterium suspension into a petri dish, and immersing the eggplant explants in the Agrobacterium solution for 10 minutes, shaking the petri dish occasionally. After transformation, the explants had been co-cultivated on the medium (MS+0.1 mg/L IAA+1.0 mg/L ZT) for 2 days, then cultured on the antibacterial screening medium (MS+0.1 mg/L IAA+1.0 mg/L ZT+ 400 mg/L Tim+30 mg/L Kan). The small shoots were cut and then cultured on the rooting medium (MS+30 mg/L Kan).

[0051] 2.5 Application of SmFLS gene

[0052] 1) PCR detection of transgenic plants

[0053] The genomic DNA of the transformed plants was extracted, which was used as a template. The FLSmq-F and FLSmq-R were used as primers for PCR amplification, and the DNA of non- transgenic eggplant was used as a negative control. The results indicate that the SmFLS gene has been integrated into the eggplant genome (Fig. 2).

[0054] 2) Determination of flavonol content in transgenic plants

[0055] The wild-type plants in the same period were used as controls.

[0056] Extraction of flavonols and determination of their components (HPLC): Referring to the method of Tan Xiaoliang et al. (2015) for flavonol extraction. Using Shimadzu high-performance liquid chromatograph for sample injection, selecting UV detector and chromatographic column is reversed phase C18 column. Column temperature is set to 35 °C and wavelength is 330 nm. Mobile phase A is methanol and mobile phase B is 0.2% of phosphoric acid aqueous solution. Injection volume is 10 uL and flow rate is 1 mL/min. Gradient elution conditions: 0 min, 5% of B; 10 min, 10% of B; 15 min, 38% of B; 25 min, 68% of B; 30 min, 48% of B; 35 min, 5% of B; 43 min, 5% of B.

[0057] The results are shown in Fig. 3. Compared with the non-transformed plants (CK), the flavonol content of the transformed plants increased significantly, indicating that SmFLS promoted the accumulation of flavonols.

[0058] The above are only preferred examples of the present invention, and are not intended to limit the protection scope of the present invention. -6-

BL-5204 LU102583

Sequence list

<110> ML Huaihua University

<120> RRÉGEX SmFLS VEN FE Gene SMFLS for peel coloring and use of the same

<160> 2

<210> 1

<211> 1014

<212> cDNA

<213> Di Eggplant

<400> 1

1 atggaggtggcgaggotacaagegatatcgtcaataacgagatgcatggacacaatteca

61 tcggaatatattcgttcagagaacgaacaacctgtggccacaacgctgcacggagtagtt

121 cttgaggigccagtcategacataggtacttetetcgatgaggaaaaaatage gaaagag

181 atagctgaggctagcaaagagtggggtatttttcaagtgataaatcatgggattccagat

241 gagetgattgegaatttacagaaagttgggaaggagttetttgaggaagteccacaagaa

301 gagaaagagttgattgcgaagaagccaggggc gcagagtatagaagggtatgggacttet

361 llgcagaaggaagtggaagegaaaaaagettgggtggatcatttetttcataagatttgg | 421 cctecticagecatcaactategttattggcctaaaaaccetectttctacagggaagea 481 aatgaggaatacgcaaagaagttgcgegatgttggagataagatgtttaggagcttgteg 541 cttgggcttggtttggaageecatgaaatgattgaggeagetggtagtgaagacatagtt 601 tacatgttaaagatcaattattacccaccatgeccacggecagatttggetcttggagtt 661 gtggctcatacagatatgtcctacatcaccettettgtgecaaatgaagtccaaggecte 721 Caagtctttaaagatggccattggtatgatgtcagttacataccaaatgcaataattgta 781 Cacattggtgaccaaattgagattcttagcaatgggaaatataagagtgtgtaccaccgg 841 acaacagtgaacaaggacaagacaagaatetcatgecctettttettggageccccacca 901 gagcatgaagtigggccaatgectaaactgattaatgaggccaacccacccaaattcaag 961 accaagaagtacaaggattatgtctattgtaagcttaacaaacttcctcagtga

1

BL-5204 LU102583

<210> 2

<211> 337

<212> ZEEE Amino acid

<213> Eggplant

<400> 2

1 MEVARVQAISSITRCMDTIPSEYIRSENEQPVATTLHGVV

41 LEVPVIDIGTCVDEEKIAKEIAEASKEWGIF QVINHGIPD

81 EVIANLQKVGKEF FEEVPQEEKELIAKKPGAQSIEGYGTS

121 LQKEVEGKKGWVDHLFHKIWPPSAINYRY WPKNPPFYREA 161 NEEYAKKLRDVGDKMFRSLSLGLGLEAHEMIEAAGSEDIV 201 YMLKINYYPPCPRPDLALGVVAHTDMSYITLLVPNEVQGL 241 QVFKDGHWYDVSYIPNAIIVHIGDQIEILSNGKYKSVYHR 281 TTVNKDKTRMSWPVFLEPPPEHEVGPMPKLINEANPPKFK 321 TKKYKDYVYCKLNKLPQ

2

Claims (6)

BL-5204 Claims: LU102583

1. A gene SmFLS for peel coloring cloned from eggplant, wherein the gene SmFLS encodes the amino acid sequence shown in SEQ ID NO: 2.

2. The gene SmFLS for peel coloring according to claim 1, wherein its nucleic acid sequence is shown in SEQ ID NO:1.

3. The gene SmFLS for peel coloring according to claim 1, wherein the gene SmFLS encodes 337 amino acids and has two conserved domains, H-X-D and R-X-S.

4. Use of the gene SmFLS for peel coloring according to any of claims 1 to 3 in regulating the flavonol content of eggplant peels.

5. A vector containing the gene SmFLS for peel coloring according to any one of claims 1to3.

6. A host cell containing the gene SmFLS for peel coloring according to any one of claims 1 to 3.

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