CN118086331A - Clone of lily anthocyanin synthesis regulating gene LhERF061 and application thereof - Google Patents

Abstract

The invention discloses a clone and application of lily anthocyanin synthesis regulatory gene LhERF061, wherein the nucleotide sequence of LhERF061 gene is shown as SEQ ID NO. 1. ERF061 genes in other crops have not been reported to be involved in anthocyanin synthesis. The anthocyanin content in the arabidopsis thaliana, the tobacco and the lily which are subjected to over-expression LhERF061 genes is reduced, which shows that the lily LhERF061 genes are responsible for regulating and controlling the synthesis of anthocyanin. The LhERF061 gene provided by the invention can be used for cultivating lily rich flower color varieties, and has a broad prospect in lily flower color molecular breeding.

Description

Clone of lily anthocyanin synthesis regulating gene LhERF061 and application thereof

Technical Field

The invention relates to the technical fields of molecular biology and plant engineering, in particular to cloning and application of lily anthocyanin synthesis regulatory gene LhERF 061.

Background

The lily is a perennial herb bulb flower of the genus Lilium (Lilium spp.) of the family Liliaceae (LILIACEAE). The cultivation history is long, the flower color types are rich, and the flower color varieties are various. Flower color is one of the most important ornamental traits. Anthocyanin is used as a main color-developing substance of lily flower color, is widely existing in petals and fruits of plants, is a main color-developing substance of Asian lily quilt sheet, and can cause the flower quilt sheet to generate pink, purple and other flower color types. Anthocyanin synthesis pathway is one of the more thoroughly studied secondary metabolic pathways of plants. The synthesis uses phenylalanine as precursor, and the synthesis of key structural genes, such as C4H(Cinnamate4-hydroxylase)、CHS(Chalcone synthase)、CHI(Chalcone isomerize)、F3H(Flavanone 3-hydroxylase)、F3'5'H(Flavonoid 3',5'-hydroxylase)、F3'H(Flavonoid 3'-hydroxylase)、DFR(Dihydroflavonol 4-reductase)、ANS(Anthocyanin synthase)、UFGT(UDP-glucose:flavonoid-3-O-glucosyltransferase) structural genes, is catalyzed by enzyme on endoplasmic reticulum. Synthetic pathway genes are regulated by multiple factors such as transcription level, post-transcriptional level, epigenetic level, and environment, e.g., MBW complexes, DNA methylation, histone modification, etc.

The AP2/ERF type transcription factor is a type of transcription factor specific to plants. AP2/ERF family member ERF61 regulates synthesis of osmanthus fragrans flower fragrance in osmanthus fragrans, responds to cadmium stress in liquorice, and has not been reported to participate in anthocyanin synthesis. Earlier analysis of lily transcriptome data and qRT-PCR results show that AP2/ERF transcription factor family gene LhERF061 may play an important role in anthocyanin accumulation. Therefore, the separation of anthocyanin synthesis related regulatory gene LhERF to 061 from lily and the elucidation of the function thereof are key links for researching the color mechanism of lily quilt pieces, and will lay a theoretical foundation for developing lily color regulation and color molecular breeding.

Disclosure of Invention

The invention aims to provide a clone of lily anthocyanin synthesis regulatory gene LhERF061 and application thereof.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In a first aspect, a lily anthocyanin synthesis regulatory gene LhERF061 is provided, and the CDS region sequence of the gene is shown as SEQ ID No. 1.

In a second aspect, a lily anthocyanin synthesis regulatory gene LhERF and a protein coded by the gene LhERF are provided, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

In a third aspect, a cloning method of a lily anthocyanin synthesis regulatory gene LhERF061 is provided, which includes the following steps:

(1) Extracting total RNA of lily quilt sample and synthesizing cDNA;

(2) Designing a primer according to transcriptome data, taking cDNA as a template, and carrying out PCR amplification to obtain a CDS sequence, wherein the primer for amplifying the CDS sequence is as follows:

5' end primer: 5'-ATGAGCATGATGAATCAATTC-3' A

3' -Terminal primer: 5'-CTAATTAGCCAGTACTTCCCA-3' A

The amplified sequence is compared with the transcriptome sequence after sequencing verification, and the sequence of 705bp of CDS region of LhERF061 gene is obtained, which is shown as SEQ ID NO. 1.

In a fourth aspect, a recombinant overexpression vector is provided, comprising lily LhERF061 gene.

Further, according to CDS sequence of LhERF061 gene, a joint primer is designed, and PCR amplified gene primer is as follows:

5' end primer: 5'-CACGGGGGACTCTTGACCATGGATGAGCATGATG-3' A

3' -Terminal primer: 5'-GGGGAAATTCGAGCTGGTCACCCTAATTAGCCAG-3' A

And (3) carrying out PCR amplification to obtain a CDS sequence with a joint, and carrying out recombination on a linear vector fragment obtained by using the PCAMBIA3301 double-enzyme-digested vectors of the Nco I and the BstE II and the CDS sequence with the joint by using a homologous recombination method to finally obtain a recombinant vector.

In a fifth aspect, an application of lily anthocyanin synthesis regulatory gene LhERF061 in controlling lily anthocyanin synthesis is provided.

The beneficial effects of the invention are as follows:

ERF061 genes in other crops are not reported to participate in anthocyanin synthesis, and the lily LhERF061 genes provided by the invention are unknown in lily plants and participate in lily quilt anthocyanin synthesis. The invention successfully clones LhERF to 061 gene, provides the nucleotide sequence and the coding protein amino acid sequence of the gene, and provides theoretical basis and application reference value for germplasm creation of lily flower color molecular breeding.

Drawings

FIG. 1 shows LhERF A061 gene CDS region amplification electrophoresis, M: DNA MARKER;

FIG. 2 is a diagram showing amino acid sequence alignment of LhERF to other ERF061 homologous proteins;

FIG. 3 is a diagram of a LhERF061 phylogenetic tree analysis;

FIG. 4 is a LhERF061 expression pattern diagram; a is a schematic diagram of different development stages of the festoon sheet; b is LhERF to be a schematic diagram of the relative expression condition of the perianth at different development stages of the perianth; c is a schematic diagram of different tissues of lily; d is LhERF and the relative expression condition of the 061 in different tissues is shown in the schematic diagram; values in the figure are mean ± SDs (n=3), lower case letters a-e indicate significant differences at P <0.05 level, asterisks indicate statistically significant differences (< 0.05; P <0.01, P < 0.001);

FIG. 5 is subcellular localization of LhERF061 protein; a is a schematic diagram of subcellular localization vector construction; b is a subcellular localization result plot, bar=20 μm;

FIG. 6 is a schematic diagram of a pCAMBIA3301 vector;

FIG. 7 is a schematic representation of phenotypic characteristics and results analysis of LhERF A. Heterologous over-expression Arabidopsis; a is a phenotype schematic diagram of accumulation of anthocyanin on hypocotyl and cotyledon of transgenic Arabidopsis after LhERF061 is stably and overexpressed; b is a schematic diagram of anthocyanin content in transgenic arabidopsis after LhERF061 is stably and overexpressed; the scale in the figure is equal to 5mm; c is a transgenic Arabidopsis identification gel diagram; d is an expression analysis schematic diagram of an arabidopsis anthocyanin synthesis pathway related gene; values in the figures are mean ± SDs (n=3), lower case letters a-e indicate significant differences at P <0.05 level;

FIG. 8 is a schematic diagram showing phenotypic characteristics and results analysis of LhERF061 heterologous overexpressed tobacco flower and quilt pieces; a is a phenotype schematic diagram of anthocyanin accumulation of transgenic tobacco festive sheets after LhERF061 is stably and overexpressed; b is a schematic diagram of anthocyanin content in transgenic tobacco festive sheets after LhERF061 is stably and overexpressed; c is a transgenic tobacco identification glue graph; d is an expression analysis schematic diagram of genes related to anthocyanin synthesis pathways of tobacco; values in the figures are mean ± SDs (n=3), lower case letters a-e indicate significant differences at P <0.05 level;

FIG. 9 is a schematic representation of phenotype and anthocyanin content of lilium brownii sheets after LhERF061 transient overexpression; a is a phenotype detection schematic diagram; b is an expression analysis schematic diagram of a lily anthocyanin synthesis pathway related gene; values in the figures are mean ± SDs (n=3), asterisks indicate statistically significant differences (×p <0.05; ×p <0.01;, < 0.001).

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

Examples

The research material lily TINY PADHYE' is planted in a glass greenhouse of vegetable and flower research institute of China academy of agricultural sciences and can be issued to the public. The reagent kit involved in the experiment is carried out according to the reagent instruction. RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit and high-purity plasmid small extraction kit are purchased from Tiangen biochemical technology (Beijing) limited company, common agarose gel DNA recovery kit and homologous recombination kitSeamLess Cloning and Assembly Kit, reverse transcription kit TRANSSCRIPTII ONE-Step gDNA Removal AND CDNA SYNTHESIS Supermix was purchased from Beijing all gold Biotechnology Co. pTOPO-TA/Blunt is available from Edley biosystems. Ampicillin sodium salt (AMPICILLIN), kanamycin (KANAMYCIN), rifampicin (RIFAMPICIN), magnesium chloride (MgCL ₂), 2-morpholinoethanesulfonic acid (MES), acetosyringone (AS), RNase-free water, and the like were purchased from Soy Bao technology (Beijing) Inc. Premix hot start high fidelity enzyme KAPAHiFi HotStart ReadyMix was purchased from KAPA and restriction endonucleases Ncol I, bstE II, ecoR I, xba I and Xma I were purchased from NEB. Reverse transcription kit II 1st Strand cDNA Synthesis SuperMix for qPCR (GDNADIGESTER PLUS), fluorescence quantification kit HIEFFTM QPCR GREEN MASTER Mix (No Rox) were purchased from Shanghai Biotech Inc. of the next year. Primer synthesis and sequencing were performed by the company Shanghai, inc.

Example 1 cloning of LhERF061 Gene and bioinformatics analysis

1. RNA extraction and cDNA Synthesis

RNA extraction: tissue of the upper and lower parts of the lily 'TINYPADHYE' festive sheet is taken, and 0.2g of lily festive sheet is taken and rapidly ground into powder in liquid nitrogen. Extracting plant total RNA by using a polysaccharide polyphenol plant total RNA extraction kit of Tiangen biochemical technology (Beijing) limited company RNAprepPure, wherein the extraction method is carried out according to the specification.

CDNA synthesis: cDNA synthesis was performed using a Beijing full gold Biotechnology Co., ltd TransScriptIIOne-Step gDNA Removal AND CDNASYNTHESIS Supermix (first strand cDNA Synthesis and gDNA removal) reverse transcription kit, and the procedure was as follows:

(1) Preparing the following mixture in a centrifuge tube of RNAse-free;

(2) Gently stirring and mixing the mixed solution by using a pipettor, and carrying out the following reaction in a PCR instrument for 30min at 50 ℃;85 ℃,5s.

And (3) PCR amplification: the sequence primers for amplifying the CDS region of LhERF061 gene were designed with PRIMER PREMIER 6.0.0 based on transcriptome data as follows:

5' end primer: 5'-ATGAGCATGATGAATCAATTC-3' A

3' -Terminal primer: 5'-CTAATTAGCCAGTACTTCCCA-3'.

Amplifying cDNA of a lily 'TINYPADHYE' inner envelope as a template, adding the following components,

The mixture was gently swirled with a pipette and subjected to the following reaction in a PCR instrument. Pre-denaturation at 95℃for 3min; heating and denaturing at 98 ℃ for 20sec, annealing at 60 ℃ for 15sec, extending at 72 ℃ for 45sec,35 cycles; extending at 72 deg.C for 5min.

And (3) performing electrophoresis detection on the obtained PCR amplification product in 1% agarose gel, and purifying and recovering the amplified product fragment by using a Beijing full-type gold biotechnology Co-Ltd common agarose gel DNA recovery kit.

2. The PCR product was ligated with the vector pTOPO-Blunt (commercially available). The connection system is shown in the following table,

After gently stirring and mixing by a pipetting gun, the mixture was connected at room temperature for 5min. The ligation product was transferred into E.coli competent DH 5. Alpha. (Shanghai only) according to the heat shock method, and the monoclonal was subjected to PCR detection and sent to the Biochemical company for sequencing.

3. Bioinformatics analysis:

The open reading frame of LhERF061 and the amino acid sequence encoded by it were analyzed using the online software ORF Finder (http:// www.ncbi.nlm.nih.gov/projects/gorf /). And carrying out homologous sequence alignment on the LhERF061 coded amino acid and other ERF061s protein amino acid sequences by utilizing DNAMAN software. The amino acid sequence of LhERF061 and the amino acid sequence of ERF061 of Arabidopsis AP2/ERF transcription factor family and other species are constructed into a Neighbor-training phylogenetic tree by MEGA6.0 software, and the repetition number of bootstrap is 1000.

4. Analysis of results:

705bp is shown in SEQ ID NO.1, and 234 amino acids are coded in SEQ ID NO.2. The LhERF061 protein was found to have an AP2 domain by NCBI Conserved Domain Search belonging to the AP2/ERF transcription factor family (FIG. 2). The sequence was constructed into phylogenetic trees with the Arabidopsis ERF family proteins by the MEGA7.0 software Neighbor Joining (NJ) method, respectively. As a result, lhERF061,061 was found to belong to group A-6, namely the DREB subfamily. (FIG. 3).

Example 2 LhERF analysis of Gene expression

RNA extraction:

The method comprises the steps of taking a perianth tablet of lily 'TINYPADHYE' in the same development period and other different tissues (scales, leaves, stems, stigmas, anthers, filaments and ovaries), extracting plant total RNA by using a polysaccharide polyphenol plant total RNA extraction kit of Tiangen biochemical technology (Beijing) limited company RNAprep Pure, and performing the extraction method according to the specification.

CDNA synthesis:

cDNA synthesis was performed using the following St.John's Biotechnology (Shanghai) Inc. II 1st Strand cDNASynthesis SuperMix for qPCR (GDNADIGESTER PLUS) reverse transcription kit. The method comprises the following steps:

Quantitative Real-time RT-PCR：

Based on LhERF061 sequences obtained by PCR amplification, PRIMERPREMIER software was used to design specific primers qlilyERF061-F/R for performing real-time fluorescent quantitative PCR:

5' end primer: 5'-CATACGACGTACCAGATTACGC-3' the process of the preparation of the pharmaceutical composition,

3' -Terminal primer: 5'-TAGGCATTGCCTCAGTAGCGA-3'.

LILYACTIN (GenBank: 7X 826390) is taken as an internal reference gene, and fluorescent quantitative primers are QLILYACTIN-F/R:

5' end primer: 5'-GCATCACACCTTCTACAACG-3' the process of the preparation of the pharmaceutical composition,

3' -Terminal primer: 5'-GAAGAGCATAACCCTCATAGA-3'.

Fluorescent quantitative PCR was performed using the assist Saint Co HIEFF TM QPCR GREEN MASTER Mix (NoRox) kit, the components were as follows:

The amplification procedure was as follows: 3min at 95 ℃;95℃for 20s,60℃for 10s,72℃for 20s,40 cycles. Primer amplification efficiency was analyzed using CFX ManagerTM software v3.1 (Bio-Rad). The relative expression levels of the different genes were calculated using the 2 ^-ΔΔCt method. All qRT-PCR experiments contained 3 biological replicates and 3 technical replicates.

Expression analysis of LhERF061 gene:

The present example divides the lily quilt piece color forming process into 4 periods (S1-S4), wherein the flower bud development stage (St) is as follows: stage 1 (S1: bud length about 1 cm without anthocyanin accumulation), stage 2 (S2: anthocyanin starts to accumulate at the basal part of the flower quilt), stage 3 (S3: day before flowering, anthocyanin accumulates in a large amount at the lower part of the flower quilt), and stage 4 (S4: day of flowering) (FIG. 4A). The expression level of LhERF and 061 in this process was analyzed, and as shown in fig. 4B, lhERF and 061 showed that the expression level of the top non-colored region in the S2 period of asian lily was significantly higher than that of the top and bottom portions in the basal colored region and other periods, and this was inversely related to the structural gene and anthocyanin content and expression level. Tissue-specific expression results showed that LhERF061 was expressed in the perianth (Petal) at the highest level and significantly higher than other tissues and organs, and also expressed in trace amounts in young leaves and ovaries, but hardly expressed in other organs, tissue filaments (filaments), bulbs (Bulb), stem segments (stem), stigma (Stigma), anthers (Anther) (fig. 4c,4 d). Therefore, it is presumed that the transcription factor LhERF061 plays a regulating role mainly on the flower organ, and further, it is confirmed that it is a flower color regulating gene.

Example 3, lhERF, subcellular localization

LhERF061 construction of subcellular localization vector: based on the CDS sequence of LhERF061 gene (stop codon removed), a vector linker primer was designed:

5' end primer:

5′-ATTTGGAGAGGACAGGGTACATGAGCATGATGAA-3′，

3' -terminal primer:

5′-CCATGGTACTAGTGTCGACTCTAGATTAGCCAGTA-3′。

The target fragment was obtained by PCR amplification, the target band was separated by agarose gel electrophoresis, and the target band was recovered using a gel recovery kit. The pCAMBIA2300 vector is subjected to SmaI and XbaI digestion and then is recovered for standby. The LhERF061 linker product was ligated with the pCAMBIA2300 double cleavage product using homologous recombinase. The molar ratio of vector to insert was 1:5. After being gently blown and evenly mixed by a pipetting gun, the mixture is recombined in a PCR instrument at 50 ℃ for 15-20min, the recombined product is transferred into competent transformation of escherichia coli DH5 alpha, and the monoclonal is sequenced after being detected positive. Obtaining the pCAMBIA2300 with successful construction: lhERF 061A 061-GFP vector plasmid. The positive plasmid with correct sequence is transferred into an agrobacterium competent GV3101 strain (only, shanghai) by a freeze thawing method, and the steps are as follows;

(1) 1ng of plasmid was added to 100. Mu.L of GV3101 competent cells, followed by ice-bath for 5min, liquid nitrogen quick-freezing for 5min, water-bath at 37℃for 5min, and ice-bath for 5min.

(2) 700. Mu.L of LB liquid medium was added thereto, and the culture was continued at 28℃for 2-3 hours with shaking at 200 rpm.

(3) Centrifuging at 5000rpm for 1min, sucking out supernatant, collecting 100 μl, mixing, spreading on LB solid medium containing rifampicin (25 mg/L) and kanamycin (50 mg/L), and culturing at 28deg.C for 48 hr.

Selecting monoclonal for colony PCR detection, selecting correct monoclonal in LB liquid medium containing rifampicin (25 mg/L) and kanamycin (50 mg/L), shake culturing at 28deg.C and 200rpm for 24-48h, propagating, storing in 50% sterile glycerol, and freezing at-80deg.C for use.

Agrobacterium transformation tobacco:

Well-grown 3-4 weeks of Nicotiana benthamiana (Nicotiana benthamiana) was selected for subcellular localization. The specific operation steps are as follows:

a. Bacterial liquid is activated, and the recombinant plasmid pCAMBIA2300 is contained in pCAMBIA-2300: the GV3101 strain LhERF-GFP and mCherry (the nuclear marker) was streaked onto LB plates (containing Rif and Kan) and cultured upside down at 28℃until a single clone was obtained. Single colonies were picked in 1mL LB liquid medium, cultured at 28℃and 200rpm overnight with shaking. 1mL of the bacterial liquid was added to 50mL of YEB medium, and the culture was performed at 28℃and 200rpm with shaking until OD 600 = 0.5.

B. Suspension was prepared, centrifuged at 6000rpm for 10min, the supernatant was discarded, resuspended with permeation buffer (10mM MES+10mM MgCl ₂ +200. Mu.M acetosyringone), and OD 600 = 0.6 was adjusted and incubated in the dark for 2-3h at room temperature.

E. tobacco leaves were infected, the back of the tobacco leaves was injected using a 1mL syringe, and the leaves were collected after 72h of dark culture.

C. GFP fluorescence was observed and images were acquired using a confocal laser scanning microscope (Zeiss LSM 510).

Experimental results: as shown in FIG. 5, lhERF061-GFP green fluorescent signal was found to be coincident with the nuclear stain DAPI expression site in the nucleus, indicating that LhERF061 protein was nuclear localization.

Example 4 acquisition and phenotypic analysis of transgenic Arabidopsis thaliana

LhERF061 construction of recombinant overexpression vector:

according to the sequence of LhERF061 gene, designing a linker primer with enzyme cutting site:

5' end primer: 5'-CACGGGGGACTCTTGACCATGGATGAGCATGATG-3' the process of the preparation of the pharmaceutical composition,

3' -Terminal primer: 5'-GGGGAAATTCGAGCTGGTCACCCTAATTAGCCAG-3'.

The target bands were amplified by PCR and separated by agarose gel electrophoresis, and recovered using a gel recovery kit. Vector pCAMBIA3301 was double digested with restriction enzymes NcoI and BstEII and the linear target band was recovered. The map of pCAMBIA3301 is shown in figure 6, the target fragment and the vector enzyme cutting sequence are connected by a homologous recombination method, the connected product is transferred into the competence of escherichia coli DH5a by a heat shock method, and the correct positive bacterial liquid is detected and sent to sequencing. The recombinant plasmid with correct sequence was transferred into GV3101 Agrobacterium competence by freeze thawing.

Agrobacterium transformation of Arabidopsis thaliana:

arabidopsis thaliana (Arabidopsis thaliana) with good growth is selected, and heterologous expression is performed when the buds are exposed to white. The specific operation steps are as follows:

a. Agrobacterium, which identified the correct recombinant vector, was added with 50% glycerol and stored at-80 ℃. The agrobacterium of the recombinant vector is coated on LB plate medium containing rifampicin (25 mg/L) and kanamycin (50 mg/L), and monoclonal is selected and activated in LB liquid medium containing rifampicin (25 mg/L) and kanamycin (50 mg/L), and the bacteria liquid is prepared: LB liquid medium = 1:100 (V/V) and at 28 ℃,200rpm are used for shake propagation for 2d until the bacterial liquid OD600 value=0.8-1.2, and the infection activity is optimal.

B. the bacterial solution was centrifuged at 7000rpm for 5min, and a 2 Xresuspension Buffer (10% sucrose solution: 0.1% inversion Buffer-Sliwet L-77) was prepared, and 1/2 of the sterilized water-suspended Agrobacterium strain was added during inversion, and then 1/2 of the 2 Xresuspension Buffer was added for resuspension.

C. during infection, fungus droplets are sucked by a dropper on flower buds of wild (col.) Arabidopsis thaliana, and the infected plants are placed in a light-proof and moisture-preserving environment for 16-24 hours after infection. After being protected from light, the seeds are cultivated in normal environment at 20-22 ℃ until the seeds are mature.

D. Seeds harvested from transformed plants were first dried for 1 week, vernalized (dormancy breaking) at 4℃for 2-4 days, sterilized with 75% ethanol for 5min, washed 3-5 times with sterile water, and screened on a 1/2MS plate containing glufosinate-ammonium resistance (Basta). Until T3 generation seeds were harvested, identified by phenotypic observation and molecular level.

Experimental results: the LhERF transgenic arabidopsis plant seeds and the wild type arabidopsis plant seeds are respectively inoculated in the same 1/2MS culture medium plate under the aseptic condition, the normal culture is carried out for 14 days under the same environment, the phenotype of the two arabidopsis plants is observed and compared, the accumulation of anthocyanin on hypocotyls and leaves of the wild type arabidopsis plant is found, the accumulation of anthocyanin on hypocotyls and cotyledons of the LhERF061 transgenic arabidopsis plant is reduced, the anthocyanin is not accumulated on hypocotyls and cotyledons of even the hypocotyls of the other transgenic arabidopsis plant (figure 7A), and then the total anthocyanin content of Wild Type (WT), empty load control (EV) and 5 transgenic lines is detected, and the result shows that the anthocyanin content in the WT and EV is obviously higher than that of the LhERF transgenic arabidopsis plant (figure 7B). The PCR assay showed that only positive plants had the target band (FIG. 7C). According to anthocyanin related structural gene sequences in arabidopsis, real-time quantitative PCR primers are designed, and the arabidopsis AtActin genes are used as internal reference genes, and the WT, EV and cDNA of transgenic arabidopsis plants are used as templates, so that the expression quantity changes of the structural genes are subjected to real-time fluorescent quantitative PCR analysis. The results show (as in fig. 7D) that the expression level of anthocyanin structural gene is significantly reduced in transgenic arabidopsis, in particular AtDFR gene, compared with the expression level in wild type and empty arabidopsis plants, which indicates that the overexpression of LhERF061 transcription factor inhibits the expression of downstream structural gene in arabidopsis plants, so that the expression level is reduced, and anthocyanin biosynthesis is inhibited.

Example 4 acquisition and phenotypic analysis of transgenic tobacco

LhERF061 construction of recombinant overexpression vector:

According to the obtained LhERF061 gene sequence, designing a linker primer with an enzyme cutting site:

5' end primer: 5'-CACGGGGGACTCTTGACCATGGATGAGCATGATG-3' the process of the preparation of the pharmaceutical composition,

3' -Terminal primer: 5'-GGGGAAATTCGAGCTGGTCACCCTAATTAGCCAG-3'.

The target bands were amplified by PCR and separated by agarose gel electrophoresis, and recovered using a gel recovery kit. Vector pCAMBIA3301 was double digested with restriction enzymes NcoI and BstEII and the linear target band was recovered. The map of pCAMBIA3301 is shown in figure 6, the target fragment and the vector enzyme cutting sequence are connected by a homologous recombination method, the connected product is transferred into the competence of escherichia coli DH5a by a heat shock method, and the correct positive bacterial liquid is detected and sent to sequencing. The recombinant plasmid with correct sequence was transferred into GV3101 Agrobacterium competence by freeze thawing.

Agrobacterium transformation tobacco:

well-grown tobacco (Nicotiana tabacum) for 3-4 weeks was selected for heterologous expression. The specific operation steps are as follows:

a. Inoculating: planting tobacco in a centrifuge tube, sterilizing the surface of 75% ethanol for 60s, continuously flushing with sterile water for 3-5 times after sterilization, flushing the surface of the tobacco with ethanol, and inoculating seeds in an MS inoculation culture medium by using sterile bamboo sticks;

b. pre-culturing: when 4 true leaves grow out, cutting off edges of tobacco leaves in a culture flask, placing the tobacco leaves on sterile water-wetted filter paper, spreading the tobacco leaves on an MS differentiation medium flat plate with the near leaf axis facing downwards, and culturing in darkness at 25 ℃ for 2-3d;

c. Co-cultivation: taking the explant in agrobacterium tumefaciens liquid (the bacterial liquid is resuspended by MS liquid culture medium, and the dilution OD600 = 0.4-0.6) of the recombinant vector, treating for 5-10min, sucking the excessive bacterial liquid by sterile filter paper to avoid damaging plants by the agrobacterium tumefaciens liquid, inoculating back to a differentiation culture medium for continuous culture, and co-culturing for 2-4d under the dark condition of 25 ℃;

d. And (3) degerming and culturing: transferring the co-cultured leaves into a degerming culture medium without selective pressure, carrying out delay screening, and carrying out illumination culture at 25 ℃ for 6-7d;

e. Selection and culture: transferring the explant to 0.2% basta degerming culture medium (250-500 mg/L cephalosporin, inhibiting the growth of agrobacterium), gradually reducing the concentration of cephalosporin (Cef), illuminating at 25 ℃ for 16 hours, culturing in darkness for 8 hours, and carrying out secondary culture every two weeks (0 days of delayed screening, the higher the conversion rate) (the concentration gradient of Cef in the culture medium can be reduced from high, and the concentration can be reduced when the agrobacterium is inhibited);

f. Rooting culture: after the adventitious bud is more than 1cm, cutting the adventitious bud and placing the adventitious bud into a rooting culture medium for rooting (containing 0.2% basta of selective pressure), transplanting the adventitious bud into a matrix for domestication and further culturing after the adventitious bud grows out (about two weeks). The tobacco phenotype was then observed and a digital photograph taken.

Experimental results:

Observing LhERF transgenic tobacco strain phenotype, the petal colors of 5 strain tobacco are reduced to different degrees compared with wild type and injection empty petals, and the reduction amplitude is large overall, so that the petal colors are almost white. Wherein petals of LhERF061-1 strain have a plurality of light pink lines in the vascular bundles extending through the midvein under a white background, and other strains have slight pigmentation at the midvein. Individual transgenic lines do not have the same color intensity. The negative control of the transformed empty vector has no difference with the color of the wild petals (figure 8A), and the influence of other experimental variables on experimental results is eliminated, which shows that the overexpression of LhERF061 leads to the lightening of the color of the tobacco petals. The total anthocyanin content of the corolla was reduced in all 5 transgenic tobacco lines compared to wild type and empty (FIG. 8B).

The use of LhERF061 primers in the crown cdnas of 5 transgenic tobacco lines amplified bands of the corresponding length, indicated that LhERF061 was successfully transferred into transgenic tobacco and transcribed (fig. 8C). In transgenic tobacco, significant downregulation of anthocyanin structural gene expression was observed (FIG. 8D).

Example 5 overexpression of LhERF061 Gene in Lily quilt pieces

LhERF061 construction of recombinant overexpression vector

According to the obtained LhERF061 gene sequence, designing a linker primer with an enzyme cutting site:

5' end primer: 5'-CACGGGGGACTCTTGACCATGGATGAGCATGATG-3' the process of the preparation of the pharmaceutical composition,

3' -Terminal primer: 5'-GGGGAAATTCGAGCTGGTCACCCTAATTAGCCAG-3'.

The target bands were amplified by PCR and separated by agarose gel electrophoresis, and recovered using a gel recovery kit. Vector pCAMBIA3301 was double digested with restriction enzymes NcoI and BstEII and the linear target band was recovered. The map of pCAMBIA3301 is shown in figure 6, the target fragment and the vector enzyme cutting sequence are connected by a homologous recombination method, the connected product is transferred into the competence of escherichia coli DH5a by a heat shock method, and the correct positive bacterial liquid is detected and sent to sequencing. The recombinant plasmid with correct sequence is transferred into EHA105 agrobacterium competence by freeze thawing method.

Agrobacteria infects the lily sheet:

Lily plants well grown in the greenhouse were selected for the test. The specific operation steps are as follows:

(1) The bacterial liquid is activated, 2 days before infection, agrobacterium EHA105 containing different plasmids pCAMBIA3301 and pCAMBIA3301-LhERF061 is streaked on LB solid medium (containing 25mg/L rifampicin and 50mg/L kanamycin) and cultured for 48h at 28 ℃. The monoclonals were picked up in 1mL LB liquid medium (containing 25mg/L rifampicin and 50mg/L kanamycin), cultured overnight at 28℃at 200 rpm.

(2) Cultures were added to 50mL LB liquid medium (containing 25mg/L rifampicin and 50mg/L kanamycin) at a 1:50 ratio and amplified overnight to an OD value between 0.8-1.0.

(3) Preparing a suspension, and centrifuging the bacterial liquid in the step (2) at 6000rpm for 10 minutes. The supernatant was discarded, the prepared suspension was added for suspension, acetosyringone AS was added to the MMA suspension to a final concentration of 200. Mu.M/L, and 2- (N-morpholino) ethanesulfonic acid Mes buffer solution was added at 10mM/L until each bacterial solution OD 600 = 0.8-1.0, and the mixture was allowed to stand at room temperature in a dark place for 4 hours.

(4) Prior to infiltration, agrobacterium EHA105 cultures containing pCAMBIA3301, pCAMBIA3301-LhERF061 were injected with a 1mL sterile syringe to approximate an open, external perianth of the flower bud was a green, uncolored 'Robina' (about 6-7 cm) bud. Agrobacterium (EHA 105) colonies carrying the empty 35S: GUS vector (pCAMBIA 3301) were used as negative controls. Each treatment included at least three biological replicates, each replicate including at least 10 strains. The infiltrated plants were phenotyped in a growth chamber (22 ℃,60% humidity, 16 hours light/8 hours dark). Digital photographs were taken 1 week after infiltration.

Experimental results: as shown in fig. 9, after one week of culture, the festooned sheet phenotypes of each group were observed, and the anthocyanin content of the festooned sheet overexpressed by LhERF061 gene was significantly lower than that of the negative control (the festooned sheet injected with the pCAMBIA3301 empty vector). Meanwhile, the expression quantity of LhERF061 is in negative correlation with anthocyanin positive regulation genes and anthocyanin structural genes, and the structural gene expression quantity of the festoon sheet over-expressing LhERF061 genes is obviously reduced, so that the festoon sheet base is almost free from anthocyanin coloration.

The lily LhERF061 gene provided by the invention is unknown in lily plants and participates in synthesis of anthocyanin in lily angiosperm. The invention successfully clones LhERF to 061 gene, provides the nucleotide sequence and the coding protein amino acid sequence of the gene, and provides theoretical basis and application reference value for germplasm creation of lily flower color molecular breeding.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (6)

1. A lily anthocyanin synthesis regulatory gene LhERF061 is characterized in that the CDS region sequence is shown as SEQ.ID.NO. 1.

2. A lily anthocyanin synthesis regulatory gene LhERF061 encoded protein according to claim 1, characterized in that the amino acid sequence is shown in seq id No. 2.

3. A method for cloning the lily anthocyanin synthesis regulatory gene LhERF061 according to claim 1, comprising the steps of:

(1) Extracting total RNA of lily quilt sample and synthesizing cDNA;

(2) Designing a primer according to transcriptome data, taking cDNA as a template, and carrying out PCR amplification to obtain a gene CDS sequence, wherein the amplified sequence primer is as follows:

5' end primer: 5'-ATGAGCATGATGAATCAATTC-3' A

3' -Terminal primer: 5'-CTAATTAGCCAGTACTTCCCA-3' A

The amplified sequence is compared with the transcriptome sequence after sequencing verification, and the sequence of 705bp of CDS region of LhERF061 gene is obtained, which is shown as SEQ ID NO. 1.

4. A recombinant overexpression vector comprising the lily anthocyanin synthesis control gene LhERF061 according to claim 1.

5. The recombinant overexpression vector according to claim 4, wherein the adaptor primer is designed according to CDS sequence of LhERF061 gene, and the 5 'primer and 3' primer of PCR amplified gene are as follows:

5' end primer: 5'-CACGGGGGACTCTTGACCATGGATGAGCATGATG-3' A

3' -Terminal primer: 5'-GGGGAAATTCGAGCTGGTCACCCTAATTAGCCAG-3' A

And (3) carrying out PCR amplification to obtain a CDS sequence with a joint, and carrying out recombination on a linear vector fragment obtained by using the PCAMBIA3301 double-enzyme-digested vectors of the Nco I and the BstE II and the CDS sequence with the joint by using a homologous recombination method to finally obtain the recombinant overexpression vector.

6. The use of lily anthocyanin synthesis regulatory gene LhERF061 in controlling lily anthocyanin synthesis according to claim 1.