CN112391398A - Apple flavone ketotransferase gene MdGT1 and application thereof - Google Patents

Abstract

The invention discloses an apple flavone ketotransferase gene MdGT1, which is derived from the leaves of malus baccata, the nucleotide sequence of which is shown in SEQ ID No.1, and the amino acid sequence coded by the nucleotide sequence of which is shown in SEQ ID No. 2. In vitro enzymology experiments and in vivo transgenic apple callus experiments prove that when anthocyanin substrates are added to the genes, anthocyanin metabolism is promoted, and salt resistance of callus can be enhanced. The invention identifies an apple anthocyanin glycosyltransferase gene MdGT1 for the first time, provides a preliminary explanation for the function of the gene, and lays a theoretical and practical foundation for the selection and acquisition of apple germplasm resources in the future.

Description

Apple flavone ketotransferase gene MdGT1 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an apple xanthoxylulotransferase gene MdGT1 and application thereof.

Background

Flavonoids are a polyphenolic compound that is widely present in various plants, are numerous and occupy the first place of the natural compound list. Currently, 7000 more flavonoids are found in nature, mainly including flavonols, flavones, isoflavones and anthocyanidins. The synthetic pathway of flavonoids is mainly accomplished through this metabolic pathway of propane. First, chalcone synthase (CHS) catalyzes 4-coumarin-CoA and malonyl CoA to generate chalcone, and then chalcone undergoes a series of enzymatic reactions to generate flavanonol (including dihydrophenol and dihydroquercetin), and then dihydroflavonol is generated under the action of flavonoid synthase (FLS) to generate quercetin and kaempferol. Anthocyanins are produced by hydroxyflavone reductase (DFR). Finally, active quercetin, kaempferol and anthocyanins are modified by various sugar groups in the plant and transported to other sites in the form of inactive glycosides or stored for later use.

Flavonoids are in a wide variety, and many compounds have the same metabolic pathway, so that the metabolic regulation of flavonoids is very complicated and is regulated by various factors. At present, the transcriptional regulation of flavonoid metabolism in Arabidopsis has been well studied. Three major transcription factors are involved in the complex, namely MYB transcription factor, bHLH transcription factor and WD40 protein. These transcription factors can regulate the expression of structural genes by binding to the promoters of these genes for flavonoid metabolism. Similar regulatory mechanisms have also been found in petunia, maize, apple and rice plants. For example, apple has a series of transcription factors that regulate the formation of fruit color. In 2012, Shexing et al found that the transcription factor MdbHLH3 was upregulated at low temperatures. And it can interact with another important transcription factor, MdMYB1, regulate fruit color, and bind to the promoter of MdMYB1 to activate its expression. The two transcription factors can also be directly combined with a promoter of an anthocyanin synthesis structural gene to activate the expression of the anthocyanin synthesis structural gene, so that the apple fruit coloring is promoted.

The flavonoids and the secondary metabolites thereof play an important role in the growth and development of plants. According to research, the physiological effects of flavonoids and anthocyanins in plants are mainly shown in the following aspects. (1) Participating in the stress of plants. Under the adverse UV-B radiation, the content of the flavonoid compounds in the plants can be obviously increased, and the increasing degree of the flavonoid compounds can be changed according to the type, variety and quantity of the radiation. (2) Participate in the reproductive development of plants. In studies with corn and petunia, it was found that pollen fails to form functional pollen tubes when certain flavonoids are deficient. If a certain amount of flavonol is applied to the stigma in the pollen tube medium or during pollination, the pollen function will be restored. (3) Participate in the polar transport of auxin. The research shows that the flavonoid compound can also regulate the polar transportation and distribution of the auxin and compete with an inhibitor NPA, so that the transportation of the auxin is inhibited. (4) Participate in the formation of plant color. Meyer et al introduced the DFR gene from maize into morning glory, which presented a new color. Vanderkrolar et al transferred the antisense CHS gene to petunias, which turned purple petunias white.

In plants, glycosyltransferases are capable of transferring sugar groups to small molecule substances. Such small molecule substances include plant hormones, polypeptides, proteins and secondary metabolites. After glycosyl transfer occurs, the properties of the receptor molecule can be changed, and plant growth and environmental reactions are further influenced. Although considerable progress has been made in recent years in the study of the function of plant glycosyltransferases, major research has focused on the model plant Arabidopsis thaliana. The functions of plant glycosyltransferases are summarized below. (1) Involved in abiotic stress responses in plants. Li et al found that Arabidopsis glycosyltransferase UGT76C2 overexpressing lines exhibited a sensitive phenotype at seedling stage, but a drought resistant phenotype at later stages. (2) Regulating balance of plant hormones. Glycosylation can reduce or even completely eliminate the biological activity of plant hormones, but the mechanism of action is not clear. Studies have speculated that glycosylation might alter the recognition of hormones by receptors. And (III) participating in plant secondary metabolism. Forest et al demonstrated that glycosyltransferase UGT72B1 can glycosylate important precursors of lignin synthesis pathway through in vitro enzymatic reactions and studies of plant mutants, and mutant strains show accumulation of lignin, cell wall thickening and reduced-fertility plant phenotypes. (4) Participate in the biological stress response and detoxification response of plants. The glycosyltransferase gene, TOGT, was overexpressed in tobacco by Matros and Mock et al, and it was found that overexpression enhanced tobacco resistance to potato virus Y. Poppenberger et al found that the Arabidopsis glycosyltransferase UGT73C5 glycosylates the toxin Deoxynorcetophenol (DON) and that the toxicity is lost after glycosylation.

In 2010, Velasco et al sequenced the genome of apples, and laid a foundation for the study of apple gene functions in the future. However, as apples have long growth period, complicated metabolism and difficult gene cloning, studies on functional genes of apples have been limited so far, and studies on genes related to flavonoid metabolic pathways and flavone glycosyltransferase in apples have been less. The research reports of related genes are less. In 2016, Yahyaa et al identified a phloretin-4 '-O-glycosyltransferase gene (MDPH-4' -OGT) from apple. In 2017, Dare et al identified a phloretin-specific glycosyltransferase that interfered with phenylpropane biosynthesis and plant development. In 2017, Weekly et al identified a glycosyltransferase in apples that converts phloretin to phlorizin. In 2019, Elejalde-Palmett et al identified and biochemically analyzed the entire genome of the apple UGT88F subfamily.

The physiological balance of ion concentration between plant cells is the basis for maintaining normal physiological functions of living plant cells. Generally, under normal growth conditions, there is always a high concentration of K + ions and a low concentration of Na + ions in plant cells. However, under salt stress conditions, high concentrations of Na + ions can hinder plant photosynthesis and cause damage to plant cell membranes.

According to the current research, the salt tolerance mechanism of plants mainly comprises the following aspects: (1) regulating the osmotic pressure of the plants. Plants may maintain osmotic balance of plant cells by decreasing the uptake of salt by the plant through dormancy or slow growth and development. Likewise, plants can maintain normal osmotic balance within the cell by increasing the content of soluble sugars, proline and other small molecules in the body. (2) Modulating ion uptake and migration in plant cells. There are many ion channels in plants that regulate the uptake and transport of Na + and K + ions. (3) Eliminating residual active oxygen in plant cells. Active oxygen has great harm to the normal development of plants. Thus, antioxidants that can remove active oxygen include in plants: superoxide dismutase (SOD), Catalase (CAT), ascorbic acid, glutathione, Peroxidase (POD), Glutathione Reductase (GR), and the like. (4) Regulation of plant hormones. Under salt stress, the content of many hormones in plants changes. For example, the plant hormones ABA, ABA content is increased under stress, thereby enhancing the resistance of plants to stress. ABA can also induce the expression of some important genes in a salt-tolerant signal path and promote the closure of stomata.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an apple ketosyl transferase gene MdGT1 and application thereof. A glycosyltransferase gene, MdGT1, was obtained by transcriptome sequencing in apple. In vitro enzymology experiments and in vivo transgenic apple callus experiments prove that MdGT1 participates in the metabolic pathway of anthocyanin and the formation of apple leaf color. The invention identifies an apple anthocyanin glycosyltransferase gene MdGT1 for the first time, provides a preliminary explanation for the function of the gene, and lays a theoretical and practical foundation for the selection and acquisition of apple germplasm resources in the future.

The specific technical scheme is as follows:

one of the purposes of the invention is to provide an apple ketotransferase gene MdGT1, the nucleotide sequence of which is shown in SEQ ID No. 1.

The amino acid sequence coded by the nucleotide sequence of the gene is shown in SEQ ID No. 2.

The subject group discovers a mutant strain of yellow-leaf apple in northern tribune base of Qingdao agricultural science research institute, and analysis of transcriptome sequencing results shows that the flavone metabolic pathway has very obvious change. Through bioinformatics analysis, 1 glycosyltransferase gene MD09G1064900_ T01(MdGT1) was screened. The spatiotemporal expression mode shows that MdGT1 is mainly expressed in stems and leaves; real-time PCR and HPLC analysis respectively find that the anthocyanin-induced MdGT1 is obviously up-regulated, and has stronger anthocyanin enzyme activity in vitro.

Further, the gene is derived from the yellow leaves of Malus baccata (Malus baccata).

The invention also aims to provide application of the apple ketosyl transferase gene MDGT1 in regulation and control of apple anthocyanin metabolism.

Furthermore, the apple xanthose transferase gene MdGT1 is applied to promoting the metabolism of anthocyanin in apple callus.

It is known from research that MdGT1 glycosylates anthocyanins. The invention constructs an MdGT1 plant expression vector and expresses the MdGT1 plant expression vector in apple fruit callus. High performance liquid chromatography analysis shows that the anthocyanin content in the over-expressed callus is obviously reduced; real-time PCR proves that the expression of the anthocyanin-related gene is obviously reduced. Experimental results prove that the flavone ketotransferase MdGT1 in the apples can promote the metabolism of anthocyanin in callus tissues of the apples.

Phenotype observation shows that the over-expressed callus grows worse after NaCl treatment than the control group, and grows better with the addition of exogenous anthocyanin; real-time PCR and physiological data measurement find that MdGT1 participates in salt stress and is closely related to antioxidant pathway. Experimental results show that when anthocyanin is added in vitro, the salt tolerance of apple callus can be improved by MdGT 1.

The invention has the following beneficial effects:

the invention screens out a flavone ketotransferase gene MdGT1 from apples. In vitro enzymology experiments and in vivo transgenic apple callus experiments prove that the gene can be applied to promoting the metabolism of anthocyanin in apple callus. The invention identifies an apple anthocyanin glycosyltransferase gene MdGT1 for the first time, provides a preliminary explanation for the function of the gene, and lays a theoretical and practical foundation for the selection and acquisition of apple germplasm resources in the future.

Drawings

FIG. 1 is the expression of genes involved in the flavonoid biosynthetic pathway in example 3;

FIG. 2 is an alignment of the amino acid sequence of MdGT1 with other plant species in example 4;

FIG. 3 is the spatiotemporal expression pattern of MdGT1 in example 4;

FIG. 4 shows the induced expression of MdGT1 in example 5;

FIG. 5 is an HPLC analysis of the reaction product of MdGT1 catalyzed anthocyanin, quercetin and kaempferol in example 5;

FIG. 6 shows the expression intensity and phenotype of the overexpression line of MdGT1 in example 8;

FIG. 7 is a phenotypic observation of the MDGT1 overexpression line under salt stress and anthocyanin in example 9;

FIG. 8 is a phenotypic analysis of the salt stress and anthocyanin-below MDGT1 overexpression line in example 9;

FIG. 9 is the gene expression associated with the abiotic stress and antioxidant pathways of example 10;

in fig. 3: the horizontal coordinate is sequentially root, stem, young leaf, mature leaf, flower, young fruit skin, mature fruit skin, young seed and mature seed from left to right;

in fig. 4: part A is Anthocyanin (Anthocynin), Quercetin (Quercetin) and Kaempferol (Kaempferol) to induce the expression of MdGT 1; part B is abiotic stress induced expression of MdGT 1;

in fig. 5: part A is MdGT1 catalyzed by anthocyanin, part B is MdGT1 catalyzed by quercetin, and part C is MdGT1 catalyzed by mannitol;

in fig. 6: the part B is total flavonoids of an MdGT1 overexpression system analyzed by HPLC, the part C is total anthocyanins of an MDGT1 overexpression system measured by an ultraviolet spectrophotometer, and the part D is related gene expression of flavonoid biosynthesis pathways;

in fig. 8: b part is biological accumulation statistics, C part is anthocyanin content determination, D part is active oxygen content, E part is antioxidant activity, F part is glutathione content, and G part is ascorbic acid content;

in each figure: anthocy is Anthocyanin, Quercetin is Quercetin, Kaempferol is Kaempferol, Mannitol is Mannitol, Control is contrast, Glucose is Glucose, Relative Expression is Relative Expression, and Wild Type is Wild Type.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

In specific embodiments, the reagents related to molecular biology are purchased from tacara, ltd (japan); the carrier construction reagent was purchased from Tiangen Biochemical technology, Inc. (Beijing, China); chromatography-related reagents and consumables were purchased from Thermo Fisher Science (china); the others are conventional reagents and consumables.

In a specific embodiment, plant material is prepared as follows:

the bud of the Malus baccata (Malus baccata) is naturally mutated into Yellow Leaf (YL), which is found in northern residential base of Qingdao agricultural science research institute in 2014. Other germinating tillers are normal green leaves. In autumn of the year, branches of yellow Green Leaves (GL) are cut off and grafted on Malus hupehensis (Malus hupenensis). Germination of YL and GL was observed in spring 2015, and the length and width of the third leaf selected from the top of the new germination were measured. And the length and thickness of the petiole (diameter) was measured. The stem diameter (diameter) and internode length between the third and fourth leaves were determined from the new shoots. Leaves with the same growth state and without any disease are rapidly preserved in liquid nitrogen, and then transcriptome analysis sequencing is carried out. Three samples were sent per sample. Sequencing was repeated 3 times.

The material used in the space-time expression mode is purple-leaf apple resource grown in 10 years at the north homestead of Qingdao agricultural science research institute. Apple callus was stored in a plant growth chamber and cultured in a dark room at 25 ℃ with passage every 3 weeks. Taking the skin and seeds of the new root, annual branch (stem), young leaf and mature leaf, flower (full bloom), fruit 30 days and 120 days after flowering (without bagging). Immediately after sampling, all materials were placed in liquid, rapidly frozen in nitrogen, and stored in an ultra-low temperature freezer at-80 ℃ for later use.

In a specific embodiment, the various experimental methods are as follows:

1. detection of Reactive Oxygen Species (ROS) levels: the active oxygen level of the callus was measured with a reactive oxygen species measurement Kit (Plant ROS ELISA Kit, Beijing Dongge, CK-E0071P) according to the protocol.

2. And (3) measuring the total antioxidant capacity: the total antioxidant capacity of the callus is measured by using a total antioxidant capacity detection kit (FRAP method, production batch S0116), and the measuring method is operated according to the instruction.

3. And (3) proline content determination: reference is made to the international general method. Determination of soluble sugar content: reference is made to the international general method anthrone process.

4. And (4) HPLC detection: the HPLC is Shimadzu LC-20AT (Shimadzu, Japan). Conditions for HPLC analysis: the mobile phase of the flavonoid compound is acetonitrile (mobile phase A) and 0.1% trifluoroacetic acid (mobile phase B). Eluting with binary high pressure gradient method at flow rate of 1ml/min for 35min, setting procedure of 0min, phase B90%, 20min, phase B25%, 22min, phase B90%, and 35min, and detecting flavone at wavelength of 270 nm. The LC-MC analyzer is a MSQ (American) single quadrupole liquid chromatography mass spectrometer.

Example 1

And (4) determining the contents of anthocyanin and chlorophyll in the apple yellow leaf mutant and the green leaf.

The plants were grown in the northern tribe of the institute of agricultural science, Qingdao, using yellow leaves and green leaves in the preparation material. Leaves were harvested at 2016, 4 months, 15 days, flash frozen in liquid nitrogen, and stored at-80 ℃ until further processing.

Total anthocyanin and chlorophyll contents in leaves were determined by the method proposed by Wang et al (Wang, L., S.ZHao, C.Gu, Y.ZHou, H.ZHou, J.Ma, J.Cheng & Y.Han (2013) Deep RNA-Seq inverters the peach transgenic plant biology,83, 365-. The leaves were ground and mixed for three replicates. Anthocyanins and chlorophyll were extracted in 10 ml methanol (containing 1% hydrochloric acid) for 2 hours at 4 ℃ in the dark. Anthocyanin and chlorophyll contents were measured at 553 and 600nm and the results are shown in Table 1.

TABLE 1 anthocyanin and chlorophyll content in Green and yellow leaves of apple

As shown in Table 1, the contents of chlorophyll a, chlorophyll b and carotenoids in Green Leaves (GL) were significantly higher than in Yellow Leaves (YL). In YL, the chlorophyll a content is only 1.9% of GL, the chlorophyll b content is 7.9% of GL, and the carotenoid content is 10.7% of GL. The average YL anthocyanin content was 17 μ g/g, 30% of GL content, with a significant difference.

Example 2

Extracting total RNA, constructing library and sequencing transcriptome.

1. Total RNA extraction:

extracting total RNA from Green Leaf (GL) and Yellow Leaf (YL) of apple respectively by modified CTAB method.

RNA quantity and quality (purity and integrity) were analyzed with a nanophotometric scoring photometer (IMPLEN, Westlake Village, CA, USA) and Agilent bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively.

2. Library construction and transcriptome sequencing:

equal amounts of total RNA in the samples were mixed as a mixing pool. Enrichment of eukaryotic mRNA with magnetic beads carrying oligo (dT) (if prokaryotic, mRNA was enriched by removal of rRNA via kit); adding an interrupting reagent to break mRNA into short segments, synthesizing first-strand cDNA by using a hexabasic random primer by using the broken mRNA as a template, preparing a second-strand reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA by using a kit; carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed library was qualified by quality inspection using an Agilent 2100Bioanalyzer and then sequenced using a sequencer such as Illumina HiSeqTM 2500 or Illumina HiSeq X Ten to generate 125bp or 150bp paired end data. And after the quality test is qualified, sequencing by using an Illumina sequencer.

Two cDNA libraries were constructed from total RNA from Green (GL) and Yellow (YL) leaves. Genes expressing differently in the two samples were identified and screened to give corrected P values <0.005 and log2 (fold change) values > 1. We compared the DEGs (differentially expressed genes) between varieties at different stages within the varieties and within a particular stage. The number of DEGs varied among the comparisons; approximately 250-5000 single genes showed significant changes in expression, with an average number of 1877.

To identify the biological pathways activated in apple leaves, the present invention maps the annotated sequences to reference pathways in the KEGG database. From these pathways we focus on the "biosynthetic other secondary metabolites" class and leaf pigmentation. KEGG pathway analysis also showed that 328 single genes were divided into 12 subclasses within the category of "biosynthesis of other secondary metabolites". Among these, the cluster of "phenylpropane biosynthesis" is the largest group, followed by "flavonoid biosynthesis" and "flavone and flavonol biosynthesis". 857 single genes are found in genetic information processing, including folding, classification and degradation, replication and repair, transcription and translation. In addition, 5873 monogenes were found in the biological information processing, 58 monogenes were found in the cellular process, and 587 monogenes were found in the organic system.

The present invention selects and studies single genes involved in flavonoid biosynthetic pathways. A total of 80 single genes encoding 14 enzymes were assigned to the flavonoid biosynthetic pathway based on the KEGG pathway assignment.

Example 3

qRT-PCR analysis was performed on the above genes.

The qRT-PCR validation was performed by selecting 12 single genes, which included MdSOD, MdRD22, MdRD29A, MdRD29B, MdAREB1A, MdDREB2B, MdHAR, MdCHS, MdCHI, MdF3H, MdDFR, in addition to MdGT 1. Specific primer pairs for the selected genes described above for qRT-PCR were designed. The primers for MdGT1 are shown in Table 2. Wherein the amplification primers of MdGT1 are shown in SEQ.ID.NO.3(cMdGT1-F) and SEQ.ID.NO.4(cMdGT1-R), and the qRT-PCR primers of MdGT1 are shown in SEQ.ID.NO.5(qMdGT1-F) and SEQ.ID.NO.6(qMdGT 1-R).

First strand cDNA synthesis was prepared using a cDNA reverse transcription synthesis kit (Thermo, USA) (1. mu.g of total RNA was reacted in a 20. mu.L reaction system to obtain cDNA). The rapid real-time detection system ABI7500 (applied biosystems) and Ultra SYBR Mix (ROX) (CWBIO, Beijing, China) are adopted to carry out qRT-PCR. SYBR Green RT-PCR reaction program: 10min at 95 ℃; at 95 ℃ for 15s and 55 ℃ for 1min, for 40 cycles. Three experiments were performed with reference and selected genes in duplicate. The reference gene (. beta. -ACTIN) was normalized. The expression levels of the different genes were analyzed by the comparative CT method (2 Δ Δ CT method).

TABLE 2 amplification primers and qRT-PCR primers for MdGT1

To confirm the single genes obtained by sequencing and further analyze the difference in expression profiles of yellow and green leaves during maturation, five single genes associated with flavonoid synthesis were selected for qRT-PCR analysis. Yellow leaves expressed much less in most of the selected single genes than green leaves, whereas the MdGT1 gene (the sequence of which is shown in seq.id No. 1) showed high expression. These qRT-PCR results are consistent with those obtained from DGE expression profiles, as shown in figure 1.

Example 4

Bioinformatics analysis and prediction was performed to determine the apple flavone ketotransferase candidate gene MdGT 1.

The nucleotide sequence of the glycosyltransferase gene MdGT1 screened from the apple is shown in SEQ ID No.1, and the amino acid sequence coded by the nucleotide sequence is shown in SEQ ID No. 2.

Carrying out complete protein secondary structure analysis by SOPMA online software; predicting a three-dimensional structure using a SWISS MODEL; downloading amino acid sequences of other glycosyltransferase genes from the NCBI gene bank; the amino acid sequence homology and phylogenetic tree construction comparisons of MD09G1141700, UGT79B2, UGT78D2, UGT84A2 and CsUGT78A14 were performed with MEGA7 software.

Amino acid sequence homology comparison shows that glycosyltransferase UGT79B2 of glycoside flavonoids in arabidopsis thaliana and tea leaves; UGT78D 2; UGT84a 2; CsUGT78A14, shown in FIG. 2, shows that all amino acids containing the alkali transferase conserved sequence are 44 (see the box in FIG. 2 for details), and the conserved amino acid sequence is shown in SEQ. ID. NO. 7. Glycosyltransferases in arabidopsis (including MdGT1 and MD09G1141700 in apple), UGT79B2, were analyzed using phylogenetic tree analysis software EMGA 7.0; UGT78D 2; UGT84a2 and CsUGT78a14 in tea leaf). The results show that MdGT1 and UGT79B 2; UGT78D 2; UGT84a 2; CsUGT78A14 is some distance away. This result suggests that the function of MDGT1 may differ functionally from the four published genes. The protein structure of MDGT1 was predicted and the results indicated that the protein structure consisted primarily of alpha-helices and irregular coils.

As shown in fig. 3, the spatiotemporal expression pattern found that MdGT1 was expressed predominantly in stems and leaves, a result indicating that MdFLS1 may play a role in apple leaves and stems.

Example 5

Induction experiments were performed.

And (3) flavonoid induction treatment: apple tree seedlings which normally grow to 6 leaves are taken (note that the plants are ensured to be complete when taking materials, particularly roots are not damaged), and the roots are respectively put into 100 mu mol/L Quercetin (Quercetin), Kaempferol (Kaempferol) and Anthocyanin (Anthocynin) solutions for treatment for 1h,3h,6h,12h and 24 h. After the treatment, all the materials are sampled and then are quickly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at minus 80 ℃ for later use.

Abiotic stress induction treatment: apple tree seedlings that grew normally to 6 leaves were taken (note: the plants were guaranteed to be intact at harvest time, in particular the roots were not damaged) and the roots were treated for 1h,3h,6h,12h,24h at 150mM NaCl, 100. mu.M ABA, 250mM Mannitol (Mannitol) and 4 ℃ respectively. After treatment, all materials were sampled and frozen quickly in liquid nitrogen and stored in an ultra low temperature freezer at-80 ℃ for later use.

In vitro enzyme reaction substrate identification and enzyme activity analysis (same conditions as example 7) were performed, and the results are shown in FIG. 4. As shown in fig. 4A, quercetin, kaempferol, and anthocyanins were most significant in upregulation of the MdGT1 gene. This result suggests that MdGT1 may be involved in flavonoid metabolic pathways. The results are shown in FIG. 4B, where gene MdGT1 was upregulated by NaCl, ABA, mannitol, and 4 ℃ with NaCl upregulation being most significant. This result suggests that MDGT1 may be associated with salt stress.

The induced product was subjected to HPLC detection. As a result, as shown in fig. 5 and table 3, MdGT1 can glycosidate anthocyanins, quercetin, and kaempferol of the phenylpropane family in vitro, but cannot glycosidate other compounds of the phenylpropane family. The specific enzyme activity of quercetin and kaempferol to anthocyanin was about 3.47. From these results, we demonstrated that apple xanthose transferase MdGT1 is likely involved in anthocyanin metabolic pathways.

TABLE 3 results of enzyme Activity analysis of MdGT1

Example 6

Carrying out vector construction

1. Cloning genes: all reaction solutions were prepared on ice. The specific steps are shown in the specification. The kit for gene cloning was purchased from Takara bioengineering, Inc., Dalian. The specific product catalog number is R011.

2. Connecting: first, a gene cloning fragment was recovered by gel. The specific method refers to gel recovery kit purchased from Tiangen Biotechnology Ltd (Beijing), and the specific product catalog number is DP 208/209. The ligation of gene fragments was performed. The intermediate vector pZeroBluntSimple is available from Beijing Quanshi gold Biotechnology Ltd, with the product catalog number CB 101. The PCR product was recovered in 4ul, the Blunt vector in 1ul, and ligated for 5min at room temperature.

3. And (3) transformation: coli and agrobacterium were purchased from the company, Transgene, beijing, and the specifications were referred to for the specific experimental procedures.

4. Plasmid extraction: the extraction of Escherichia coli plasmid refers to Tiangen plasmid extraction kit, and is purchased from Tiangen Biochemical technology Co., Ltd (Beijing). The catalogue number of the escherichia coli plasmid extraction kit is DP 103-03.

Example 7

Protein expression and purification were performed.

1. And (3) bacteria collection: first, a protein-expressing strain containing the desired gene was inoculated into 5mL of LB medium, 50. mu.g/mL of AMP (ampicillin, purchased from Changsheng biotech Co., Ltd., Kyoto, Beijing) was cultured overnight at 37 ℃ for activation of the strain. Then, the above activated bacteria solution was taken out in 2 XYT medium (containing 50ug/mL AMP) and amplified at 20 ℃. The culture was carried out until OD600 became 1.0, IPTG was added to a final concentration of 1mM, and the culture was continued at 20 ℃ for 24 hours. Finally the bacteria were collected by centrifugation at 4500rpm for 10 minutes.

2. Protein purification: the GST-tagged protein on the prokaryotic expression vector can be successfully eluted by binding to glutathione Sepharose 4B Sepharose beads (purchased from general electric company). Specific methods include methods such as Li (Li P, Li YJ, Zhang FJ, Zhang GZ, Jiang XY, Yu HM, et al. the Arabidopsis UDP-glycosylation transfer UGT79B2 and UGT79B3 construct to cool, salt and moisture strain tissue modification anticancer in culture. plant Journal,2017,89:85-103)), and finally active enzyme protein is obtained.

3. In vitro enzymatic reaction: in vitro enzymatic reaction systems are shown in Table 4, and specific procedures are described with reference to methods of Li and the like (Li YJ, Wang B, Dong RR, Hou BK. AtUGT76C2, an Arabidopsis cytokine inactivation lysine in gravity addition adaptation. plant Science,2015,236: 157-167).

4. Kinetic constant analysis: first, the enzyme protein is analyzed for a time-linear relationship with the substrate. According to the above reaction system, the reaction is carried out at optimum temperature and pH value at different times or with different substrate concentrations in a water bath at 30 ℃. Enzyme activity was analyzed according to HPLC peak.

TABLE 4 in vitro enzymatic reaction System

0.5M Tris-HCL(pH＝8.0)	20ul
		50mM MgSO4	10ul
200mM KCl	10ul
		0.1mol/L UDP-glucose	5ul
10% beta-mercaptoethanol	2ul
		100mmol/L substrate	2ul
Purified UGT protein	2-4ug
		ddH2O	To 200ul

Example 8

And carrying out callus transformation on the apple fruits.

From the above studies, MdGT1 glycosylated anthocyanin was known. To further validate the role of glycosyltransferase gene MdGT1 in anthocyanin pigmentation, we overexpressed MdGT1 in apple fruit calli.

Before transformation, the required callus should be prepared, and the callus cultured 10-15 days after subculture is selected for transformation. Transferring the plant expression vector to agrobacterium, screening positive clones by PCR (LB liquid culture medium is used, corresponding antibiotics and rifampicin are added), shaking to be golden yellow (OD600 is about 0.8), activating once, taking out 1-2mL bacterial liquid to 40mL culture medium, adding acetylacetone to 100uM, continuing shaking to be golden yellow, starting infection: (1) pouring the bacterial liquid into a 50mL sterile centrifuge tube, centrifuging at 5000rpm for 5 minutes at room temperature; (2) pouring waste liquid, adding 2mL of sterile water to resuspend bacteria, and then continuing adding water to 20 mL; (3) centrifuging at 5000rpm for 5min at room temperature; (4) discarding the waste liquid, adding 2mL of sterile water, and suspending the bacteria for later use; (5) adding 40mL of sterile water into a sterile Erlenmeyer flask, adding 500uL of 300-one bacteria solution, shaking up, and adjusting OD600 to 0.6-0.8; (6) taking fresh callus, kneading the callus by using forceps, transferring the callus into diluted bacterial liquid, and shaking the callus for 10 minutes at room temperature; (7) filtering the callus on a pre-prepared sterile gauze, collecting the callus, absorbing water on a sterile filter paper, and transferring to a new filter paper; (8) after 2 days of dark culture, transferring the callus onto a callus culture medium by using corresponding antibiotics, and uniformly diffusing; (9) after about 30 days of dark culture, successfully transformed calli will grow a clump on resistant medium, transfer calli to new resistant medium, when they grow to a certain amount, extract DNA and perform PCR identification. The calli identified by PCR were further quantified and relatively high expressing calli (labeled as OE4 and OE15) were selected for further experiments.

The results of the experiments are shown in fig. 6, where OE4 and OE15 were overexpressed in subsequent experimental studies, as shown in fig. 6A. Total anthocyanins were extracted from each callus as shown in FIGS. 6B and 6C. The total anthocyanin content in OE4 and OE15 was indeed significantly lower than in the control group as determined by hplc analysis. Next, the expression of each gene in the anthocyanin synthesis pathway was verified using real-time PCR technology. As shown in fig. 6D, the anthocyanin synthesis pathway-associated genes in OE4 and OE15 were significantly down-regulated.

These results indicate that the flavone ketotransferase MdGT1 in apple can promote the accumulation of anthocyanin in apple callus, which further demonstrates that MdGT1 is indeed involved in the metabolic pathway of anthocyanin.

Example 9

And (5) carrying out stress treatment on the callus.

From the above studies, it is known that MDGT1 is caused by salt stress. In order to directly prove that the increase of anthocyanin improves the salt tolerance of apple callus, low-content anthocyanin is added into the culture medium.

Specifically, the uniformly grown apple calli obtained in example 8 were cultured for 3-4 weeks on a medium containing 100mM NaCl, 10. mu.M anthocyanin, 100mM NaCl and 10. mu.M anthocyanin. And (3) observing and comparing the growth conditions of the calluses by taking a normal culture medium as a control, and determining the fresh weights of different calluses.

The results of the experiment are shown in FIGS. 7 and 8. FIGS. 7 and 8B (fresh weight) show that there is no difference in the growth state of calli on NaCl-free medium; however, on media containing NaCl, OE4 and OE15 grew significantly worse than non-transgenic calli; after adding low content of anthocyanin in the culture medium, the growth of each callus is obviously improved. The anthocyanin was analyzed by HPLC, and as shown in fig. 8C (anthocyanin content), the results were consistent with phenotype, and the anthocyanin content was higher in well-grown calli.

These results indicate that anthocyanin accumulation can improve the salt tolerance of apple callus.

The flavonoids are natural antioxidants in plants and can remove active oxygen. As a result of measuring active oxygen in each callus, as shown in FIG. 8D (active oxygen content), the active oxygen level in the callus was low under normal conditions, but the active oxygen level increased after the treatment with 100mM NaCl, and the active oxygen level in the callus decreased after the addition of anthocyanin to the medium. This result indicates that the increased salt tolerance of the callus is due to the active oxygen scavenging activity of anthocyanins.

The total antioxidant capacity of each callus was measured, and the results are shown in fig. 8E (antioxidant activity). In contrast to the results of active oxygen content in callus, the total antioxidant capacity of over-expressed callus OE4 and OE15 was significantly reduced. Glutathione and ascorbic acid were measured in each callus by the kit. As a result, as shown in FIGS. 8F (glutathione content) and 8G (ascorbic acid content), there was almost no difference in the glutathione and ascorbic acid contents in each callus.

Example 10

And carrying out data statistics and analysis.

All experiments were performed in three independent biological replicates, at least three technical replicates each time were established. Significance of differences between each experimental and control sample, P <0.05, very P <0.01, according to the student test.

In the above studies, it is known that MdGT1 is involved in plant stress by affecting the body's antioxidant capacity. To further study the mechanism, we analyzed the expression of some stress-related functional genes in the MdGT1 calli using real-time PCR. These genes include: MD SOD, MDRD22, MDRD29A, MDRD29B, mdmdmddha, MDDREB1A, MDDREB 2B. MDDREB1A and MDDREB2B are ABA-independent pathway genes and are regulated by salt induction; RD29A is related gene of ABA dependent pathway and is regulated and controlled by salt induction; other genes are related to oxidative stress pathways and are induced and regulated by salt stress. The results are shown in FIG. 9. Under normal conditions, the expression levels of all genes are not greatly different, but stress related genes are all up-regulated after salt stress, the up-regulation of non-transgenic callus is more obvious, anthocyanin is continuously added into a culture medium, and the up-regulation of the related genes in OE4 and OE15 is obvious. This is probably because anthocyanins can promote the expression of stress-related genes, reduce the content of active oxygen in vivo, and increase the ability to resist stress.

In our study, we propose a working model for the apple fructosyltransferase MdGT 1. When plants are stressed by adversity, the adversity stress transcription factor quickly senses the adversity stress environment and is induced to be up-regulated, and then is combined with a promoter region of the gene MdGT1 to regulate and control the expression of the gene MdGT 1. Then, MdGT1 glycosylates the anthocyanin, making it produce a large amount of 3-O-glucose anthocyanin, resulting in less accumulation of anthocyanin content in the plant, but the anthocyanin can remove harmful active oxygen in the plant, ultimately reducing the plant's ability to resist salt stress. In this study, we identified for the first time a flavoketotransferase, MdGT1, from apple, which is involved in the regulation of plant stress through oxidative stress pathways.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (5)

1. An apple xanthophylls transferase gene MdGT1 is characterized in that the nucleotide sequence is shown in SEQ.ID.NO. 1.

2. The maltulose transferase gene, MdGT1, of claim 1, wherein the nucleotide sequence encodes an amino acid sequence set forth in seq id No. 2.

3. The apple ketose transferase gene, MdGT1, according to claim 1, which is derived from the yellow leaves of malus baccata.

4. Use of the apple ketoulotransferase gene MdGT1 of any of claims 1-3 in regulating apple anthocyanin metabolism.

5. The use of claim 1, wherein the maltulose transferase gene MdGT1 is used to promote the metabolism of anthocyanin in apple calli.

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