Protein encoded by safflower CtHA02 gene and its application

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to a protein encoded by the safflower CtHA02 gene and its application. The technical problem to be solved by the present invention is to provide a new direction for exploring the synthesis of safflower HSYA. The technical solution of the present invention is a protein encoded by the safflower CtHA02 gene, and its amino acid sequence is shown in SEQ ID NO.2. The present invention also provides a method for promoting the synthesis of flavonoids in plants, which is achieved by increasing the expression level of the CtHA02 gene in the plant. The present invention provides a new option for the synthesis of medicinally active flavonoids.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a protein encoded by a safflower CtHA02 gene and an application thereof. Background Art

[0002] Safflower (Carthamus tinctorius L.) is also known as golden thread flower and thorny safflower. Since the 21st century, safflower, as a high-quality medicinal and edible crop, has been planted in my country with an area of ​​more than 40,000 hectares. The main producing areas of safflower in my country include Xinjiang Uygur Autonomous Region, Zhejiang, Sichuan, Henan and Yunnan. As a traditional medicinal crop, safflower filaments are rich in flavonoids and have significant medicinal effects. Studies have shown that safflower has multiple functions, including anti-oxidation, inhibition of cancer cell proliferation, prevention of chronic inflammation and relief of diabetes. It can also protect the myocardium and treat cardiovascular and cerebrovascular diseasesADDIN CNKISM.Ref.{E0C06AC2AE9D489cA158793425443985} [14-6] ; Studies have found that more than 200 compounds have been found in safflower filaments, including more than 60 flavonoids, such as chalcone glycosides, safflower yellow, kaempferol glycosides and hydroxysafflor yellow A (HSYA). The pharmacological effects of safflower are mainly attributed to its rich flavonoids. HSYA, which is unique to safflower, has shown significant effects in various clinical treatments. Since HSYA was first isolated from safflower in 1993 and found to have significant pharmacological activity, it has been regarded as an important standard component for evaluating the medicinal value of safflower by the Chinese Pharmacopoeia. Modern studies have shown that HSYA has significant pharmacological activity and is widely used to treat or prevent a variety of diseases. The saffron yellow injection approved by the China Food and Drug Administration (containing 45 mg HSYA per 50 mg) is often used to treat cardiovascular and cerebrovascular diseases such as angina pectoris and cerebral infarction, and is recognized as a new type of cardiovascular drug. In addition, HSYA is also widely used to treat myocardial infarction, myocardial ischemia, coronary heart disease and vascular damage, and has anti-cancer, anti-oxidation, blood pressure lowering, liver cell and nerve cell protection functions. As one of the medicinal active ingredients in safflower, HSYA has shown important research and application value in pharmacology and clinical applications.

[0003] Safflower is rich in a variety of flavonoids, which are important indicators for evaluating the medicinal value of safflower and also have significant pharmacological effects. At present, scholars have discovered multiple genes that regulate the biosynthesis of flavonoids in safflower, including flavanone 3-hydroxylase (F3H), UDP-glucuronic acid transferase (UDP-glucuronic acid, UGT), chalcone synthetase / isomerase (CHS / CHI) and anthocyanin synthase (ANS). The expression levels of CHS, CHI and ANS at different flowering stages of safflower are closely related to the synthesis and content of flavonoids. Based on the transcriptome data of safflower, it was found that the upstream genes CHS, CHI and hydroxycinnamyl transferase (HCT) in the flavonoid synthesis pathway were upregulated under MeJA treatment, and the downstream genes anthocyanin reductase (ANR) and ANS were downregulated, thereby promoting the biosynthesis of flavonoids. In addition, two CHI genes (CHI1 and CHI2) were cloned in safflower, and the accumulation pattern of HSYA and the expression pattern of CHI1 gene during the flowering period had a similar trend; the transient transformation of tobacco leaves showed that the CHI2 gene may affect the accumulation of flavonoids in different flowering stages of safflower. In the transcriptome data analysis of safflower corolla, 20 reductase genes related to flavonoids were found, among which the overexpression of CtSDR3 gene could significantly increase the content of HSYA, Scutellarin and D-Phenylalanine compounds in the overexpression strain. Recent studies have shown that C-glucuronic acid (CGT) and P450 enzymes may be involved in the biosynthesis of HSYA; however, the genes in the safflower HSYA biosynthesis pathway have not yet been fully reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new direction for exploring the synthesis of safflower HSYA.

[0005] The technical solution of the present invention is a protein encoded by the safflower CtHA02 gene, and its amino acid sequence is shown in SEQ ID NO.2.

[0006] Furthermore, the nucleotide sequence of the CtHA02 gene is shown in SEQ ID NO.1.

[0007] The present invention also provides an expression framework, element, vector or host for expressing the protein encoded by the safflower CtHA02 gene.

[0008] Wherein, the vector is a plant expression vector.

[0009] Preferably, the backbone vector of the plant expression vector is pAMBIA1301.

[0010] The present invention also provides the use of a protein encoded by the safflower CtHA02 gene, an expression framework, an element, a vector or a host for expressing the protein encoded by the safflower CtHA02 gene in regulating the synthesis of plant flavonoid compounds.

[0011] Specifically, the flavonoid compound is genistein, echinatin, dihydrokaempferol, naringenin chalcone, corylin, isorhamnetin, kaempferol, hydroxysaffron yellow A (HSYA), eriocytoside, quercetin, spiraeoside, luteolin and / or apigenin.

[0012] Particularly, the plant is Carthamus tinctorius or Arabidopsis thaliana.

[0013] Furthermore, the regulation is positive regulation.

[0014] Wherein, the positive regulation is achieved by increasing the expression level of the CtHA02 gene in the plant.

[0015] Furthermore, the method for increasing the expression level of the CtHA02 gene in plants is: constructing a plant expression vector expressing the CtHA02 gene, and transforming the plant.

[0016] Preferably, the backbone vector of the plant expression vector is pAMBIA1301.

[0017] The present invention also provides a method for promoting the synthesis of flavonoid compounds in plants, which is achieved by increasing the expression level of the CtHA02 gene in the plant; the amino acid sequence of the protein encoded by the CtHA02 gene is shown in SEQ ID NO.2.

[0018] Specifically, the flavonoid compound is genistein, echinatin, dihydrokaempferol, naringenin chalcone, corylin, isorhamnetin, kaempferol, hydroxysaffron yellow A (HSYA), eriocytoside, quercetin, spiraeoside, luteolin and / or apigenin.

[0019] Particularly, the plant is Carthamus tinctorius or Arabidopsis thaliana.

[0020] Furthermore, the method for increasing the expression level of the CtHA02 gene in plants is: constructing a plant expression vector expressing the CtHA02 gene, and transforming the plant.

[0021] Preferably, the backbone vector of the plant expression vector is pAMBIA1301.

[0022] Beneficial effects of the present invention: The present invention cloned and obtained the safflower CtHA02 gene for the first time, and verified the function of the gene in plants through transgenic technology. The results showed that the contents of most medicinal flavonoids (genistein, echinatin, dihydrokaempferol, naringenin chalcone, corylin, isorhamnetin, kaempferol, hydroxysafflower yellow A HSYA, eriocytoside, quercetin, spiraeoside, luteolin and / or apigenin) in the leaves overexpressing CtHA02 were significantly increased compared with the control. In particular, the contents of medicinal active flavonoids such as HSYA, kaempferol and quercetin were significantly increased, further confirming that the CtHA02 gene can promote the accumulation of medicinal active flavonoids. The HSYA content in the leaves of transiently transformed safflower increased by 2.1 times, and the HSYA content in transgenic Arabidopsis increased by 1.8 times. The present invention provides a new option for the synthesis of medicinal active flavonoid compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the linkage disequilibrium and chromosome location map of the safflower CtHA02 gene.

[0024] Figure 2 Phylogenetic tree of the amino acid sequence encoded by the CtHA02 gene of safflower and its alignment with other related genes.

[0025] Figure 3 The relative expression of safflower CtHA02 gene in different tissues of safflower (A), the relative expression of safflower CtHA02 gene in flowers at different flowering stages (B), and the sampling diagram of safflower at different times during flowering (C). T1: 1h of flowering, T2: 24h of flowering, T3: 48h of flowering, T4: 60h of flowering. In the figure, a, b, c, and d are used to indicate the statistical differences between different groups of data. If the same letter is marked above the column, it means that there is no significant difference between the corresponding two groups of data; if different letters are marked, it means that there is a significant difference between the corresponding two groups of data.

[0026] Figure 4 Schematic diagram of pCAMBIA1301g-CpFT vector.

[0027] Figure 5 This is the detection diagram of HSYA content in safflower cotyledons after CtHA02 transient transformation.

[0028] Figure 6 PCR detection of CtHA02 gene in transgenic Arabidopsis. M: DNAmaker DL5000; 1-9: resistant strains; WT: wild type; CK: negative control.

[0029] Figure 7 Real-time fluorescence quantitative PCR was used to detect the relative expression level of the CtHA02 gene in different individuals of the T2 generation of transgenic Arabidopsis thaliana, with Actin as an internal reference.

[0030] Figure 8 This is a graph showing the detection of HSYA content in the T2 generation of CtHA02 transgenic Arabidopsis plants.

[0031] Fig. 9 It is a phylogenetic tree of 163 related genes and flavonoid biosynthesis genes; ● represents the genes involved in regulating flavonoid biosynthesis in published articles.

[0032] Fig.10 This is a heat map of the expression levels of 163 related genes in different tissues of safflower.

[0033] Fig.11 qRT-PCR analysis of 12 candidate genes in 4 varieties with different HSYA contents: 23B002 / 23A005: low HSYA content varieties; 23A012 / 23A024: high HSYA content varieties.

[0034] Fig.12qRT-PCR analysis of 12 candidate genes in different tissues of safflower.

[0035] Fig.13 qRT-PCR analysis of 12 candidate genes at different flowering stages in two varieties with different HSYA content; 23A005: variety with low HSYA content, 23A0012: variety with high HSYA content; T1: 1 hour after flowering, T2: 24 hours after flowering, T3: 48 hours after flowering, T4: 60 hours after flowering.

[0036] Fig.14 The tertiary structure of the protein encoded by the CtHA02 gene from Carthamus tinctorius. DETAILED DESCRIPTION

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are based on conventional experimental conditions.

[0038] Main reagents: Phanta Max Super-Fidelity DNA Polymerase (Phanta high-fidelity DNA polymerase (single reagent), Nanjing Novozymes Biotechnology Co., Ltd.).

[0039] Kits: RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN, DP441); FastKing cDNA First-Strand Synthesis Kit (Genomic Depleted) (TIANGEN, KR116); SuperReal Fluorescence Quantification Premix Enhanced Kit (SYBR Green) (TIANGEN, FP205); Nanjing Novozyme Biotechnology Company C114 Kit; GUS Staining Kit (Coolaber).

[0040] Example 1 Preliminary Analysis

[0041] The MLM model was used to perform GWAS analysis on HSYA content. Under the condition of significant P value of 6.61 (-log(0.1 / 406,698)≈6.61), 12, 14 and 11 SNP loci significantly associated with HSYA content were detected in the three environments, respectively. A total of 18 SNP loci were detected, which were unevenly distributed on the 12 chromosomes of safflower. Among them, qHA4, qHA8.2, qHA8.4, qHA10.2, qHA11.2, qHA12.1, qHA12.2 and qHA12.3 loci were commonly associated in two environments; qHA7.1, qHA8.1, qHA9, qHA10.1 and qHA11.1 loci were commonly associated in three environments; and finally 163 related genes were obtained.

[0042] By comparing and analyzing the 163 related genes in the association sites with the genes involved in the biosynthesis of flavonoids, a phylogenetic tree of the 163 related genes and the genes involved in the biosynthesis of flavonoids was constructed using MEGA software ( Fig. 9 ). The results showed that some related genes were clustered into similar branches with flavonoid biosynthesis genes. Based on the correlation between function and evolution, it is speculated that these genes with similar branches are related to the biosynthesis of flavonoids.

[0043] We further used the NCBI-SRA database to obtain RNA-seq data from different tissues of safflower, and analyzed the expression patterns of 163 related genes in different tissues of safflower ( Fig.10 ). The results showed that most of the related genes were expressed in the root, stem, leaf and flower tissues of safflower, and their expression levels in different tissues were significantly different. Among them, 125 genes were expressed in roots; 123 genes were expressed in leaves; 126 genes were expressed in stems; and 130 genes were expressed in flowers. Combining the results of phylogenetic tree and expression pattern analysis, this study preliminarily screened out 12 candidate genes, which were close to the flavonoid biosynthesis gene branch and had higher expression levels in flowers than in other tissues, indicating that these candidate genes may play an important role in the synthesis of HSYA (Table 1). The candidate gene CtHA07 (evm.TU.Hic_asm_8.1150) encodes UDP-glucosyltransferase / flavonol 3-O-glucosyltransferase, which is widely involved in the biosynthesis pathway of plant flavonoids and affects the accumulation and distribution of flavonoids in plants.

[0044] Table 1 Functional annotation of preliminary screened candidate genes

[0045]

[0046] To further verify the above research results, four safflower varieties with significant differences in HSYA content were selected, and the expression patterns of 12 candidate genes in different safflower varieties, different tissues and different flowering periods were analyzed by qRT-PCR technology. Fluorescence quantitative analysis of the four varieties with different HSYA content found that, except for the CtHA05, CtHA09 and CtHA10 genes, the expression levels of other candidate genes in varieties with high HSYA content were significantly higher than those in varieties with low HSYA content ( Fig.11 Among them, the expression levels of CtHA01 and CtHA02 genes in high HSYA content varieties were more than 4 times higher than those in low HSYA content varieties, and the expression levels of CtHA03, CtHA04, CtHA05, CtHA06, CtHA11 and CtHA12 genes in high HSYA content varieties were more than 8 times higher than those in low HSYA content varieties.

[0047] Tissue-specific analysis revealed that the expression levels of the 12 candidate genes in different safflower tissues were significantly different ( Fig.12 Among them, the expression levels of CtHA01, CtHA02, CtHA07 and CtHA08 genes in flower tissues were significantly higher than those of other candidate genes, and their expression levels in flowers were more than 4 times higher than those of other candidate genes.

[0048] Fluorescence quantitative analysis of 12 varieties with different HSYA content at different flowering stages found that the expression levels of the 12 candidate genes at different flowering stages were significantly different, and the expression levels of most candidate genes at different flowering stages in varieties with high HSYA content were significantly higher than those in varieties with low HSYA content ( Figure 3-13 ). As the flowering period progressed, the expression level of the CtHA02 gene showed a rapid growth trend, reaching a peak at the peak of flowering, and was significantly higher than its expression level in low HSYA content varieties, which was similar to the accumulation pattern of HSYA during the flowering period of red flowers ( Fig.13 ). In summary, this study confirmed the important candidate gene of CtHA02. Figure 1 As shown, the location of CtHA02 gene on chromosome is: between 26654060 and 26658331 on chromosome Chr8. After determination, the cDNA sequence of Carthamus tinctorius CtHA02 is shown as SEQ ID No.1, and the encoded amino acid sequence is shown as SEQ ID No.2.

[0049] SEQ ID No.1 Carthamus tinctorius CtHA02 cDNA sequence:

[0050] atgaaaaccc taaatcccaa atgctacttc aactccccac tttaccacct ttctctacttcccatcg cttcctcgta acaatcgcac tttctcaac ctcccaaca ccacaaacc tctcccagatataaata cggtcttg tcgtacca gggcgattaggactcg tcatccgccggtggcgatc aggtgaagcc aatcagctca acaggttatt gattcactc ggaatccatg acaaaagtatgcattgtgt atctgttagg tttcgtatct gctcgctc tttctagggt taggtatca tcgatcgtt gttgtttttttcccag atttgggatt gtgaatgggg ggcatttgaatgaggttatg agtatattcg ggggtaatgg tggtaagaa aacaagaaga aaggattcag agatgatgaggttaggttt cgattgaaa cttgaggaat tggtggagt tgttaaatgg gttgatgta atattgaatcagtagtag tagtagtag tag aattagtaaa agagatttag tagttatgttgaggtgta gatcgattg aacaagcttt ttgcaagcta aaggtatgat tgaggttat attgatatatggtagttga cacaatgaa actctgaaag cttcgaatca gaacctagt gatcaaaga aagaggtc taggaggattcattcagg ggggaagatc tttgggtcg aggcctagtgaattgaaaga tagtgtcaag atgattchca gatcttcaga ggctagcgat catggttcag haaacgtcatggatccctt tgttgaaggcaagacgacga aatctattcc aagtgatgat ataaacacat ataacgacaatgatgttagg gttaaagaaa catatggagg aaaagcagtg gtagaatggc taaaatgact ttcatggagatggatactgg tgctaaacaa gtttttgaca gagatgagta tgtcattaag gataacaat tgcggatctta tcgacattc caataggaat ccggttcaga gatgggagtc tgatgatggttttcttgatt cggagttcag tgccagcctt aagcaaaaaa gaacagagga atttttcag caagaacaaagaaagttcaa gaaaactaat gggaataata tgctttatga gaatctggaa tcaagggaaa agatgaatgaatgactgactgact ttcctctccg tcttccgaga tatcaagtga tatgcttttcaacgagtgtg taaccgaggc aaatgcactt ctgaagcaag cgagagagtg gttgagacat agaggtatcgaagaaaatg agaaaattg ttgtatagat caaccgaatt gctctctaaa gccgtagaaaa gcgtagaatt agccatgcatt acacttacct tcttcatgga gaattgaaac tgaaactcagccgtaaactg agagctctac ttgcacagag tgactccgat gtactagaaa gcctagatga tgaggtttctaacagagaaa agtttgttga ttatcttgtt aacgttttga agagtgtgaa gaactccttac cgggaatgat atgagagcat tgtacaactggggtcttgct ctctccttca gggctcaatt gatagcagacatcggacctg aagcagcttt ggatgctgacaaggtttact tggcggccat tgacaagttt gacgcaatga tgtccaaaag caatgctcac acaccagatgctcttttcag atggggtgtg gcgttacaa caaagatctc gcttacgaaa caagaaactt tagagagaaggttaaattgt tgagccaggc gaaaaggctt tacgaggacg cacttctcat ggactccagc aatctccaagtgaaagaagc cctatcgact tgtgtatccg aactcaggtac aagaattact actag

[0051] SEQ ID No.2 Amino acid sequence of the protein encoded by the safflower CtHA02 gene:

[0052] MKTLIPNATSTPHFTTFISNFPSLPRNNRTFLNLPNTTKPLPRYKITVLCSYPADYGGWADLTSSAGGDSGEANQLNRLLNSLGIHDKRYAFVYLLGFVSALALSRVRVSSIVVFPACFLVFAIGFSFGIVNGGHLNEVMSIFGGNGGKKNKKKGFRDDEVRVSIENLRNLVELLNG FDVNIVNLKNSMRKDVDSNRISKRDLVSYVEGVESIEQAFLQAKGMIEGYIDNMVVDNNETLKASNQKPSDQKKEVKGKRFDVSQFIDGFRGRSFGSRPSELKDSVKNDSRSSEASDHGSANVMDPLVEGKTTKSIPSDDINTYNDNDVRVKETYGGKAVGRMAKMTFMEMDTGAKQV FDRDEYVIKDNKLRFMKSRRQSFDMNNRNPVQRWESDDGFLDSEFSASLKQKRTEEFFQQEQRKFKKTNGNNMLYENLESREKEAFDELQNENGFASSPSSEISSDMLFNECVTEANALLKQAREWLRHRGIEENAENMLYRSTELLSKAVEMKPMSLLAVGQLGNTYLLHGELKLKL SRKLRALLAQSDSDVLESLDDEVSNREKFVDYLVNVCEECEELLVKAGRKYRLALSIDGNDMRALYNWGLALSFRAQLIADIGPEAALDADKVYLAAIDKFDAMMSKSNAHTPDALFRWGVALQQRSRLRTRNFREKVKLLSQAKRLYEDALLMDSSNLQVKEALSTCVSELRYKNYY

[0053] Multiple sequence alignment and phylogenetic tree construction of amino acid coding sequences

[0054] The phylogenetic tree was constructed by using MEGA software to compare the safflower CtHA02 gene with genes regulating flavonoids in reported articles. Figure 2 As shown, the CtHA02 gene and flavonoid synthesis genes CHI1, CHS2, etc. are clustered into similar branches. Based on the correlation between function and evolution, it is speculated that CtHA02 is related to the biosynthesis of flavonoids.

[0055] Example 2 Cloning of Carthamus tinctorius CtHA02 gene

[0056] CDS sequence and primer design

[0057] According to the annotation information of the safflower cDNA library constructed in this laboratory, the cDNA sequence of the safflower CtHA02 gene containing the complete ORF frame was detected. The experimental material of the cDNA library is the A017 safflower variety grown in the laboratory, and the flowers of different periods are taken as materials for construction. According to the obtained cDNA sequence, specific primers with homology arms and specific restriction sites were designed using the CED esign V1.04 online tool. The primer sequences are as follows:

[0058] CtHA02-F: CGGGGATCCTCTAGAGTCGACATGAAAACCCTAATCCCAAATGC (SEQ ID No. 3);

[0059] CtHA02-R: GCCAAGCTTGCATGCCTGCAGCTAGTAGTAATTCTTGTACCTGAGTTCGG (SEQ ID No. 4).

[0060] Template and cloning vector construction

[0061] Using safflower cDNA as a template, PCR was performed using Phanta high-fidelity DNA polymerase and specific primers to amplify a fragment of about 2,100 bp. The target fragment was recovered, and then the CtHA02 gene was connected to the pCAMBIA1301 linearized empty load after double restriction digestion using the C114 kit of Nanjing Novozyme Biotechnology Co., Ltd. Then, the ligation product was transformed into Escherichia coli Dh5α competent cells, and the recombinant plasmid was screened and sequenced. It was determined that the safflower CtHA02 cDNA sequence is shown in SEQ ID No.1, and the encoded amino acid sequence is shown in SEQ ID No.2.

[0062] Example 2 Analysis of Carthamus tinctorius CtHA02 Gene Expression

[0063] The roots, stems, young leaves, mature flowers of A017 safflower (all collected from the plant room of Xinjiang Agricultural Science and Economic Crops Institute and under routine maintenance and management) and filament materials at different flowering periods (T1: 1 hour after flowering, T2: 24 hours after flowering, T3: 48 hours after flowering, T4: 60 hours after flowering, all collected from the plant room of Xinjiang Agricultural Science and Economic Crops Institute and under routine maintenance and management) were taken.

[0064] Primer design, internal reference gene

[0065] The EF-1a and Actin genes of safflower were selected as the double internal reference genes for the real-time fluorescence quantification of the safflower CtHA02 gene. The fluorescence quantitative specific primers of the internal reference gene and the target gene CtHA02 were designed by the NCBI-Primer-BLAST online tool and then sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The primer names and sequences are shown in Table 2.

[0066] Table 2 Primer names and sequences

[0067]

[0068] 2. Analysis of gene expression characteristics of Carthamus tinctorius CtHA02

[0069] (1) Acquisition of materials from different tissues

[0070] When the 23A005 and 23A012 varieties of safflower (provided by the Xinjiang Agricultural Science Economic Crops Research Institute, the HSYA content in the filaments of the 23A005 variety was 0.4%; and the HSYA content in the filaments of the 23A012 variety was 5.9%, and both varieties were bred by conventional breeding methods) grew to maturity, the roots, stems and leaves of mature safflowers, as well as filaments of four flowering periods (T1: 1 h of flowering, T2: 24 h of flowering, T3: 48 h of flowering, and T4: 60 h of flowering) were obtained. A total of 1 g (0.1 g / bag) of each tissue was taken, marked, quick-frozen in liquid nitrogen, and stored at -80°C.

[0071] (2) Extraction of RNA from safflower roots, stems, leaves and filaments

[0072] 1) Take out 0.1 g of safflower roots, stems, leaves and filaments from -80°C and immediately freeze them in liquid nitrogen;

[0073] 2) Pre-cool the mortar and grinding rod with liquid nitrogen to a temperature that cannot be touched by fingers, and then pour each tissue into the mortar and grind it thoroughly (the mortar and materials should be kept at ultra-low temperatures and not thawed during this period);

[0074] 3) After quick freezing the medicine spoon in liquid nitrogen, use the medicine spoon to transfer each ground tissue into a 1.5 mL RNase-free centrifuge tube, immediately add 500 μL of lysis buffer SL (containing β-mercaptoethanol) into each centrifuge tube, and centrifuge at 12,000 rpm (or 13,400 × g) for 2 min;

[0075] 4) Transfer the supernatant to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12,000 rpm (or 13,400 × g) for 2 min, and carefully pipette the supernatant in the collection tube into a new RNase-Free centrifuge tube, trying to avoid the pipette tip from contacting the cell debris precipitate in the collection tube;

[0076] 5) Slowly add 0.4 times the volume of the supernatant anhydrous ethanol, mix well (precipitate may appear at this time), transfer the resulting solution and precipitate into the adsorption column CR3, centrifuge at 12,000 rpm (or 13,400 × g) for 15 sec, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0077] 6) Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm (or 13,400 × g) for 15 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0078] 7) Preparation of DNaseel working solution: Take 10 μL of DNaseel storage solution and put it into a new RNase-Free centrifuge tube, add 70 μL of RDD buffer and mix gently;

[0079] 8) Add 80 μL of DNaseI working solution to the center of the adsorption column CR3. After standing at room temperature for 15 min, add 350 μL of deproteinization solution RW1 to the adsorption column CR3. Centrifuge at 12,000 rpm (or 13,400 × g) for 15 sec. Pour away the waste liquid in the collection tube and put the adsorption column CR3 back into the collection tube.

[0080] 9) Add 500 μL of rinse solution RW to the adsorption column CR3 (please check whether ethanol has been added before use), centrifuge at 12,000 rpm (or 13,400 × g) for 15 sec, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0081] 10) Repeat step 9;

[0082] 11) Centrifuge at 12,000 rpm (or 13,400 × g) for 2 min, place the adsorption column CR3 in a new RNase-Free centrifuge tube, add 30-50 μL RNase-Free ddH2O to the middle of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm (or 13,400 × g) for 1 min to obtain RNA solution;

[0083] 12) Measure the concentration and check the RNA integrity by agarose gel electrophoresis. The remaining RNA can be used for reverse transcription immediately or stored at -80°C.

[0084] (3) Detection of RNA concentration and integrity in various tissues of safflower

[0085] 1) Use a micro-spectrophotometer to measure the concentration of RNA, the ratio of A260 / 280 and A260 / 230. A260 / 280 of 2.0 is the best, and A260 / 230 should be between 2.0 and 2.5.

[0086] 2) Agarose gel electrophoresis was used to detect RNA integrity. Complete RNA should have two bands, of which 28S is twice as bright as 18S.

[0087] (4) Reverse transcription of RNA from safflower roots, stems, leaves and filaments (operated on ice throughout the process to avoid RNase contamination)

[0088] 1) Please perform the following steps on ice. Thaw the template RNA on ice; thaw 5×gDNA Buffer, FQ-RTPrimer Mix, 10×King RT Buffer, and RNase-Free ddH2O at room temperature and quickly place on ice after thawing. Vortex each solution to mix before use, and briefly centrifuge to collect the liquid remaining on the tube wall;

[0089] 2) Prepare the genomic DNA removal system. Prepare the mixture and mix thoroughly. Centrifuge briefly and incubate at 42°C for 3 min. The system is as follows: 4 μL of 5×gDNA Buffer, 2 μg of RNA from safflower roots, stems, leaves and filaments, add RNase free ddH2O to 20 μL;

[0090] 3) Prepare the reverse transcription reaction system. Add the mixed solution from the reverse transcription reaction to the reaction solution from the gDNA removal step and mix thoroughly. The reaction system is 10×King RT Buffer 2 μL, FQ-RT Primer Mix 2 μL, FastKing RT Enzyme Mix 1 μL; RNase-Free ddH2O 8 μL.

[0091] 4) Incubate at 42°C for 15 min; incubate at 95°C for 3 min and place on ice. The resulting cDNA is stored at -20°C for reverse transcription.

[0092] (5) qRT-PCR

[0093] 1) Using the cDNA of safflower root, stem, leaf and filament stored at -20°C as template, the primers are qCtHA02-F / R and internal reference qCtEF-1a-F / R and qCtActin-F / R, prepare 20 μL reaction system on ice. Prepare the target gene reaction system in a 1.5 mL centrifuge tube as follows: 2×SuperReal PreMix Plus 10 μL, forward primer (10 μM) 0.6 μL, reverse primer (10 μM) 0.6 μL, cDNA template 1.0 μL, RNase-free ddH2O 7.8 μL.

[0094] 2) Three-step reaction procedure: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing at 60-65°C for 20 s, extension at 72°C for 30 s, 40 cycles.

[0095] 3) Via CFX96 Touch TM Real-Time PCR System (Bio-Rad, USA) was used for qRT-PCR analysis. -ΔΔCt The relative gene expression was calculated.

[0096] 3. Analysis of the expression characteristics of the safflower CtHA02 gene in different tissues of safflower

[0097] The expression characteristics of the CtHA02 gene in different tissues of safflower were analyzed by real-time fluorescence quantitative PCR. It was found that the gene was expressed in the roots, stems, leaves and flowers of safflower during the mature period. The expression level of the CtHA02 gene in flowers was relatively the highest, followed by the expression level in leaves, and the lowest in stems and roots. Its expression level in flowers was 10.2 times that in roots and 3.6 times that in stems ( Figure 3 A).

[0098] 4. Analysis of the expression characteristics of the safflower CtHA02 gene in different flowering stages

[0099] The expression characteristics of the CtHA02 gene in flowers of safflower at different flowering stages were analyzed by real-time fluorescence quantitative PCR. It was found that the expression of the gene increased with the advancement of the flowering period, with the highest expression level in the T4 period, which was about 8.8 times that of the T1 period. At the same time, the expression level of the gene in the high HSYA content variety (23A012) was significantly higher than that in the low HSYA content variety (23A005), and was similar to the accumulation pattern of HSYA in the flowering period of safflower ( Figure 3 B).

[0100] Example 3 Transient transformation of safflower CtHA02 gene and differential analysis of metabolites

[0101] 1. Construction of the Carthamus tinctorius CtHA02 gene overexpression vector

[0102] GUS was used as a reporter gene. First, the CtHA02 gene was connected to the pCAMBIA1301 linearized empty vector after double enzyme digestion using the C114 kit of Nanjing Novozymes Biotech Co., Ltd. Then, the ligation product was transformed into Escherichia coli Dh5α competent cells, spread on LB solid culture medium containing kanamycin (Kana), and cultured at 37°C for 12-14 h. A single colony was picked up with a sterile pipette tip for PCR and double enzyme digestion identification. After correct identification, it was sent to Beijing Bomade Gene Technology Co., Ltd. for sequencing to obtain the correctly sequenced pCAMBIA1301-CtHA02-GUS vector. The vector schematic diagram is shown in Figure 4 shown.

[0103] 2. Transformation of safflower cotyledons by Agrobacterium injection

[0104] (1) Agrobacterium transformation

[0105] The constructed plasmid vector was transformed into Agrobacterium GV3101 competent cells by heat shock transformation method, spread on LB solid medium containing kanamycin (Kana) and rifampicin (Rif), and cultured at 28°C for 48 h. The colonies were screened by colony PCR method to confirm the positive single colony. The positive single colony was transferred to LB liquid medium (LB+Kan+Rif) for expansion and cultured at 28°C with shaking until the OD600 value was 1.0. Then 500 uL of bacterial solution was taken and expanded in LB liquid medium (LB+Kan+Rif) until the OD600 value was 1.0, centrifuged at 3,950 rpm for 10 min to precipitate the bacteria, and washed with resuspension solution twice. Finally, the Agrobacterium cells were resuspended to different OD 600 Spare (OD 600 = 0.6, 0.8, 1.0 and 1.2).

[0106] (2) Injection and GUS staining of safflower cotyledons

[0107] The cotyledons of safflower seedlings of 23A005 variety that were cultivated for 7 days and in good growth condition were used as experimental materials. They were watered well the night before the experiment. First, a 1 mL sterile syringe needle was used to gently scratch the epidermis at the branch of the veins on the back of the cotyledon. Be careful not to pierce the epidermis of the cotyledon. The sterile syringe needle was removed and the concentration of OD was injected into different samples. 600= 0.6, 0.8, 1.0 and 1.2 suspension. Then, the injected safflower seedlings were cultured at 28℃ in the dark for 2 days. Finally, the treated cotyledons were immersed in GUS staining solution and infected at 37℃ in the dark for 24 hours. After taking out, remove the GUS staining solution and wash with 75% ethanol three times. If the stained area of ​​the cotyledon exceeds 2 / 3, it is judged as positive.

[0108] (3) Detection of positive cotyledon flavonoid compounds

[0109] Select 3 to 6 plants with the same growth from the injected plants and the control plants as the source of a biological replicate sample. Select 3 to 4 leaves (the number is adjusted according to the size) from the same position of the different plants corresponding to the source of the replicate sample, mix them, cut them into 1-2 cm lengths, and crush the oversized leaves slightly after quick freezing in liquid nitrogen, put them into a 10 ml centrifuge tube, quick freeze them in liquid nitrogen for 5-10 minutes, and store them in a -80 ° C refrigerator. Each sample has 5 biological replicates, each replicate is 0.6g. Then send them to Wuhan Mattwell Biological Co., Ltd. for quantitative detection of flavonoids.

[0110] 3. Determination of flavonoid content in cotyledons of safflower CtHA02 gene overexpression

[0111] The quantitative detection of the content of brass compounds in the cotyledons of the control CK and CtHA02 gene overexpression showed that the content of most medicinal flavonoids in the leaves of CtHA02 overexpression was significantly increased compared with the control ( Figure 5 ). Among them, the contents of Genistein, Echinatin, Dihydrokaempferol, Naringenin chalcone, Corylin and Isorhamnetin in the overexpressed leaves ranged from 0.096 to 0.224 nmol / g, which was significantly increased by 1.21 to 15.89 times compared with the control CK; the contents of Kaempferol, HSYA, Eriodictyol, Quercetin, Spiraeoside, Luteolin and Apigenin in the overexpressed leaves ranged from 1.665 to 5.970 nmol / g, which was significantly increased by 0.66 to 15.28 times compared with the control CK. This indicates that overexpression of the CtHA02 gene significantly affects the content of flavonoids in safflower, especially the content of medicinal active flavonoids such as HSYA, Kaempferol and Quercetin increased significantly, further confirming that the CtHA02 gene can promote the accumulation of medicinal active flavonoids.

[0112] Example 4 Genetic transformation of safflower CtHA02 gene into Arabidopsis thaliana and differential analysis of metabolites

[0113] 1. Construction of the Carthamus tinctorius CtHA02 gene overexpression vector

[0114] The vector is the safflower CtHA02 gene overexpression vector pAMBIA1301-CtHA02-GUS constructed in Example 3. The vector schematic diagram is shown in Figure 5 shown.

[0115] 2. Arabidopsis transformation by flower bud dip method

[0116] (1) Arabidopsis thaliana cultivation

[0117] The planting experimental material is the wild-type WT material. Take an appropriate amount of Arabidopsis seeds and put them into 1.5 mL centrifuge tubes. Disinfect the surface of Arabidopsis seeds with 10% sodium hypochlorite (NaClO) for 15 min. Wash them with ultrapure water 4-5 times and then place the Arabidopsis seeds on 1 / 2MS culture medium. After vernalization at 4℃ in the dark for 3 days, culture them at 22℃, 16 h light and 8 h dark. After the seedlings grow true leaves, they are transplanted to nutrient soil: vermiculite = 3:1 for culture. After about 3 weeks of culture, when the scape length is about 5 cm, the Arabidopsis can be infected with Agrobacterium.

[0118] (2) Arabidopsis transformation by inflorescence infection method

[0119] 1) Inoculate the overexpression vector pAMBIA1301-CtHA02-GUS Agrobacterium engineering bacteria into the K ＋ and S ＋ 600 mL YEP liquid culture was added and cultured in a 28°C constant temperature shaker until the OD value was 0.8-1.0;

[0120] 2) Take the engineered Agrobacterium with an OD value of 1.0-1.2 in a 50 mL centrifuge tube and centrifuge at 12,000 rpm for 15 minutes at room temperature;

[0121] 3) Use the prepared infection solution to resuspend the centrifuged bacteria, adjust the OD value to 1.0-1.2 with dip solution, add 200 μL of Swilet-77 reagent, mix well and transfer to a beaker for later use;

[0122] 4) Place the wild-type Arabidopsis plants to be infected upside down in the infection solution. The timer is 5 min / pot. Each infection solution can infect up to 4 pots.

[0123] 5) Place the infected Arabidopsis in a dark place for 24 hours;

[0124] 6) After being placed away from light, the Arabidopsis thaliana was taken out and cultured normally in a climate chamber to collect T1 transgenic Arabidopsis thaliana seeds;

[0125] 7) Sow T1 generation Arabidopsis seeds, wait until they grow to 4-6 leaves, and spray 1% Basta for screening;

[0126] 8) Transplant the surviving Arabidopsis lines into individual plants and collect T2 transgenic Arabidopsis seeds;

[0127] 9) Repeat steps 7 and 8, and the seeds received are T3 generation transgenic Arabidopsis seeds.

[0128] (3) PCR identification of transgenic Arabidopsis

[0129] According to the instructions of the genome kit, DNA of WT and transgenic Arabidopsis genome was extracted. PCR detection was performed with safflower CtHA02 gene specific primers qCtHA02-F and qCtHA02-R. It was found that except for wild-type Arabidopsis WT and overexpression strain 5, the other strains amplified bands with the same size as the target fragment, indicating that the target CtHA02 gene has been successfully inserted into the Arabidopsis genome ( Figure 6 ). The primer names and sequences are as follows:

[0130] qCtHA02-1-F: CAGTGTCGACATGAAAACCCTAATCCCAAATGC (SEQ ID No. 11)

[0131] qCtHA02-1-R: CAGTCTGCAGCTAGTAGTAATTCTTGTACCTGAGTTC (SEQ ID No. 12)

[0132] PCR reaction system: 2×SuperReal PreMix Plus 10μL, forward primer (10μM) 0.5μL, reverse primer (10μM) 0.5μL, cDNA template 1μL, RNase-free ddH2O 8μL.

[0133] PCR reaction program: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing at 60-65°C for 20 s, extension at 72°C for 30 s, 35 cycles.

[0134] Real-time fluorescence quantitative PCR detection of transgenic Arabidopsis

[0135] In order to further detect and analyze the expression level of the safflower CtHA02 gene in transgenic Arabidopsis, total RNA was extracted from the T2 generation homozygous transgenic Arabidopsis and wild-type Arabidopsis WT seedlings grown about 14 days after sowing. Real-time fluorescence quantitative PCR detection and analysis were performed using the reverse transcribed cDNA as a template, the Arabidopsis Actin gene as an internal reference (primer sequences are shown in Table 3), and the wild-type Arabidopsis WT as a control ( Figure 7 ). The results showed that the expression levels of the safflower CtHA02 gene in different strains were different. The expression level of the CtHA02 gene was relatively high in strain 2, among which the highest expression level was in strain 2-2, followed by strains 2-1 and 2-8. The expression level of strains 1-7 in strain 1 was relatively high. The lowest expression level was in strain 3.

[0136] Table 3 Real-time fluorescence quantitative PCR primer information

[0137]

[0138] Differential analysis of metabolites in transgenic Arabidopsis

[0139] (1) Collection of transgenic Arabidopsis plant samples

[0140] Select the young parts of overexpressed Arabidopsis and WT Arabidopsis with normal growth and development, and select plants with relatively high CtHA02 gene expression and consistent growth as the source of a biological replicate sample. Take the petals of different plants from the source of the replicate sample and mix them to form a sample, put them into a 10 mL centrifuge tube, freeze them in liquid nitrogen for 5-10 min, and store them in a -80℃ refrigerator. Five biological replicates, 0.6 g each. Then perform quantitative detection of flavonoids.

[0141] (2) Detection of HSYA content in transgenic Arabidopsis plants

[0142] The HSYA content of five biological replicate samples of Arabidopsis overexpressing the CtHA02 gene was quantitatively detected, and it was found that the HSYA content in most CtHA02 overexpressed Arabidopsis was significantly higher than that in WT. Among them, the HSAY content in the overexpressed samples of OE 2-1, OE 2-4 and OE 2-8 was relatively the highest. There was no significant difference in the HSYA content of the three, but they were all significantly higher than WT and OE 3-4. Among them, the HSAY content in the overexpressed samples of OE 2-1, OE 2-4 and OE 2-8 was more than 1.8 times that of WT and OE 3-4 ( Figure 8 ). The results showed that the CtHA02 gene promoted the synthesis and accumulation of HSYA content.

Claims

1. A protein encoded by the safflower CtHA02 gene, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. Carthamus tinctorius CtHA02 gene, characterized in that: The nucleotide sequence of the CtHA02 gene is shown in SEQ ID NO.

1.

3. An expression framework, vector or host cell for expressing the protein of claim 1 or the protein encoded by the safflower CtHA02 gene of claim 2, characterized in that: The host cell is of non-animal or plant variety.

4. Use of the protein encoded by the safflower CtHA02 gene of claim 1, the safflower CtHA02 gene of claim 2, and the expression framework, vector or host cell expressing the protein encoded by the safflower CtHA02 gene of claim 3 in regulating the synthesis of plant flavonoids, characterized in that: The flavonoids are genistein, echinatin, dihydrokaempferol, naringeninchalcone, corylin, isorhamnetin, kaempferol, hydroxysaffron yellow A (HSYA), eriocytoside, quercetin, spiraeoside, luteolin and / or apigenin; the regulation is to promote the synthesis of flavonoids by plants by increasing the expression of CtHA02 gene in plants; the plants are safflower or Arabidopsis thaliana.

5. The application according to claim 4, characterized in that: The method for increasing the expression level of the CtHA02 gene in plants is as follows: constructing a plant expression vector for expressing the CtHA02 gene and transforming the plants.

6. The use according to claim 5, characterized in that: The backbone vector of the plant expression vector is pCAMBIA1301.

7. A method for promoting the synthesis of flavonoids in plants, characterized in that: The method is achieved by increasing the expression level of the CtHA02 gene in the plant; the amino acid sequence of the protein encoded by the CtHA02 gene is shown in SEQ ID NO.2; the flavonoids are genistein, echinatin, dihydrokaempferol, naringenin chalcone, corylin, isorhamnetin, kaempferol, hydroxysaffron yellow A (HSYA), eriocytoside, quercetin, spiraeoside, luteolin and / or apigenin; the plant is safflower or Arabidopsis thaliana.

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