CN116606865B - Red skin longan DlMYBrp gene and application thereof - Google Patents
Red skin longan DlMYBrp gene and application thereof Download PDFInfo
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- CN116606865B CN116606865B CN202310706700.1A CN202310706700A CN116606865B CN 116606865 B CN116606865 B CN 116606865B CN 202310706700 A CN202310706700 A CN 202310706700A CN 116606865 B CN116606865 B CN 116606865B
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Abstract
The invention provides a DlMYBrp gene which can accumulate anthocyanin, and analysis of cDNA sequences of RP longan and SX longan finds that 3 basic groups of mismatch exists in an R3 conserved structure domain, so that two amino acids are replaced, the difference of the RP longan and the SX longan in accumulating anthocyanin is caused, and experiments show that the DlMYBrp gene of the RP longan and the DlMYBrp gene of the SX longan can improve the expression quantity of AtCHS and the like, and can remarkably accumulate anthocyanin. The invention obtains SNP loci for identifying heterozygous longan groups based on the base difference of DlMYBrp genes of RP longan and SX longan in an R3 conserved structural domain, thereby providing a functional marker for identifying positive filial generation by subsequent hybridization breeding of RP longan.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a red skin longan DlMYBrp gene and application thereof.
Background
Longan of the genus longanDimocarpus longanLour.) is a genus of longan (Dimocarpus) of the family Sapindaceae (sapindacenae)Lour) evergreen trees are cultivated in many areas of the tropical subtropical zone. Therefore, the continuous and efficient development of the longan industry has important significance for promoting the economic development of hot areas and increasing the production and income of fruit growers. In China, the culture history of longan cultivation is over 2000, and the number of longan varieties (lines) is over 300, wherein nearly 40 cultivars are cultivated, so that different region types with local characteristics are formed (2015 such as Han Dongmei and 2019 such as Zheng Shaoquan). Although longan varieties (lines) are numerous, the appearance and luster of fruit peel are mainly regulated by yellow brown or yellow gray in varieties with commercial value of longan at present. The development of diversity of biological characteristics of longan cultivation resources and the competitive potential of diversified markets are seriously hindered by the relative single color, so that the excavation of special longan resources and the utilization of excellent characters become the emphasis of longan breeding work.
Consumer behavior habits are gradually shifted from price sensitivity to quality sensitivity as people's income increases and consumption upgrades. While fruit color is the most intuitive quality, even for very good flavor varieties, rich color is needed to meet the increasingly diverse consumer markets. The problem group introduces red skin (RP) longan resources from abroad in the early stage, breaks through the recognition that only anthocyanin is not accumulated in the traditional cultivated longan, and more importantly, the longan is used as a specific precious parent material, thereby providing a new breeding scheme for creating excellent varieties of longan with commodity value. Using a multi-pack analysis, it has been clarified that longan fruit color is mainly due to anthocyanin accumulation. However, the research on the anthocyanin accumulation control mechanism of red skin longan is still blank at present. In view of the important effect of anthocyanin on the appearance quality improvement of longan, the molecular mechanism of metabolism regulation is the key point of the improvement of longan varieties and the creation of new varieties, so that the research on the molecular mechanism of the metabolism regulation of anthocyanin of longan has important theoretical and practical significance for improving the appearance quality of longan fruits and cultivating red skin longan varieties with commodity value.
The research takes characteristic longan resources RP longan and main cultivated variety 'Dan' (SX) longan collected by a subject group as research materials, analyzes the regulation and control mechanism of RP longan anthocyanin, aims at providing a molecular theory basis for improving the quality of longan fruits, and develops molecular markers related to red characters for auxiliary breeding according to the theory basis.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a red skin longan DlMYBrp gene and application thereof.
A first aspect of the invention provides a method ofDlMYBrpThe CDS sequence of the gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.
A second aspect of the invention provides a method as described in the first aspect of the inventionDlMYBrpA protein encoded by the gene.
A third aspect of the invention provides a kit comprising the first aspect of the inventionDlMYBrpRecombinant vectors or hosts or expression cassettes for the CDS sequences of genes.
Wherein, the recombinant vector original vector can adopt vectors commonly used in the field of gene recombination, such as viruses, plasmids and the like. The invention is not limited in this regard.
A fourth aspect of the invention provides a method as described in the first aspect of the inventionDlMYBrpThe gene, or the protein according to the second aspect of the invention, or the recombinant vector or host bacterium or expression cassette according to the third aspect of the invention, is improvedAtCHSAnd/orAtCHIAnd/orAtF3HAnd/orAtDFR、And/orAtANSAnd/orAtUFGTIs used in the expression level of (a).
A fifth aspect of the invention provides a method as described in the first aspect of the inventionDlMYBrpUse of a gene, or a protein according to the second aspect of the invention, or a recombinant vector or host bacterium or expression cassette according to the third aspect of the invention, for modulating plant accumulation of anthocyanins, wherein the recombinant vector or host bacterium or expression cassette comprisesDlMYBrpCDS sequences of genes, for example.
DlMYBrpThe gene can promote the plant to accumulate anthocyanin, especially the anthocyanin shown in SEQ ID NO. 1DlMYBrpThe effect of promoting plant to accumulate anthocyanin by the gene is obvious.
In a sixth aspect of the present invention, there is provided a SNP marker, which is SNP1, SNP2 or SNP3; SNP1 is positioned at 197 th position of a nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the base is T or G; SNP2 is positioned at 317 th site of a nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the base is G or A; SNP3 is positioned at 318 th site of the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the base is A or T.
A seventh aspect of the present invention is to provide a SNP marker combination comprising SNP2 and SNP3; SNP2 is positioned at 317 th site of a nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the base is G or A; SNP3 is positioned at 318 th site of the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the base is A or T.
An eighth aspect of the present invention provides a primer set for amplifying a nucleotide sequence containing the SNP marker combination according to the eighth aspect of the invention, comprising: mybrpamms-af: 5 '-GCTCATGCTTTGATGTCTGGTC-3', and mybrpamms-ar: 5' -CTTTGCATTGTCTTCTTCTGTAC-3.
A ninth aspect of the invention provides a method for amplifying a nucleic acid as described in the first aspect of the inventionDlMYBrpA primer set for a gene, the primer set comprising: inf-DlMYBrp-F: cGGTACCCGGGGATCCATGGAGGGTCACGTAGGAGTT, and Inf-DlMYBrp-R: TGCTCACCATGTCGACCTTTGCATTGTCTTCTTCTGTACTTAGAAGATTCC.
In a tenth aspect, the present invention provides a primer set for identifying longan, the primer set comprising: mybrpamms-af: 5 '-GCTCATGCTTTGATGTCTGGTC-3', mybrpas-ar: 5 '-CTTTGCATTGTCTTCTTCTGTAC-3', ybrparms-bf:5 '-TCAAGAACTATTGGAACACACAT-3', and mybrpamms-br: 5 '-GCAACAGCTTTTTTGCGTAATC-3'.
An eleventh aspect of the present invention provides the use of the SNP marker according to the seventh aspect of the present invention, or the SNP marker combination according to the eighth aspect of the present invention, or the primer set according to the ninth aspect of the present invention, or the primer set according to the tenth aspect of the present invention, or the primer set according to the eleventh aspect of the present invention, for the identification of longan.
In a twelfth aspect, the present invention provides a method for identifying longan, which detects the SNP marker of the seventh aspect or the SNP marker combination of the eighth aspect of the invention to obtain the genotype of the longan variety to be detected. If the genotype of SNP1 locus is TT, it is homozygous red skin longan, if GG, it is homozygous Dan longan, if GT, it is heterozygous longan (hybrid eye of the invention refers to hybrid offspring of red skin longan and Dan longan); if the genotype of the SNP2 locus is GG, the locus is homozygous red skin longan, if the locus is AA, the locus is homozygous Dan longan, and if the locus is GA, the locus is heterozygous longan; if the genotype of SNP3 locus is AA, it is homozygous red skin longan, if it is TT, it is homozygous Dan longan, if it is AT, it is heterozygous longan.
The thirteenth aspect of the invention provides another identification method of longan, which comprises the steps of carrying out PCR amplification on the longan variety to be detected by adopting the primer group of the eleventh aspect of the invention, amplifying the 716bp and 253bp bands to obtain homozygous red skin longan, amplifying the 716bp and 506bp bands to obtain homozygous Dan longan, and amplifying the 716bp, 506bp and 253bp bands to obtain heterozygous longan.
The invention provides a kind ofDlMYBrpGene capable of accumulating anthocyanin for RP longan and SX longanDlMYBrpAnalysis of cDNA sequence of the gene shows that there is 3 base mismatch in R3 conserved structure domain, resulting in substitution of two amino acids, which results in difference of RP longan and SX longan in accumulation of anthocyanin, and experiments show that RP longan and SX longanDlMYBrpThe gene can be improvedAtCHS、AtCHI、AtF3H、AtDFR、AtANS、AtUFGTExpression level of (A), especially SX longanDlMYBrpThe gene has obvious effect of promoting the expression of the genes and can accumulate anthocyanin obviously. The invention is based on RP longan and SX longanDlMYBrpThe base difference of the gene in the R3 conserved structural domain obtains SNP loci for identifying the heterozygous group of longan, which provides a functional marker for identifying positive filial generation by subsequent hybridization breeding of RP longan.
Drawings
Fig. 1 shows the phenotypic differences between RP longan and SX longan peel and leaf. (A) morphological characteristics of mature fruits of SX longan. (B) morphological characteristics of mature fruits of RP longan. (C) morphological characteristics of SX longan mature leaves. (D) morphological characteristics of mature RP longan leaves.
FIG. 2 is an alignment of DlMYBrprp and DlMYBrpsx proteins. The R2 and R3 conserved domains are represented by black lines, and asterisks indicate differences between the conserved domains of RP longan and SX longan.
FIG. 3 is a phylogenetic analysis.
FIG. 4 shows DlMYBrprp and DlMYBrpsx subcellular localization.
FIG. 5 shows the time-space expression profile of anthocyanin content and DlMYBrp at the developmental stage of red skin longan fruits. (A) Anthocyanin (G1-G5 represent 15, 35, 55, 75 and 95 DAA respectively) in the fruit development stage of RP longan. (B)DlMYBrpExpression level of RP longan at different development stages. (C) And (5) analyzing the expression quantity of DlMYBrp in different tissues of RP longan.
FIG. 6 is the establishment of longan callus genetic system.
FIG. 7 is a transient expression in tobacco leavesDlMYBrp。（A）DlMYBrpThe phenotypic character of transient transformation in tobacco leaves was used as a negative control with pCAMBIA 2300-35S. And (B) transiently expressing the total anthocyanin content of the tobacco leaves.
FIG. 8 is a diagram ofDlMYBrpPhenotype of stable expression in Arabidopsis and related gene expression level. (A) Arabidopsis phenotype. a. b and c represent Arabidopsis thaliana flower tissues transformed with empty, 35S: dlMYBrpsx and 35S: dlMYBrprp, respectively. EV: and (5) no load is applied. (B)DlMYBrpExpression level in Arabidopsis thaliana. (C)DlMYBrpAnd (5) analyzing the expression level of anthocyanin biosynthesis related genes in the superlist system.
FIG. 9 shows the phenotype and related gene expression levels of DlMYBrprp site-directed mutagenesis in tobacco. (A) DlMYBrprp site-directed mutagenesis transient phenotype in tobacco. (B) And (C) the expression level of the genes related to anthocyanin biosynthesis in different point mutation types.
FIG. 10 shows the amplification results of mybrpamms functional markers in the progeny of crosses and in different longan varieties. And (A) positive identification of hybrid offspring of RP longan and SX longan. (B) 21 parts of germplasm resource PCR result. M represents 2000 bp marker, red arrow represents RP longan specific band. BY represents Bayian, DBL represents Dabinlong, BQ represents white dry Jiao Long, MJ represents Minjiao, SN represents Shuin No. 1, DWY represents Dawuyuan, SF represents Pinus, ZB represents early white jiao, YT represents oil Tan Ben, DB represents east wall, LL represents Linglong, LQ represents Liqing No. 2, GM represents Gui Ming No. 1, KT represents Kang Tan, CS represents punishment, QYM represents green round woody, XP represents Xideas, GH represents sweet osmanthus flavor, FG represents Feng Guang, lee represents Lee, SX represents Dan, RP represents red-skin longan.
FIG. 11 DlMYB15 and DlMYBrp temporal and spatial expression profiles.DlMYB15AndDlMYBrpexpression levels at different stages of RP longan (G1-G5 represent 15, 35, 55, 75 and 95DPA, respectively).
FIG. 12 is a protein interaction analysis.
FIG. 13 shows the transient expression of tobacco leaves and the determination of total anthocyanin content. (A) The phenotype characteristics of DlMYB15, dlMYBrp, dlbHLH and the transient transformation of various combinations in tobacco leaves were used as negative controls with pCAMBIA 2300-35S. (B) DlMYB15, dlMYBrp, dlbHLH3 and total anthocyanin content of different combinations.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Abbreviation vocabulary
| Abbreviation symbol | English holonomy scale | Chinese holonomic scale |
| Abbr. | English full name | Chinses full name |
| BBGS | the beginning biosynthetic genes | Initial biosynthesis genes |
| EBGs | the early biosynthetic genes | Early biosynthesis genes |
| LBGs | the late biosynthesis genes | Late biosynthetic genes |
| PAL | Phenylalanine ammonialyase | Phenylalanine ammonia lyase |
| C4H | Cinnamate 4-hydroxylase | Cinnamic acid hydroxylase |
| 4CL | 4-coumarate: CoA ligase | 4-coumaroyl-CoA ligase |
| CHS | Chalcone synthase | Chalcone synthase |
| CHI | Chalcone isomerase | Chalcone isomerase |
| F3H | Favone 3-hydroxylase | Flavanone-3-hydroxylase |
| F3′ H | Flavonol 3′-hydroxylase | Flavonoid-3-hydroxylase |
| DFR | Dihydroflavonol-4-reductase | Dihydroflavonol 4-reductase |
| ANS | Athocyanidin synthase | Anthocyanin synthase |
| UFGT | Uridine diphosphate (UDP)-glucose: flavonoid 3-O-glycosyl-transferase | UDP-glucose flavonoid-3-OGlycosyltransferase |
| GST | Glutathione S-transferase | Glutathione S-transferase |
| PCR | Polymerase chain reaction | Polymerase chain reaction |
| qRT-PCR | Real-Time Quantitative RT-PCR | Real-time fluorescent quantitative PCR |
| bp | Base pair | Base pairing |
| GFP | Green Fluorescence Protein | Green fluorescent protein |
| 35S | Cauliflower mosaic virus promoter | Cauliflower mosaic virus promoter |
| rpm | Revolutions per minute | Revolutions per minute |
| DPA | days post-anthesis | Days after flowering |
| RP | Red pericarp | Red skin |
| SX | ‘Shixia’ | 'Dan' |
| FW | Fresh weight | Fresh weight |
| Y2H | Yeast two-hybrid | Yeast double hybridization |
| DDO | SD/-Leu/-Trp | |
| QDO/3AT | SD/-Leu-/Trp/-His/-Ade/3AT | |
| QDO/X/3AT | SD/-Leu-/Trp/-His/-Ade/X-α-Gal/3AT |
1. Plant material
The 'Dan' longan (SX longan) and red-skin longan (RP longan) used in the research are planted in litchi longan germplasm resource nursery of the national academy of tropical agricultural sciences of south and subtropical crop institute. The peel of SX longan is yellow brown as most cultivars, while the inner and outer surfaces of RP longan peel are dark red, which is very rare in longan cultivars. In SX longan, cotyledons do not accumulate anthocyanin, while RP longan cotyledons accumulate a small amount of anthocyanin; the pseudoseed coats of both are typically milky white (FIGS. 1A & B). The front and back sides of the leaf of SX longan are green, and the veins are light yellow (figure 1C); the front surfaces of the RP longan leaves are all dark red, and the back surfaces of the RP longan leaves are dark green (figure 1D).
The transcript analysis collects the pericarp and mature leaf of 95d (After 95 days post-anthesis, DPA) After the two longan seed flowers. The red skin longan fruits at the development stage (15, 35, 55, 75 and 95 DPA) are collected by the samples of pericarp, seed, pulp and different tissue parts (leaf and flower) and immediately frozen by liquid nitrogen and stored at-80 ℃ for standby.
2 primers required for experiments
TABLE 1 primers required for experiments
| Gene Gene | Forward primer | Reverse primer |
| Gene cloning | ||
| Inf-DlMYBrp | cggtacccggggatccATGGAGGGTCACGTAGGAGTT | tgctcaccatgtcgacCTTTGCATTGTCTTCTTCTGTACTTAGAAGATTCC |
| DlbHLH3 | cggtacccggggatccATGGCTACTACTGGGGTTCAAAG | tgctcaccatgtcgacACACTTCCAAATGACTCTGTCAAGTGA |
| DlMYB15 | cggtacccggggatccATGGGGAGGAGCCCATGT | tgctcaccatgtcgacGGGCCACTCATCGGAATCAAG |
| Point mutation bridging | ||
| DlMYBrp -197 | CAAAAGAGGGGAATTCAgGGAAGATGAAGTTGATC | GATCAACTTCATCTTCCcTGAATTCCCCTCTTTTG |
| DlMYBrp -317 | CTATTGGAACACACatTTACGCAAAAAAGC | GCTTTTTTGCGTAAatGTGTGTTCCAATAG |
| Yeast experiments | ||
| pGBKT7- DlMYBrp | catggaggccgaattcATGGAGGGTCACGTAGGAGTT | gccgctgcaggtcgacCTACTTTGCATTGTCTTCTTCTGTACTTAGAAGAT |
| pGBKT7- DlMYB15 | catggaggccgaattcATGGGGAGGAGCCCATGT | gccgctgcaggtcgacTCAGGGCCACTCATCGGAATCA |
| pGADT7- DlMYB15 | ggaggccagtgaattcATGGGGAGGAGCCCATGT | cgagctcgatggatccTCAGGGCCACTCATCGGAATCA |
| pGADT7- DlbHLH3 | ggaggccagtgaattcATGGCTACTACTGGGGTTCAAAG | cgagctcgatggatccCTAACACTTCCAAATGACTCTGTCAAGTGA |
| Molecular markers | ||
| mybrparms-a | GCTCATGCTTTGATGTCTGGTC | CTTTGCATTGTCTTCTTCTGTAC |
| mybrparms-b | TCAAGAACTATTGGAACACACAT | GCAACAGCTTTTTTGCGTAATC |
| qPCR | ||
| NtActin | AATGATCGGAATGGAAGCTG | TGGTACCACCACTGAGGACA |
| NtCHS | TGACACCCACTTGGATAGTTTAG | CGACCTCTGGAATTGGATCAG |
| NtCHI | CTTTTCTCGCCGCTAAATG | TTTCTGCCACCTTCTCTG |
| NtF3H | CAAGGCATGTGTGGATATGG | TGTGTCGTTTCAGTCCAAGG |
| NtF3′H | AGGCTCAACACTTCTCGT | CATCAACTTTGGGCTTCT |
| NtDFR | AACCAACAGTCAGGGGAATG | TTGGACATCGACAGTTCCAG |
| NtANS | TGGCGTTGAAGCTCATACTG | GGAATTAGGCACACACTTTGC |
| NtUFGT | GAGTGCATTGGATGCCTTTT | CCAGCTCCATTAGGTCCTTG |
| AtCHS | ATCTTGGCTATTGGCACTG | CGCTTGAACTTCTCCTTGA |
| AtCHI | CTCCTCCAATCCATTATTCCT | CTTCCACTTGACAGATAGAGA |
| AtF3H | CATCGTCTCTAGTCACCTCCAG | CTCACTATACTCCTCCGTCACTT |
| AtF3'H | CGACAGGAAGAGGTTGGA | CCTAGAGCGTTGACTACAC |
| AtDFR | GGTCGGTCCATTCATCAC | GCACATACTGTCCTTGTCTTA |
| AtANS | GAGATGGTGAGTGTTGAGT | CAATTCCTCTTGTTCCTTCC |
| AtUFGT | TGATATGGCGACGGAGATA | TGTGTAGAGATGAGCAGAGA |
| AtActin | GGAACTGGAATGGTGAAGGCTG | CGATTGGATACTTCAGAGTGAGGA |
| Yang test | ||
| p35S | CTATCCTTCGCAAGACCCTTCC |
Example 1
1、DlMYBrpGene cloning, transformation and sequence analysis
(1) Cloning and recovery of fragments of interest
RNA was extracted from SX longan and RP pericarp tissues, respectively, and cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit with DNase I (Thermo Scientific, USA) reverse transcription kit.
The reference sequence of the gene is obtained by longan reference genome, and a CDS region of the target gene is amplified from longan peel cDNA of red skin and 'Dan' longan peel by designing a primer with 15 bp homology arm specificity. Primers were synthesized by Guangzhou Ai Ji Biolimited. The resulting primers were used with sterile ddH 2 O was dissolved and diluted to a working concentration of 10. Mu.M for use. Primer Inf-DlMYBrp-F was used: 5 '-cGGTACCCGGGGATCCATGGAGGGTCACGTAGGAGTT-3', and Inf-DlMYBrp-R:5 '-TGCTCACCATGTCGACCTTTGCATTGTCTTCTTCTGTACTTAGAAGATTCC-3' by PCR amplification with high-fidelity DNA polymerase KOD. The PCR reaction system is as follows:
| reagent(s) | Usage amount |
| 10×buffer | 2 μL |
| dNTP (2 mM/L) | 2 μL |
| MgSO 4 | 0.8 μL |
| Inf-DlMYBrp-F | 0.4 μL |
| Inf-DlMYBrp-R | 0.4 μL |
| cDNA | 0.4 μL |
| KOD | 0.4 μL |
| ddH 2 O | 13.6 μL |
PCR amplification procedure: 94. 2 min at the temperature; 94. 15 s at the temperature of 62 ℃ for 30 s at the temperature of 68 ℃ for 1 min for 30 cycles; 16. preserving at the temperature. And (3) cutting and recovering the PCR amplified product by using a DNA gel recovery kit of Sigma after agarose gel electrophoresis detection, and detecting the recovered product by agarose gel electrophoresis.
(2) Vector linearization and ligation transformation of fragments of interest
The pCAMBIA2300 vector is subjected to double enzyme digestion to linearize the vector, and enzyme digestion sites areBamHⅠ、SalI, the reaction system is as follows:
| reagent(s) | Usage amount |
| Plasmid(s) | ≤2 μg |
| 10×FlyCut buffer | 5 μL |
| BamHⅠ | 1 μL |
| SalⅠ | 1 μL |
| ddH2O | to 50 μL |
The mixture was incubated at 37℃for 2 h and 80℃for 20 min in PCR. The result of the enzyme digestion is detected by agarose gel electrophoresis, and the linearized vector is recovered by gel digestion. In-Fusion Snap Assembly Cloning Kit (Takara, japan) seamless cloning ligation kit was used to insert the Inf-DlMYBrp fragment with 15 bp homology arm into the linearized pCAMBIA2300 vector, the ligation system was as follows (15 min at 50 ℃):
| reagent(s) | Usage amount |
| Gene fragment of interest | 1 μL |
| Linearization carrier | 2 μL |
| 5× In-Fusion Snap Assembly Master Mix | 1 μL |
| ddH 2 O | 1 μL |
Taking 2 mu L of the connection product in 50 mu L of DH5 alpha competent cells in a semi-melted state, lightly stirring the bottom of the tube and carrying out ice bath for 25 min; then carrying out heat shock at 42 ℃ for 30 s, and standing in ice for 2 min; 700. Mu.L of antibiotic-free LB medium is added, and 1 h is recovered at 37 ℃; subsequently, the mixture was centrifuged at 4500 rpm for 1 min, 600. Mu.L of the supernatant was removed, and the resuspended broth was pipetted and spread evenly on LB (containing 100 mg/L Kana) solid plates and incubated at 37℃in the dark for 12-16 h. Single colonies were picked up in plates and cultured in 500. Mu.L LB (containing 100 mg/L Kana) broth, 3 h was used per tube broth as template, and colony positive detection was performed with vector primer p35S-F and target gene downstream primer Inf-DlMYBrp-R. The PCR reaction system is as follows:
| reagent(s) | Usage amount |
| 2×F8 mix | 10 μL |
| Bacterial liquid | 1 μL |
| p35S-F | 0.5 μL |
| DlMYBrp-R | 0.5 μL |
| ddH 2 O | 8 μL |
PCR reaction procedure: 94. 2 min at the temperature; 94. 10 s at C, 10 s at 60℃,20 s at 72℃,35 cycles; 72. preserving at 16deg.C for 5 min. The correct bacterial liquid was detected by agarose gel electrophoresis and sent to Guangzhou Ai Ji biological limited company for sequencing. The results showed that clones were obtainedDlMYBrpGene obtained from RP longanDlMYBrpThe gene is named DlMYBrprp, and the CDS sequence of the gene is shown as SEQID NO. 1, obtained from SX longanDlMYBrpThe gene is named DlMYBrpsx, and the CDS sequence of the gene is shown as SEQ ID NO. 2. Sequence analysis found that critical motifs binding to bHLH proteins were present on both R3 domains: [ D/E ]]Lx2[R/K]x3Lx6Lx 3R. However, the presence of a 3 base variation in the R3 conserved domain results in two amino acid substitutions, one at each positionDlMYBrpThe substitution of base T AT position 197 of the gene with G results in substitution of arginine with methionine, and the substitution of bases GA AT positions 317 and 318 with AT results in substitution of histidine with arginine, which is located in the third alpha helix of the R3 conserved domain (figure 2).
Amplifying and shaking the bacterial liquid with correct sequence, and extracting the recombinant plasmid according to the specification of the plasmid minibody kit.
(3) DlMYBrp gene structure, multiple sequence alignment and phylogenetic tree analysis
Protein conserved domain prediction and analysis was performed using NCBI conserved domain database (Conserved Domain Database, CDD, https:// www.ncbi.nlm.nih.gov/CDD). Related protein sequences were retrieved from NCBI and Arabidopsis genome databases and subjected to multiple sequence alignment using Genedoc software. Meanwhile, fastatree software is utilized to construct a phylogenetic tree. The results are shown in figure 3 of the drawings,D.long035496(will be based on annotation information of the swiss-port database)D.long035496Named asDlMYBrp) And (3) withLcMYB1、CsRUBY、ATMYB15、ATMYB90、ATMYB113、ATMYB114、FaMYB10、AcMYB10、AcMYB75Has higher homology.
2. Subcellular localization
The complete coding regions for DlMYBrps x and DlMYBrps (with stop codons removed) were constructed into the 5' end of GFP in the expression vector pCAMBIA2300 to form pCAMBIA2300: dlMYBrprp: GFP and pCAMBIA2300: dlMYBrpsx: GFP fusion recombinant vector, see example 1.1 for detailed procedures.
pCAMBIA2300: dlMYBrprp: GFP, pCAMBIA2300: dlMYBrpsx: GFP fusion recombinant vector and empty load are respectively added into 100 mu L of agrobacteria GV3101 in a semi-molten state, tube bottom is slightly shifted, ice bath is carried out for 5 min, liquid nitrogen is frozen for 5 min, warm water bath is carried out for 5 min at 37 ℃, and then ice bath is carried out for 5 min; 600. Mu.L of YEP medium without antibiotics was added and activated at 28℃at 200 rpm for 3 h;3000 Centrifugation at rpm for 3 min, 400. Mu.L of supernatant was discarded, 100. Mu.L of resuspended bacteria was aspirated and spread evenly on YEP (containing 100 mg/L Kana and 25 mg/L RMP) solid plates and incubated in dark at 28℃for 48-72 h.
The monoclonal positive test is selected, the correct monoclonal is amplified and shaken, the fresh onion is cut into 1 square centimeter, then the inner epidermis cells are placed in MS culture medium, and the culture is carried out in the dark at 28 ℃ for two days. Subsequently, the cells were immersed in resuspended Agrobacterium tumefaciens for 8 min, incubated in a dark medium at 28℃for 24-72℃ 72 h, and the expression of green fluorescent protein was examined using an LSM 800 laser confocal microscope (Zeiss, germany).
Subcellular localization analysis of epidermal cells of onion squama leaf revealed that no load was localized in cell membranes and nuclei, whereas DlMYBrprp-GFP and DlMYBrpsx-GFP were shown to be localized in epidermal nuclei of onion (FIG. 4), with transcription factor properties.
Example 2DlMYBrpIs a key transcription factor for regulating and controlling red skin longan coloration
1. Anthocyanin content in the development stage of red skin longan fruits,DlMYBrpAnalysis of expression level
Extracting and measuring anthocyanin by adopting a pH differential method. The method comprises the following specific steps: (1) sample preparation: grinding plant tissue into fine powder in liquid nitrogen; (2) preparation of reagent: extract (1% hydrochloric acid/methanol), ph=1.0 assay buffer (250 mM KCl buffer), ph=4.5 assay buffer (400 mM KAc buffer); (3) extraction: adding 10mL of the extract into a 0.3 g sample which is weighed in advance and placed in a 15 mL centrifuge tube, fully and uniformly mixing, performing dark extraction at 4 ℃ for 24 h, performing low-temperature centrifugation at 10000 rpm for 8 min, and transferring supernatant into another centrifuge tube (which can be stored at-20 ℃); (4) measurement: preparing 400 mu L of pH=1.0 and pH=4.5 measuring buffer solutions in two centrifuge tubes, adding 400 mu L of supernatant liquid into each centrifuge tube, uniformly mixing, standing for 15 min at a low speed Wen Biguang, measuring the absorbance at 510 nm and 700 nm by using a multifunctional enzyme-labeled instrument, and taking the measuring buffer solutions as a control; (5) calculating: anthocyanin content was calculated according to the following formula: c (mg/100 g FW) =Δa·v·n·mw·100/(ε· m), where Δa= (a 510-a 700) ph1.0- (a 510-a 700) ph4.5; v is the volume of the extract (10 mL); n is a dilution factor (2); MW is the relative molecular mass (449.2 g/mol) of anthocyanin assay standard cyanidin-3-galactoside; epsilon is the molar ratio absorption coefficient 30200 of cyanidin-3-galactoside; m is the sample mass (0.3 g).
The color of the fruit is one of the most important appearance quality of the fruit, and the RP longan fruit presents dark red color different from that of common cultivated longan. By finding the anthocyanin content of the RP longan fruits in the development stage, the red skin longan fruits do not accumulate anthocyanin 15 days after flowers, the anthocyanin is obviously accumulated in the color conversion stage (35 DPA), the anthocyanin content reaches 3.58 mg/100gFW, and the anthocyanin content of the peel is highest in the fruit maturity stage (95 DPA) and reaches 6.58 mg/100gFW (figure 5A). By measuringDlMYBrpThe expression level of different tissues in the fruit development stage can be found,DlMYBrpexpression was significantly up-regulated during the color shift phase and gradually down-regulated as the fruit matured (fig. 5B). Can be detected in pericarp, kernel and leaf of accumulated anthocyaninDlMYBrpIs not detected in flesh without anthocyanin accumulation, indicatingDlMYBrpWith tissue specificity, expression was only up-regulated in tissues accumulating anthocyanin (fig. 5C). Indicating thatDlMYBrpMay be involved in regulating the biosynthesis of RP longan anthocyanin.
2. Genetic transformation of longan callus
The genetic transformation system adopts an agrobacterium-mediated infection method, and the specific operation steps are as follows: (1) The recombinant plasmid obtained in example 1.1 was transfected into GV3101 Agrobacterium, and then GV3101 Agrobacterium containing the recombinant plasmid was shake-cultured at 28℃with 15 mL of YEP liquid medium (containing 25 mg/L rifampicin and 100 mg/L kanamycin). (2) Culturing overnight until the bacterial liquid is turbid, centrifuging at 5000 rpm for 8-10 min, and collecting bacterial cells. (3) The bacteria were resuspended by adding 15 mL sterile ddH2O and the cells were collected again (repeated once) by centrifugation at 5000 rpm for 5 min. (4) 15 mL of MS culture medium (containing 100 mg/L acetosyringone) is added again to obtain an infection liquid (sterile water can be used for replacing MS). (5) Uniformly dispersing longan callus with good growth state, immersing in the infection liquid, and vibrating for 30 min at 28 ℃. (6) Filtering with sterile filter paper, removing the invasion solution, and leaving callus. (7) The callus is sucked to dry excessive water by sterile filter paper, and the operation is gentle, so that the mechanical damage is avoided. (8) Callus was co-cultured on MS (17-18) solid medium in the dark for 2 d. (9) The co-cultured 2 d calli were washed once with sterile water, blotted to dry, plated on MS (17-18) solid medium (containing 100 mg/L kanamycin and 200 mg/L timentin) and cultured normally. (10) And then subculturing every 20 days, and raising the concentration of the timentin to 500 mg/L, and continuously subculturing for 3-5 months.
To verifyDlMYBrpIs an important regulatory factor in the process of accumulating red skin longan anthocyanin, dlMYBrpp is introduced into longan calli by means of agrobacterium infection, and the screened calli are subjected to multiple times of subculture in a certain selection pressure by screening resistance to the immersed calli, so that a stable supersurface system is finally formed (figure 6). It was found that anthocyanin accumulation in the DlMYBrprp super-surface line calli compared to the wild type clearly demonstrated that DlMYBrprp was able to cause anthocyanin accumulation. Thus, the first and second substrates are bonded together,DlMYBrpis a key transcription factor for regulating and controlling the coloration of red skin longan.
Example 2DlMYBrpVerification of the variation of the functional characterization of the sequence caused by the sequence difference
1. Transient transformation of tobacco lamina
The recombinant plasmid obtained in example 1.1 was transferred into GV3101 Agrobacterium and shake-cultured at 28℃with 15 mL of YEP liquid medium (containing 25 mg/L rifampicin and 100 mg/L kanamycin). Culturing overnight until the bacterial liquid is turbid, centrifuging at 6000 rpm for 5 min to collect bacterial cells, re-suspending twice with sterile water, and washing the redundant culture medium containing rifampicin as much as possible. Then re-suspending once by using MMA re-suspension 1mL, finally regulating the bacterial liquid OD600 to 0.5 by using MMA re-suspension, and standing for 2-3 h under dark condition. MMA heavy suspension is prepared according to the following formula:
| reagent(s) | Usage amount |
| 100 mM MES | 1 mL |
| 100 mM MgCl2·6H2O | 1 mL |
| Acetosyringone | 10 μL |
| Sterile water | 8 mL |
Taking a sterile syringe of 1-mL, taking down a needle, sucking bacterial liquid, lightly injecting the bacterial liquid from the back of tobacco leaves, marking, placing 16-24 h in a dark condition, performing normal photoperiod treatment, observing leaf phenotype by 36-48 h, and performing photographing, sampling, freezing and the like for subsequent experimental analysis.
The results show that DlMYBrprp has the ability to promote anthocyanin accumulation in tobacco lamina, whereas DlMYBrpsx and no anthocyanin accumulation in tobacco lamina is visible to the naked eye. By sampling the transient material blade and measuring the total anthocyanin content, the total anthocyanin content of injection no-load is 0, and the total anthocyanin content of injection DlMYBrpsx is 0.139 mg.100 g -1 FW, total anthocyanin content of DlMYBrprp injection is 1.298 mg.100 g -1 FW, it is seen that DlMYBrprp significantly promoted the accumulation of anthocyanin in tobacco lamina (fig. 7), and therefore, the amino acid sequence differences in DlMYBrp may be an important cause of its accumulation of anthocyanin.
2. Genetic transformation of Arabidopsis thaliana
The recombinant plasmid obtained in example 1.1 was transferred into Agrobacterium. The stable transformation system of arabidopsis thaliana is constructed by adopting an inflorescence dip-dyeing method, and agrobacterium dip-dyeing is carried out when the plant grows until most inflorescences are extracted. Selecting a successfully transformed monoclonal (same as in example 2.1) and shaking overnight in YEP (containing 50 Mg/L kanamycin and 25 Mg/L rifampicin), taking 100 mu L of fresh bacterial liquid from the medium, shaking the bacterial liquid at 28 ℃ until the bacterial liquid is yellow and turbid, centrifuging at 6000 rpm for 8 min at room temperature to collect cell supernatant, adding 250 mL osmotic culture solution (50 g/L sucrose solution) to suspend the bacterial cells, adding silwe-77 to make the concentration of the bacterial cells 200 mu L/L during the infection, soaking the arabidopsis inflorescence with the flower buds removed in the osmotic liquid at 30 s after the uniform mixing, horizontally culturing at 24 h in the dark, then normally standing and culturing, repeating the experimental steps for one time after about one week, drying the seeds until the seeds are mature (T0) and preserving at 4 ℃.
Transgenic Arabidopsis seeds were selected, T0 generation seeds were sterilized in 70% alcohol for 1 min, then sterilized with 3% sodium hypochlorite (0.05% Tween 20) for 10 min, once every two minutes with vigorous shaking, and washed 3-5 times with sterile water. Then, the seeds are resuspended by using sterile 0.05% agarose, uniformly sown in 1/2MS (containing 50 Mg/L kanamycin) solid medium, the wild seeds are sown in 1/2MS medium, and are placed in a dark culture at 4 ℃ for 3 d, then placed in a normal photoperiod incubator for culture, green seedlings are selected and planted in soil, and the seedlings are inspected after growing up. And continuously culturing until the T3 generation obtains a homozygous transgenic strain, observing the phenotype of the plant, and analyzing the expression rule of the related genes.
The phenotype of the harvested T3 generation homozygous plants was found to be that overexpression of DlMYBrpsp did not promote anthocyanin accumulation in Arabidopsis plants, whereas overexpression of DlMYBrpsp Arabidopsis plants accumulated anthocyanin in petals, at the front end of the calyx and on filaments (FIG. 8A). Further verifies that the expression level of anthocyanin synthesis pathway structural gene in arabidopsis thaliana discovers that DlMYBrpsx can induce in different degreesAtCHS(NCBI accession numberAT5G13930.1)、AtCHI(NCBI accession numberAT3G55120.1)、AtF3H(NCBI accession numberAT3G51240.1)、AtDFR(NCBI accession numberAT5G42800.1)While DlMYBrprp promotes expression ofAtCHS、AtCHI、AtF3HIsogenic expression, late synthesis genes for anthocyanin biosynthesisAtDFR、AtANS(NCBI accession numberAT4G22880.1)、AtUFGT(NCBI accession numberAT5G17050.1)The increase in expression was particularly pronounced (fig. 8C). Stable transformation of Arabidopsis experiments further demonstratedDlMYBrpSequence variations in the coding region are responsible for variations in their functional representation.
EXAMPLE 3 construction of Point mutation vector and analysis of mutation site function
Previous studies have shown thatDlMYBrpSequence variations in the coding region are responsible for variations in their functional representation, in order to verifyDlMYBrpWhether the substitution of the amino acid in the R3 conserved domain is responsible for its functional change or not was established by means of bridge PCR and linked to the plant binary expression vector pCAMBIA 2300:35S. The primers used are shown in Table 1, and the PCR reaction system is as follows:
| reagent(s) | Usage amount |
| 10×buffer | 2 μL |
| dNTP (2 mM/L) | 2 μL |
| MgSO 4 | 0.8 μL |
| Forward primer | 0.4 μL |
| Reverse primer | 0.4 μL |
| cDNA | 0.4 μL |
| KOD | 0.4 μL |
| ddH 2 O | 13.6 μL |
PCR amplification procedure: 94. 2 min at the temperature; 94. 15 s at the temperature of 62 ℃ for 30 s at the temperature of 68 ℃ for 1 min for 30 cycles; 16. preserving at the temperature. The 197 th base T of DlMYBrprp was replaced with G (the 66 th amino acid was replaced with arginine by methionine) and was named DlMYBrp -197 The method comprises the steps of carrying out a first treatment on the surface of the The 317 and 318 bases of DlMYBrprp were replaced with AT (amino acid 106 replaced with histidine by arginine) and named DlMYBrp -317 . Phenotype is observed by the method of injecting the agrobacterium into the tobacco, and the expression level of anthocyanin synthesis structural genes in the tobacco in different mutants is measured, so that DlMYBrp is found -197 The ability to promote anthocyanin accumulation in tobacco lamina is greater than that of DlMYBrp -317 (FIG. 9A), it was found that, by measuring the expression level of anthocyanin biosynthesis-related structural genes in tobacco treated with different point mutations by qRT-PCR, dlMYBrp was compared with DlMYBrp-treated leaf -197 And DlMYBrp -317 Is down-regulated in different degrees of saliency of anthocyanin structural genes and DlMYBrp -317 The effect is most pronounced (fig. 9B and fig. C).
EXAMPLE 4 longanDlMYBrpDevelopment of functional markers of gene anthocyanin accumulation mutation sites
The invention uses RP longan and SX longanDlMYBrpFunctional analysis, studies found that there were 3 base substitutions in the R3 conserved domain of DlMYBrprp and DlMYBrpsx, each with the substitution of G at position 197 for T (conversion of arginine to methionine) and 317The AT of (a) is replaced with GA (histidine to arginine) resulting in a rapid decrease in anthocyanin accumulation capacity. For this reason, molecular markers are designed aiming at the differences caused by the base mutation in the gene, four-primer amplified hindered mutation PCR (Tetra-primer ARMS-PCR) is adopted, and RP longan mutant and SX longan normal hybrid are effectively distinguished by one-time PCR amplification.
1、DlMYBrpDevelopment of gene SNP functional markers
According toDlMYBrpThe present study developed a set of mutation function markers, mybrpamms, 4 primers each, for the double mutation site (AT. Fwdarw. GA) of the R3 conserved domain. For mybrpamms, 1 pair of outer primers mybrpamms-af were first designed: 5 '-GCTCATGCTTTGATGTCTGGTC-3' and mybrpamms-ar: 5 '-CTTTGCATTGTCTTCTTCTGTAC-3' as reference control, 716bp bands can be amplified in both RP longan and SX longan, and the amplified products compriseDlMYBrpMutation site followed by designing two reverse inner primers mytrparms-bf according to mutation site: 5 '-TCAAGAACTATTGGAACACACAT-3' and mybrpamms-br: 5 '-GCAACAGCTTTTTTGCGTAATC-3', wherein mytrparms-br is matched with a mutant gene, the 3 'end of the mytrparms-br corresponds to a mutant base GA, the primer mytrparms-bf is matched with an SX longan gene, and the 3' end of the primer mytrparms-bf corresponds to a base AT. The PCR reaction system is as follows:
| reagent(s) | Usage amount |
| 2×Taq Mix | 10 μL |
| mybrparms-af | 0.8 μL |
| mybrparms-ar | 0.8 μL |
| mybrparms-bf | 0.4 μL |
| mybrparms-br | 0.4 μL |
| DNA | 1 μL |
| ddH 2 O | 6.6 μL |
PCR amplification procedure: 95. 5 min at the temperature; 95. 30 s at C, 30 s at 58℃, 54 s at 72℃,36 cycles; 16. preserving at the temperature.
Prediction by referring to Tetra-primer ARMS-PCR primer design strategy: the mytrparm s-af/mytrparm s-br can amplify a band with the size of 253bp, and is a unique band of RP longan DlMYBrprp; the mytrparm-bf/mytrparm-ar can amplify a band with 506bp, which is a band special for the DlMYBrpsx of SX longan. Thus, the RP longan can amplify two bands, and the sizes of the bands are 716bp and 253bp respectively; and two amplified bands of SX longan are 716bp and 506bp. If the hybrid offspring is obtained, three heterozygote bands of 716bp, 506bp and 253bp are amplified.
2. Verification of mytrparms functional markers
According to the amplification characteristic of mytrparms functional markers, when red skin longan is used as one of hybridization parents for hybridization breeding, if offspring can amplify two specific bands of parents and one common band through Tetra-primer ARMS-PCR, the offspring can be identified as true hybrids. Thus, the authenticity of the 8 hybrid offspring was identified and two offspring (F1-7 and F1-8) were successfully amplified to produce three heterozygous bands of 716bp, 506bp and 253bp, indicating that the two hybrid offspring were true hybrids and the remaining 6 hybrid offspring were false hybrids (FIG. 10A).
A four-primer amplified blocked mutation PCR was performed in 22 germplasm resources, and it was found that a specific 253bp band was amplified only in red skin longan in which anthocyanin was accumulated, while only a band of 506bp length was amplified specifically in the remaining 21 varieties in which anthocyanin was not accumulated, as in SX longan (FIG. 10B). Based on the results, the characteristic is used for identifying the longan hybridization group, so that early screening of longan hybridization seeds is facilitated, the breeding period is shortened, and the method has important significance for cultivating special new varieties.
Example 5 concerns the DlMYB15 Gene
1. DlMYB15 Gene cloning
And (3) extracting RNA from the red skin longan peel to obtain total RNA meeting the requirement of subsequent experiments. cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit with DNase I (Thermo Scientific, USA) reverse transcription kit. The reading frame PCR amplification primer DlMYB15-f is designed: 5'-cGGTACCCGGGGATCCATGGGGAGGAGCCCATGT-3' and DlMYB15-r:5'-TGCTCACCATGTCGACGGGCCACTCATCGGAATCAAG-3' PCR amplification was performed using the obtained cDNA as a template, and the PCR reaction system and the reaction procedure were the same as in example 1. The PCR product was recovered, and the vector linearization and ligation transformation of the target fragment, transfection, sequencing were performed as described in example 1.1 to obtain the correct DlMYB15 gene (CDS sequence of which is shown in SEQ ID NO: 4). Amplifying and shaking the bacterial liquid with correct sequence, and extracting the recombinant plasmid according to the specification of the plasmid minibody kit.
2. Tissue-specific expression of DlMYB15 genes
The expression level of the DlMYB15 gene in the fruit development stage and different tissues was measured, and the expression patterns of DlMYB15 and DlMYBrp were found to be basically consistent (FIG. 11), so we speculate that DlMYB15 also has the function of promoting anthocyanin accumulation similar to that of DlMYBrp.
Example 6 about the DlbHLH3 Gene
1. DlbHLH3 Gene cloning
And (3) extracting RNA from the red skin longan peel to obtain total RNA meeting the requirement of subsequent experiments. cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit with DNase I (Thermo Scientific, USA) reverse transcription kit. Designing an open-frame PCR amplification primer DlbHLH3-f:5'-cGGTACCCGGGGATCCATGGCTACTACTGGGGTTCAAAG-3' and DlbHLH3-r:5'-TGCTCACCATGTCGACACACTTCCAAATGACTCTGTCAAGTGA-3' PCR amplification was performed using the obtained cDNA as a template, and the PCR reaction system and the reaction procedure were the same as in example 1. The PCR product was recovered, and the vector linearization and ligation transformation of the fragment of interest, transfection, sequencing were performed as described in example 1.1 to obtain the correct DlbHLH3 gene (CDS sequence of which is shown in SEQ ID NO: 3). Amplifying and shaking the bacterial liquid with correct sequence, and extracting the recombinant plasmid according to the specification of the plasmid minibody kit.
Example 7 interaction of DlMYB15, dlbHLH3 and DlMYBrprp
1. Yeast two-hybrid experiments
And (3) extracting RNA from the red skin longan peel to obtain total RNA meeting the requirement of subsequent experiments. cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit with DNase I (Thermo Scientific, USA) reverse transcription kit. The pGBKT7-DlMYBrp primer pair, pGBKT7-DlMYB15 primer pair, pGADT7-DlMYB15 primer pair and pGADT 7-DlHLH 3 containing the enzyme cutting sites in the experimental part of the yeast in Table 1 are respectively used for amplification, and the PCR reaction system and the reaction procedure are the same as those of example 1.1, so that the corresponding enzyme cutting sites are introduced on the target genes. Then connecting the target genes into pGBKT7 and pGADT7 vectors, and respectively connecting the DlMYBrpp genes and the DlMYB15 genes to the pGBKT7 vectors through EcoRI and SalI cleavage sites to construct BD vectors (pGBKT 7-DlMYBrpp plasmids and pGBKT7-DlMYB15 plasmids in the following tables); the DlbHLH3 gene and the DlMYB15 gene were ligated into pGADT7 vector by EcoRI and BamHI cleavage sites, respectively, to construct AD vectors (pGADT 7-DlbHLH3 plasmid and pGADT7-DlMYB15 plasmid of the following Table). pGBKT7-Lam and pGADT7-T are used as negative controls; pGBKT7-53 and pGADT7-T were used as positive controls.
Streaking AH109 yeast glycerinum on YPDA plate, inversely culturing at 29 ℃ for about 3 d, picking up monoclonal in 2 mL YPDA culture medium, culturing at 220 rpm for 8 h; then, 5. Mu.L of the bacterial liquid was cultured overnight in 5 mL of YPDA medium; 700 Centrifuging for 5 min with xg, removing supernatant, and re-suspending the bacterial liquid twice with 5 mL sterile water; then, the yeast was resuspended in 1mL of a 1.1-fold TE/LiAc solution, centrifuged at 1,2000 rpm for 15 s, and again resuspended in 1mL of a 1.1-fold TE/LiAc solution and rendered yeast competent. The plasmids were then transferred into AH109 competent cells in the following table combinations:
| group of experiments | Plasmid 1 | Plasmid 2 |
| Positive control | pGBKT7-53 | pGADT7-T |
| Negative control | pGBKT7-Lam | pGADT7-T |
| Experiment group 1 | pGBKT7-DlMYBrp | pGADT7-DlMYB15 |
| Experiment group 2 | pGBKT7-DlMYBrp | pGADT7-DlbHLH3 |
| Experiment group 3 | pGBKT7-DlMYB15 | pGADT7-DlbHLH3 |
(wherein pGBKT7-DlMYBrp is a recombinant plasmid pGBKT7 carrying the DlMYBrprp gene.)
mu.L of pre-denatured Carrier DNA, 300. Mu.L of 1 XTE/LiAc/PEG 4000 solution, 50. Mu.L of yeast competent cells were added per tube reaction, and after mixing well, water bath was carried out at 30℃for 30 min; adding 20 mu L of DMSO, mixing, performing heat shock in a 42 ℃ water bath for 15 min, and performing vortex mixing once every 5 min; 700 Xg was centrifuged for 5 min, the supernatant was discarded, resuspended in 1mL YPDA medium and incubated at 28℃at 200 rpm for 1 h; the cells were resuspended by centrifugation at 1,2000 rpm for 15, 15 s, discarding the supernatant, and adding 100. Mu.L of 0.9% NaCl solution. Each system was spread with 100-fold diluted 10-fold and 100-fold bacterial solutions on DDO plates, and cultured at 28℃in an inverted manner for about 3 d to observe colony diameters. Selecting monoclone with diameter larger than 2 mm, respectively inoculating to 5 mL YPDA liquid culture medium, and culturing at 30deg.C and 220 rpm until OD600 value is 0.4-0.6; mu.L bacterial solutions are respectively inoculated to DDO (SD/-Leu/-Trp), QDO/3AT (SD/-Leu-/Trp/-His/-Ade/3 AT) and QDO/X/3AT (SD/-Leu-/Trp/-His/-Ade/X-alpha-Gal/3 AT) plates, and the plates are cultured AT 30 ℃ for about 3 d to observe the growth condition of bacterial plaques.
As shown in FIG. 12, the yeast two-hybrid experiment preliminarily proves that the DlMYB15 and the DlMYBrH 3 have no interaction relationship, but the DlMYB15 and the DlMYBrP interact, and the DlMYBrH 3 and the DlMYBrP interact.
2. Transient transformation of tobacco lamina
The recombinant plasmids obtained in example 1.1, example 5.1 and example 6.1 were taken for experiments. The experiments were divided into 8 groups: (1) An Empty Vector (Empty Vector) transfects tobacco leaves, (2) a recombinant plasmid carrying the DlMYBrprp gene (DlMYBrprp) transfects tobacco leaves; (3) Transfecting tobacco leaves with a recombinant plasmid (DlMYB 15) carrying the DlMYB15 gene; (4) Transfecting tobacco leaves with a recombinant plasmid (DlbHLH 3) carrying the DlbHLH3 gene; (5) The recombinant plasmid carrying the DlMYB15 gene and the recombinant plasmid carrying the DlMYBrprp gene (DlMYB 15/DlMYBrprp) are transfected into tobacco leaves; (6) The recombinant plasmid carrying the DlMYB15 gene and the recombinant plasmid carrying the DlbHLH3 gene (DlbHLH 3/DlMYB 15) are transfected into tobacco leaves; (7) The recombinant plasmid carrying the DlbHLH3 gene and the recombinant plasmid carrying the DlMYBrp gene (DlbHLH 3/DlMYBrp) are transfected into tobacco leaves; (8) The recombinant plasmid carrying the DlMYB15 gene, the recombinant plasmid carrying the DlMYBrprp gene and the recombinant plasmid carrying the DlMYBrprp gene (DlHLH 3/DlMYB 15/DlMYBrprp) are transfected into tobacco leaves.
Transformation and shaking methods were the same as in example 1, "2, subcellular localization". The experimental operation steps are as follows: the recombinant plasmid-containing GV3101 Agrobacterium was shake-cultured with 15 mL of YEP broth (containing 25 mg/L rifampicin and 100 mg/L kanamycin) at 28 ℃. Culturing overnight until the bacterial liquid is turbid, centrifuging at 6000 rpm for 5 min to collect bacterial cells, re-suspending twice with sterile water, and washing the redundant culture medium containing rifampicin as much as possible. Re-suspending with MMA re-suspension 1mL, and regulating bacterial liquid OD with MMA re-suspension 600 0.5, and standing under dark condition for 2-3 h. MMA heavy suspension is prepared according to the following formula:
| reagent(s) | Usage amount |
| 100 mM MES | 1 mL |
| 100 mM MgCl 2 ·6H 2 O | 1 mL |
| Acetosyringone | 10 μL |
| Sterile water | 8 mL |
Taking a sterile syringe of 1-mL, taking down a needle, sucking bacterial liquid, lightly injecting the bacterial liquid from the back of tobacco leaves, marking, placing 16-24 h in a dark condition, performing normal photoperiod treatment, observing leaf phenotype by 36-48 h, and performing photographing, sampling, freezing and the like for subsequent experimental analysis.
The results are shown in FIG. 13. The transient co-transformation tobacco experiment shows that the independent injection of DlMYB15 and the independent injection of DlbHLH3 and the co-injection of DlMYB15/DlbHLH3 can not promote the accumulation of anthocyanin; when DlMYBrp was injected alone, the anthocyanin accumulation amount was 1.298 mg.100 g -1 FW; when the tobacco leaves are co-injected with DlMYB15/DlMYBrp, the anthocyanin accumulation amount is 1.506 mg.100 g -1 FW, dlMYBrp is independently injected, and anthocyanin accumulation is obviously increased; when the DlbHLH3/DlMYBrp is co-injected into tobacco leaves, the anthocyanin accumulation amount is 2. mg.100 g -1 FW, compared with single injection of DlMYBrp, the accumulation of anthocyanin is obviously increased; especially when the DlMYB15/DlbHLH3/DlMYBrp are co-injected into tobacco leaves, the anthocyanin accumulation amount is 6.188 mg.100 g -1 FW, anthocyanin accumulation was significantly increased, 4.77 times that of DlMYBrp alone. It can be seen that DlMYB15 may enhance anthocyanin accumulation by interacting with DlMYBrp and with the complex formed by DlMYBrp and DlbHLH3, and that DlbHLH3 may enhance anthocyanin accumulation by interacting with DlMYBrp and with the complex formed by DlMYBrp and DlMYB 15.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (5)
1. The method comprises the following steps ofDlMYBrpThe gene is characterized in that the CDS sequence is shown as SEQ ID NO. 1.
2. Claim 1 is a deviceDlMYBrpA protein encoded by the gene.
3. Comprising the composition of claim 1DlMYBrpRecombinant vectors or hosts or expression cassettes for the CDS sequences of genes.
4. The method as claimed in claim 1DlMYBrpGene, or protein according to claim 2, or recombinant vector or host bacterium or expression cassette according to claim 3 in the enhancement of Arabidopsis thalianaAtCHSIts NCBI accession numberAT5G13930.1、AtCHIIts NCBI accession numberAT3G55120.1、AtF3HIts NCBI accession numberAT3G51240.1、AtDFRIts NCBI accession numberAT5G42800.1、AtANSIts NCBI accession numberAT4G22880.1Or (b)AtUFGTIts NCBI accession numberAT5G17050.1Is used in the expression level of (a).
5. The method as claimed in claim 1DlMYBrpUse of a gene, or a protein according to claim 2, or a recombinant vector or host bacterium or expression cassette according to claim 3, for regulating plant accumulation of anthocyanins.
Priority Applications (1)
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| CN114525284A (en) * | 2022-01-21 | 2022-05-24 | 长江师范学院 | Red-peel longan anthocyanin biosynthesis regulation gene DlMYB1-HP and application thereof |
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| CN103725693A (en) * | 2014-01-09 | 2014-04-16 | 华南农业大学 | Litchi R2R3-MYB gene LcMYB1 and application thereof |
| CN114525284A (en) * | 2022-01-21 | 2022-05-24 | 长江师范学院 | Red-peel longan anthocyanin biosynthesis regulation gene DlMYB1-HP and application thereof |
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