CN113881685B - Gene PpHSP20-like1 for promoting plant organ to produce red color and application thereof - Google Patents
Gene PpHSP20-like1 for promoting plant organ to produce red color and application thereof Download PDFInfo
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- CN113881685B CN113881685B CN202111177712.7A CN202111177712A CN113881685B CN 113881685 B CN113881685 B CN 113881685B CN 202111177712 A CN202111177712 A CN 202111177712A CN 113881685 B CN113881685 B CN 113881685B
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Abstract
The invention discloses a PpHSP20-like1 gene for promoting plant organs to generate red color and application thereof, wherein the nucleotide sequence of the gene is SEQ ID NO.1, and the amino acid sequence of the encoded protein is SEQ ID NO.2. The PpHSP20-like1 gene of the invention is a heat shock protein gene, the length of the complete coding frame is 1179 bases, and the coding protein contains 393 amino acids. PpHSP20-like1 heterologously transformed Arabidopsis thaliana turns red leaves and calyx, and anthocyanin content is from none to none; the homologous instant transformation white peach fruit and flesh become red, which shows that the PpHSP20-like1 gene cloned for the first time and the protein encoded by the gene promote the formation of red flesh color, and the result has important significance for the practical application of enriching the red flesh color cause basic theory, developing molecular markers, improving the defects of the existing red peach, improving the breeding efficiency of high-quality red peach and the like.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a PpHSP20-like1 gene for promoting plant organs to generate red color and application thereof.
Background
Pulp color is an important agronomic character of fruit trees, and is one of key indexes for evaluating fruit quality. Peach (Prunus persica) is originally produced in China, and has wide distribution and rich germplasm resources. The peach flesh has five colors of white, yellow, green, red and purple. The red peach is a specific resource in the peach subgenera plant, and compared with the non-red peach, the red peach has special visual impact and sensory attraction, and the anthocyanin and phenolic substances which are rich in the red peach have the functions of resisting oxidation, resisting aging, preventing cardiovascular and cerebrovascular diseases and the like, and are very beneficial to the health of human bodies. Along with the continuous improvement of the demands of people for diversification and functionalization of fruits, the nutritional value of the red-meat peaches is increasingly valued, and the demands for high-quality red-meat peaches are continuously increased. However, the existing red-meat peach resources and varieties have the defects of general sour flavor, small fruit shape and the like, and the need for breeding high-quality red-meat peach varieties is urgent. At present, although a molecular mechanism for forming the color of the peach red flesh has a certain progress in individual red flesh peach germplasm resources, the red flesh peach germplasm resources are various, the genetic rule is complex, genetic background differences exist among some germplasm, the molecular mechanism for forming the color of the red flesh of other red flesh peach germplasm resources is still unclear, and the breeding efficiency of the high-quality red flesh peach is improved by utilizing a modern molecular marking technology to a certain extent.
Heat shock proteins (heat shock protein, HSP) are commonly found in plants, animals, humans, drosophila and other organisms. Heat shock proteins are classified into several families such as HSP110, HSP90, HSP70, small molecular mass HSP (small heat shock protein, abbreviated as sHSP) and the like, according to molecular weight and homology of amino acid sequences. Since the molecular weight of sHSP family proteins is mostly 15-22 kDa, they are also called HSP20 family proteins. HSP20 family proteins are typically characterized by a conserved alpha-crystalline domain (ACD) or by a domain known as ACD-like or HSP20-like that closely resembles the ACD domain. There are also documents that separate HSP20 proteins containing HSP20-like domains into separate classes, called the HSP20-like family.
HSP20 protein can prevent protein denaturation, maintain normal physiological activity of cells and prevent the cells from being damaged by heat stress. HSP20 proteins are often used as chaperones, whose functional studies are based on experimental materials such as herbs, and play a complex and important role in a wide variety of cellular pathways under normal growth conditions and stress conditions (high temperature, cold, drought, virus attack, chemical stimulation, etc.), such as cell proliferation, differentiation, migration, signal transduction, cytoskeleton, ciliated dynamics, immunology, pathogen resistance, etc. Whereas studies in woody plants, especially peaches, are rare.
Disclosure of Invention
The invention aims to: aiming at the defects existing in the prior art, the invention provides a PpHSP20-like1 gene for positively regulating anthocyanin synthesis, which can promote the formation of red color of plant organs, such as the formation of red color of plant Arabidopsis leaves, calyx and peach pulp.
Another object of the invention is to provide PpHSP20-like1 gene and its coding protein sequence, over-expression vector, host bacterium and application of the PpHSP20-like1 gene in promoting plant organ redness. The invention constructs a PpHSP20-like1 plant over-expression vector, and the biological function of the plant over-expression vector is verified by heterologously transforming Arabidopsis thaliana and homologously transiently transforming white peach fruits by a dipping method. The cloned PpHSP20-like1 gene has the function of promoting the formation of red flesh color of fruits.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention clones out the peach local variety pheasant red by using a cloning technology to obtain a gene PpHSP20-like1 homologous to Arabidopsis thaliana HSP 20-like. The PpHSP20-like1 nucleotide sequence is shown as SEQ ID NO.1, contains 1179 bases, codes 393 amino acids, has the amino acid sequence shown as SEQ ID NO.2, has a molecular weight of 98.24997kDa, has an isoelectric point of 5.07, and is positioned at the nucleus.
The gene PpHSP20-like1 disclosed by the invention is applied to promotion of plant organs to generate red color.
The gene PpHSP20-like1 disclosed by the invention is applied to promotion of red leaves, calyx and peach pulp of arabidopsis thaliana plants.
The invention relates to application of an expression vector containing a gene PpHSP20-like1 in promoting plant organs to generate red color.
The construction of the expression vector disclosed by the invention comprises the following steps:
the cDNA of red peach variety pheasant red is used as a template, a primer is designed, a PpHSP20-like1 gene fragment is obtained by amplification, the PCR product is connected to a pENTR/D-TOPO vector after purification, and the constructed plasmid is named pENTR/D-TOPO, namely PpHSP20-like1; pENTR/D-TOPO: ppHSP20-like1 was ligated to the R05 vector by the GATEWAY method using LR enzyme, and the constructed plasmid was designated as R05-PpHSP20-like1.
The invention designs and clones the primer pair of the nucleotide open reading frame sequence of the Pplike1PpHSP20-like1, and the base sequence is shown as follows:
P1:5’-CACCATGGAAGATGACCCTCCTAAAATGTCCGTC-3’
P2:5’-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’。
utilizing agrobacterium containing recombinant vector R05-PpHSP20-like1 to carry out heterologous transformation on Arabidopsis thaliana and homologous transformation on white peach fruits to obtain red leaves and red calyx of Arabidopsis thaliana; the peach fruit has a red flesh phenotype, and the peach fruit is verified to have the function of promoting red color formation.
Recombinant expression vector and transgenic recombinant bacteria containing the genes.
The gene or the protein can be applied to improving the breeding efficiency of high-quality red-meat peaches.
The gene or the protein can be applied to the aspects of identifying red peach resources, genotyping and molecular marking.
The functional gene PpHSP20-like1 obtained by cloning from red peach variety 'pheasant red' fruits is applied to promotion of red color of plant organs, in particular to promotion of formation of red color of pulp. The applicant uses plant gene cloning technology to clone a new gene PpHSP20-like1 from the fruit of pheasant red. PpHSP20-like1 belongs to a family of HSP20 genes, having a domain called ACD-like or HSP20-like very similar to the ACD domain, expressed in fruits. PpHSP20-like1 was ligated to R05 vector containing GFP tag and transiently transformed in tobacco leaves, and it was shown that PpHSP20-like1 was located in the nucleus and was a nuclear protein. Simultaneously, the R05-PpHSP20-like1 is subjected to heterologous transformation of Arabidopsis thaliana and homologous transient transformation of white peach fruits, and the results show that: the anthocyanin content and the relative expression level of PpHSP20-like1 genes of the positive plant leaves are obviously higher than those of wild type, the positive plant leaves and the calyx are red, and high-resolution mass spectrometry (LC-QTOF/MS) analysis shows that the positive plant leaves contain anthocyanin cyanidin-3-glucoside and cyanidin-3-rutinoside; the white peach injection site turned milky white pulp red compared to the control. PpHSP20-like1 promotes the generation of red color and has wide application prospect in plant genetic engineering and genetic engineering improvement of red peach breeding.
The invention relates to a PpHSP20-like1 gene for imparting red color to plant organs. Functional research of PpHSP20-like1 reveals the expression regulation mechanism and action mechanism, and can be applied to plant genetic engineering and genetic engineering improvement of red-pulp peach breeding, and can also be used for constructing various plant expression vectors by utilizing the gene disclosed by the invention, so that the invention can be applied to agricultural biotechnology breeding to improve the color improvement efficiency of red pulp of peach fruits.
The invention discovers that a fruit red flesh color trait related gene PpHSP20-like1 is highly homologous with Arabidopsis thaliana HSP20-like chaperones superfamily protein (accession AT1G20870.1), and E-value is 2E through hybridization, QTL positioning, genome resequencing, transcriptome determination and other analysis of red flesh peach local variety 'pheasant red' and a plurality of non-red flesh peaches respectively -31 The method comprises the steps of carrying out a first treatment on the surface of the Prupe.5 in the peach 'Lovell' reference genomic sequenceG005900.1 (XM_ 007209161.2) has the highest similarity of 99.92%, and by taking Prupe.5G005900.1 sequence as a reference, designing a primer to clone the PpHSP20-like1 gene from red flesh peach variety 'pheasant red' flesh, and transforming the gene into Arabidopsis thaliana and homologous instant transformation into white flesh peach fruits, wherein the obtained transgenic Arabidopsis thaliana plant leaves and calyx are red, and the transgenic peach flesh is red. The separation of the peach flesh color and character related gene PpHSP20-like1 can further enrich the fruit quality character gene resources, and lays a foundation for the further utilization of the genes for the functional research thereof.
The beneficial effects are that: compared with the prior art, the invention has the advantages and effects that:
(1) The gene is cloned to the PpHSP20-like1 gene for promoting plant organs to generate red color for the first time, so that the red color of peach pulp can be formed, a new thought is provided for improving the breeding efficiency of red-pulp peaches, and a foundation is provided for realizing molecular breeding and saving a large amount of manpower, material resources, land fertility and other costs.
(2) Through heterologous transformation of Arabidopsis thaliana and homologous transient transformation of white peach, a red flesh color phenotype is obtained, and a functional verification result shows that the cloned gene has the function of promoting red color generation.
(3) The PpHSP20-like1 gene is related to red meat character formation, the PpHSP20-like1 is transformed into Arabidopsis through constructing a plant expression vector, and as a result, after the gene is overexpressed in a transgenic plant, compared with a wild type and empty vector control plant, leaves and calyx appear red; the white peach fruit is transformed, and compared with the control, the white peach injection part turns into milky pulp into red. The PpHSP20-like1 expression level is obviously improved. The separation of PpHSP20-like1 can further enrich the pulp red color gene resources, and the functional research lays a foundation for the further utilization of the genes.
(4) The invention is helpful for better understanding the molecular action mechanism of the formation of the red flesh color of the fruits. Cloning of PpHSP20-like1 lays a foundation for further understanding of interaction between red meat color-related gene encoding proteins and anthocyanin metabolism signal transduction pathways. The transgenic plant of the gene can be used for further analyzing how the PpHSP20-like1 transmits signals and transmits the signals, so that the mechanism that the PpHSP20-like1 participates in the anthocyanin signal transmission path of pulp is obtained. The separation and functional identification of PpHSP20-like1 lay a foundation for researching the action mechanism of pulp red color gene.
(5) The invention is applied to fruit flesh red color breeding, develops a molecular marker aiming at PpHSP20-like1, lays a foundation for eliminating non-target character seedlings in a seedling stage, greatly improves breeding efficiency, and has great application value in fruit flesh transgenic breeding.
Drawings
FIG. 1 is a graph of the red meat phenotype of the fruits of the 'pheasant red' variety at four stages of development.
FIG. 2 is an agarose gel electrophoresis of extracted total RNA of the 'pheasant red' ripe fruit.
FIG. 3 is an agarose gel electrophoresis diagram of a full-length PCR product of amplified PpHSP20-like1 gene, wherein M is Marker molecular weight standard, and the bands are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence from top to bottom; lanes 1 and 2 are all the full length of PpHSP20-like1.
FIG. 4 shows the PCR identification of positive clone agarose gel electrophoresis of pENTR/D-TOPO: ppHSP20-like1 bacterial liquid, wherein M is Marker molecular weight standard, and the bands are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence from top to bottom; lanes 1 to 8 are pENTR/D-TOPO: ppHSP20-like1 bacterial liquid PCR products 1 to 8 tubes.
FIG. 5 is a subcellular localization analysis of PpHSP20-like1; A20X objective lens was used for photographing with a rotating disc laser confocal microscope (Ultra View VOX, USA). English above the graph shows different light quality settings, and the results of Bright field (Bright field), green fluorescence (PpHSP 20-like1-GFP,488 nm), red fluorescence (Right field,561 nm) and the three conditions are overlapped together (Merge) from left to Right.
FIG. 6 is a diagram of agarose gel electrophoresis of a transgenic Arabidopsis thaliana positive strain of PpHSP20-like1, in which M is Marker molecular weight standard, and the bands are 4500bp, 30000bp, 2000bp, 1200bp, 800bp, 500bp, 200bp in order from top to bottom, for PCR detection of pENTR/D-TOPO; lanes 1 to 4 are pENTR/D-TOPO: ppHSP20-like1 transgenic strain nos. 1-4, WT being a wild-type control strain; m in the diagram B is Marker molecular weight standard, the band is 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom in sequence; lanes 5 and 6 are pENTR/D-TOPO: ppHSP20-like1 transgenic strains 5 and 6, and the empty vector is an infected R05 empty vector strain.
FIG. 7 is leaf phenotype of Arabidopsis transgenic positive lines and control lines; WT represents wild-type control, empty vector represents empty vector control, OE1 to OE5 represent over-expressed strains 1 to 5, respectively. A: an overall color phenotype; b: a calyx color phenotype on stems; c: an amplified on-stem calyx color phenotype; d: a top flower color phenotype.
FIG. 8 shows the relative expression levels of PpHSP20-like1 in leaves of Arabidopsis transgenic positive lines and control lines; WT represents the relative expression level of PpHSP20-like1 in wild-type control leaves, empty vector represents the relative expression level of PpHSP20-like1 in empty vector control leaves, and OE1 to OE5 represent the relative expression levels of PpHSP20-like1 in PpHSP20-like1 overexpressing lines 1 to 5, respectively. RPII is housekeeping gene; when the expression level of PpHSP20-like1 in the leaf of OE1 strain was 1, the relative expression levels in other strains and control strains were set.
FIG. 9 shows HPLC detection of anthocyanin composition in leaves of over-expressed PpHSP20-like1 Arabidopsis positive lines and wild type control lines; panel A represents the results of wild-type control strains and Panel B represents the results of transgenic positive strains; WT represents a wild-type control strain; OE stands for PpHSP20-like1 over-expressed strain.
FIG. 10 is a sample of LC-QTOF/MS for detection and analysis of anthocyanin components in leaves of transgenic positive lines of Arabidopsis; a represents a cyanidin-3-glucoside mass spectrum TOFMS secondary diagram, and B represents a cyanidin-3-rutinoside mass spectrum TOFMS secondary diagram.
FIG. 11 shows PpHSP20-like1 overexpressing white peach flesh color and control peach flesh color phenotype; OE1 and OE2 represent the flesh color phenotype of 2 white peaches transiently overexpressing PpHSP20-like1, CK1 represents the flesh color phenotype of an empty vector control without the PpHSP20-like1 gene, and CK2 represents the flesh color phenotype of a no-liquid control injected.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The following examples facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental consumables used in the examples, unless otherwise specified, were all purchased from conventional biochemical reagent companies.
Primer synthesis and sequencing used were both synthesized by general biosystems, inc.
The invention provides a pheasant red variety provided by Jiangsu province agricultural sciences institute (Xu Jianlan, ma Ruijuan, zhang, ding Hui, yan Juan, mingliang. Research on main character genetic rules of filial generation of different flesh color peaches. Fruit tree journal, 2019, 36 (1): 21-30).
The transiently expressed variety 'safflower' was offered by the academy of agricultural sciences of Jiangsu province (Shen Zhijun, primary core germplasm of peach, association analysis and map localization of red meat traits, 2013).
The wild type Arabidopsis thaliana (Arabidopsis thaliana) Col-0 seed and the wild type Nicotiana benthamiana (Nicotiana benthamiana) seed are both provided by the national academy of agricultural sciences of Jiangsu province.
Example 1
PpHSP20-like1 clone and sequence analysis
Cloning of peach fruit red flesh color related protein PpHSP20-like1 encoding gene PpHSP20-like1.
With reference to the reference genome prupe.5g005900.1 sequence, a primer pair (upstream primer P1 and downstream primer P2) was designed for amplifying the fruit red flesh color-related like1PpHSP20-like1 gene from red flesh peach' pheasant flesh (fig. 1).
Peach 'pheasant red' mature fruit total RNA was extracted according to the Minibest RNA Kit (Takara Shuzo Co., ltd.) protocol (FIG. 2). First Strand cDNA Synthesis kit (PrimeScript) TM II 1st Strand cDNA Synthesis Kit, bao bioengineering (Dalian) Co., ltd.) reverse transcribed into cDNA according to the protocol and diluted 10-fold and stored at-20 ℃. Using the cDNA as a modelThe plates were PCR amplified using the upstream primer P1 (5'-CACCATGGAAGATGACCCTCCTAAAATGTCCGTC-3') and the downstream primer P2 (5'-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3'). Each 50. Mu.L of PCR reaction system comprises 2x High-Fidelity Master Mix. Mu.L, 1. Mu.L (10. Mu.M) of each of the upstream and downstream primers, 2. Mu.L of cDNA and ddH 2 O21. Mu.L. The PCR reaction procedure was: 1min pre-denatured at 98 ℃,15 s annealed at 55 ℃ and 20s extended at 72 ℃, 2min at 72 ℃,35 cycles, stored at 4 ℃ for a total of 3 tubes.
The 2-tube PCR products were subjected to 1.5% agarose gel electrophoresis, and a band of about 1179bp (FIG. 3) was observed by an ultraviolet gel imager, consistent with the expected size. The same another PCR product was recovered using a PCR product purification kit (TaKaRa MiniBEST DNA Fragment Purification Kit Ver4.0, takara Bio Inc.). The recovered and purified DNA fragment was ligated with a cloning vector plasmid pENTR/D-TOPO (Semer Feichi technologies (China) Co., ltd.) to obtain a recombinant plasmid pENTR/D-TOPO: ppHSP20-like1. Recombinant plasmid pENTR/D-TOPO: ppHSP20-like1 was transformed into competent cells of E.coli DH5a (Takara Bio-engineering Co., ltd.) with kanamycin (50.0 mg.L) -1 ) The positive clones were screened for markers and bacterial liquid PCR identification (upstream primer 5'-TGTAAAACGACGGCCAGT-3' and downstream primer P2: 5'-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3') to give bands containing the expected fragment size (FIG. 4), and positive recombinant plasmids were extracted and sent to general biosystems, inc. for sequencing. Sequencing comparison results show that the ORF length of the PpHSP20-like1 is 1179bp, and the gene is named as PpHSP20-like1, and the sequence is shown as SEQ ID NO:1, which has 1 SNP variation from the reference genome 'Lovell'. The protein sequence coded by the PpHSP20-like1 gene is shown as SEQ ID NO: 2. The experimental steps are all operated according to the instruction of the kit.
SEQ ID NO：1
ATGGAAGATGACCCTCCTAAAATGTCCGTCAATCCATCGTTTGCCGACCAGCTTTTTCTACTGAACTTCGTAATGGGAAATTATTTGGGCCCTGATGTCACGTTTGATAACCTCGAATGTTCAGCATTTCAAAGGATAGCTGAAGGTTCACCTCCGTATATGTCAAGTTATTTGGGGCCTTCTTATGTTAGTGTGTCTCTTTTGGAGGGTTTATATTACTATCTCTTGAGAAATGTTCACCCTAGTCTCATTTTGAGACCAGAAATGTTGCATAAGTATTTAAAGGGCAGCCTACCTTTGCCAAATTCAGGGCTGACGAAAGACAGTTGGCAGTTCACAAATTTTTTCCCTTTGGATCTTCATGAACAGATATGGTACCCAGAAAGCTTCAGAATTGTTAAGGGGATTCTTTTTATTGATGATCCTGTTACATCATGTATGAAAGAGGACGATCTGGAAAAGTTCAAATCTTTATCAGGTATAAATAACATGAAAATTGATATAGATGAAGCCATACGATATCAACACAAGTACCTGGATAATGGGGAAAGTAAGACGAATTGCTTGAATGATGATTGCAATTTCACAAAGACAGAATTTTTTTCCAATGGAAATGGAAATTGGTCGGAAAGATTTCAGCAAAAATATAAGAGAAGGTGCTTCTCTCATTCTCCATCAAAGCCAGCATTTCCCCAAGTTGTTACTACAAAACATTTAAGCAAATATGGAGCTTCATGGAAGACCTGCAAACCAGATGGACCTGTGTTCATGCCCCTTATCTCTGTTCCTAATTTGGAAGAATGTACTTCAGATTCTTCTGTTGTTTTGTCTGGGACTGCCAGGAAAGGGGTAGTTGGGCCACCTGTTGGTGTTGTGGACATTGGTGTTAGCAAGGCTGCATATTACTTCCGGGTTGCTCTACCAGGGGTCAGGAAGGATTTTTGTCAATTCAATTGTGAGATTGAATCTAATGGCAAGATTCATCTCCAAGGCGTCACAAGTGGTGGAAATCCCATAAGGAAACGTTCGCGTGTATTCCAAATGAAATTGCAGCAATTATGCCCACCCGGACCATTCACGCTCTCGTTCAGCCTTCCAGGACCTGTTGATCCAAGGCTCTTTGCACCAAATTTTGGACCTGATGGCATTTTTGAAGGAGTTATCATAAAAGACGAGTGA
SEQ ID NO：2MEDDPPKMSVNPSFADQLFLLNFVMGNYLGPDVTFDNLECSAFQRIAEGSPPYMSSYLGPSYVSVSLLEGLYYYLLRNVHPSLILRPEMLHKYLKGSLPLPNSGLTKDSWQFTNFFPLDLHEQIWYPESFRIVKGILFIDDPVTSCMKEDDLEKFKSLSGINNMKIDIDEAIRYQHKYLDNGESKTNCLNDDCNFTKTEFFSNGNGNWSERFQQKYKRRCFSHSPSKPAFPQVVTTKHLSKYGASWKTCKPDGPVFMPLISVPNLEECTSDSSVVLSGTARKGVVGPPVGVVDIGVSKAAYYFRVALPGVRKDFCQFNCEIESNGKIHLQGVTSGGNPIRKRSRVFQMKLQQLCPPGPFTLSFSLPGPVDPRLFAPNFGPDGIFEGVIIKDE*
Example 2
Construction of PpHSP20-like1 Gene overexpression vector
The method of GATEWAY is adopted to connect PpHSP20-like1 in pENTR/D-TOPO: ppHSP20-like1 recombinant plasmid to R05 vector (Semer Feishul technology (China) Co., ltd.) according to the steps of the specification, the competent cells of E.coli DH5a (Bao bioengineering (Dalian) Co., ltd.) are transformed by freeze thawing, bacterial liquid PCR identification is carried out by picking monoclonal the next day, and 4 tubes (technical repetition) of bacterial liquid with expected band size are taken to be sent to general biological systems Co., ltd. For sequencing identification. Sequencing comparison results show that: the correct recombinant expression vector R05-PpHSP20-like1 containing the PpHSP20-like1 gene was successfully obtained. The R05-PpHSP20-like1 plasmid was recovered according to the AxyPrep plasmid recovery kit (Corning Life sciences Co., ltd.) and stored at-20 ℃.
Example 3
Agrobacterium transformation with PpHSP20-like1 gene over-expression vector
The recombinant plasmids R05-PpHSP20-like1 and R05 empty plasmid were transformed into Agrobacterium GV3101 competent cells (Takara Bio Inc.) according to the GV3101 specification procedure, and after inversion culture for 24h at 28℃the selected monoclonal was placed in a 1.5mL centrifuge tube containing 1.0mL YEB liquid medium (containing 50. Mu.g/mL gentamicin, 50. Mu.g/mL rifampicin, 100. Mu.g/mL spectinomycin) and cultured at 28℃at 200rpm with shaking overnight, PCR was performed with rTaq enzyme (Takara Bio Inc.) and vector and PpHSP20-like1 gene-specific primers (P35S (upstream primer): 5'-GACGTTCCAACCACGTCTTCAAAG-3' and P2 (downstream primer): 5'-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3') using 1. Mu.L bacterial liquid as templates, indicating that the product fragment size was consistent with the expected target fragment size, thus successfully obtaining the recombinant expression vector R05-PpHSP20-like 1-containing Agrobacterium strain.
Example 4
PpHSP20-like1 protein subcellular localization the positive clone bacterial solution containing recombinant plasmid R05-PpHSP20-like1 prepared in example 3 was expanded to 50mL of YEB liquid medium system, at 28℃and 200rpm, shake culture was continued until OD600 was 0.8-1.0, and the supernatant was discarded by centrifugation. With injection buffer (containing 10mM MES,10mM MgCl) 2 100. Mu.M acetosyringone) was washed once, and then the cells GV3101-R05-PpHSP20-like1 were resuspended to OD600 of 0.7 with the above-mentioned buffer, and allowed to stand at room temperature for 4 hours. Subsequently, selecting good-growth Nicotiana benthamiana, sucking two sides of 1mL of heavy suspension injection leaves by a disposable sterilization syringe, injecting 3 total 9 leaves, and performing dark culture on the Nicotiana benthamiana for 1 day under the conditions of 16h:8h light-dark period, 22 ℃ and 70% air humidity for 2 days. Subcellular localization of PpHSP20-like1 was observed with a rotating disc laser confocal microscope (model: ultra View VOX, perkin Elmer Co., ltd.). As shown in FIG. 5, the PpHSP20-like1 protein is located in the nucleus and is a nuclear protein.
Example 5
R05-PpHSP20-like1 recombinant expression vector for transforming Arabidopsis thaliana
1) Acquisition and identification of PpHSP20-like1 transgenic Arabidopsis lines:
the positive clone bacterial solution containing the recombinant plasmid R05-PpHSP20-like1 and the bacterial solution containing the R05 empty vector plasmid prepared in example 3 are respectively expanded to 100mL of YEB liquid culture medium system, the temperature is 28 ℃, the rpm is 200, shake culture is continued until the OD600 is 0.8-1.0, and the supernatant is removed by centrifugation. The pellet was washed once with buffer (MS-containing liquid medium, 100. Mu.M acetosyringone) and then resuspended to OD600 of 1.0 with the above buffer and allowed to stand at room temperature for 4h. The arabidopsis thaliana is transformed by a flower dipping method, specifically, agrobacterium suspension carrying recombinant plasmid R05-PpHSP20-like1 is placed in a sterilizing beaker, the bud of the arabidopsis thaliana is soaked in the liquid of the beaker for 10s, 6 plants are transfected in total, and the plant is continuously grown in a plant culture room with 16h/8h day-night photoperiod, 22 ℃ and 70% air humidity after being placed in a dark place for one day. The same method is also used for transforming the 6 strains of Arabidopsis thaliana by the thalli of the R05 empty vector plasmid. Seeds are harvested after maturation, and are sealed and preserved at 4 ℃ after drying, and are named as T0 seeds.
The wild type control was inoculated on an antibiotic-free MS medium, and the R05 empty vector control and the T0 seed were inoculated on an MS medium (containing a final concentration of 25.0 mg.L -1 Kanamycin), and the plant is planted in a square soil pot after 4 leaves grow out, and grows in a plant culture room with 16h/8h day-night photoperiod, 22 ℃ and 70% air humidity. After one month, genomic DNA of PpHSP20-like1 overexpressing, empty vector, wild type control Arabidopsis leaves was extracted, positive shoot identification was performed by PCR using primers P35S: GACGTTCCAACCACGTCTTCAAAG and P2CTCGTCTTTTATGATAACTCCTTCAAAAAT (FIG. 6), and the transgenic PpHSP20-like1 gene positive Arabidopsis lines were labeled and designated as T0 generation transgenic lines. And after the seeds are mature, harvesting the seeds for T1 generation by a single plant. The individual seeds are respectively sown in the mixture with the final concentration of 25.0mg.L -1 In MS culture medium of kanamycin, the plant of T1 generation grows true leaves and moves to a square nutrition pot for growth, and the plant is cultivated in a plant cultivation room under the same condition. Harvesting seeds of T1 generation single plant, drying, sealing at 4deg.C, and storing with additiveThe concentration is 25.0 mg.L -1 MS medium of kanamycin was screened further to observe T2 generation plant segregation. The germination rate of the T2-generation seeds on MS medium containing kanamycin is 100%. Results: the T2 generation seed is homozygous PpHSP20-like1 gene transferred strain seed, and 6 packages of stable homozygous PpHSP20-like1 gene transferred strain seed are obtained; the T2 seed was seeded and both leaves and calyx of the transgenic positive strain were red (fig. 7). Wherein: t0 represents transgenic current generation plants, T1 represents seeds generated by T0 generation selfing and plants grown by the transgenic current generation plants, and T2 represents seeds generated by T1 generation selfing and plants grown by the transgenic current generation plants.
2) Detection of expression level of PpHSP20-like1 in PpHSP20-like1 Gene-transferred Positive lines and controls
And (3) detecting the relative expression quantity of PpHSP20-like1 in the leaves of the transgenic strain and the control strain by qRT-PCR. Selecting 5T 2 generation homozygous transgenic strain seeds (OE 1, OE2, OE3, OE4 and OE 5), 1R 05 empty vector strain seeds and 2 wild type control strain seeds of the transgenic positive strain, sowing and culturing under uniform conditions, respectively extracting total RNA of leaves, respectively carrying out reverse transcription to obtain cDNA, diluting 10 times for later use (specific steps are the same as example 1), designing real-time fluorescence quantitative PCR primers (upstream primer 5'-CCTACCTTTGCCAAATTCAG-3' and downstream primer 5'-CTTTTCCAGATCGTCCTCTT-3') for the PpHSP20-like1 by taking diluted cDNA of each strain as a template, and adoptingPremix Ex Taq TM The (Tli RNaseH Plus) kit (Takara Bio Inc.) was subjected to a fluorescent quantitative PCR reaction in a reaction system of 20. Mu.L: comprises 10.0 mu L SYBR Premix Ex Taq (2X), 0.4 mu L ROX dye, 0.4 mu L upstream and downstream primers, 2.0 mu L cDNA template, ddH 2 O6.8. Mu.L was sealed in 96-well plates and the instrument used a Applied Biosystems 7500real-time PCR system. The reaction procedure was followed +.>Premix Ex Taq TM (Tli RNaseH Plus) instruction set, housekeeping gene RNA polymerase II (RP II) was used. 3 are arrangedBiological replicates and 3 technical replicates. Analysis of the relative expression level of each Gene Using 2 -ΔΔCT In the method, SPSS20.0 was used for differential significance analysis of the data, and all the data were tested by the Duncan new complex polar difference method.
qRT-PCR results showed that the PpHSP20-like1 gene was expressed in the highest amount in OE5, and that in OE3, OE2 and OE1 were repeated and in OE4 was the weakest (FIG. 8), but that in Arabidopsis positive seedlings OE 1-OE 5, the PpHSP20-like1 gene expression was significantly higher than in both the empty vector control and the wild type WT control.
Example 6
HPLC detection of anthocyanin components in leaves of Arabidopsis transgenic positive lines and wild type control lines
0.2g of fresh leaves of an arabidopsis transgenic positive strain and a wild control strain are respectively weighed by adopting a High Performance Liquid Chromatography (HPLC) method, ground into powder by liquid nitrogen, added with 2.0mL of solution (acetone: water: acetic acid=70:29.5:0.5), and leached for 12 hours in a refrigerator at 4 ℃ in a dark place. The supernatant was placed in a fresh centrifuge tube, centrifuged at 10000rpm at 4℃for 10min, filtered 3 times with a microporous filter membrane (0.22 μm) and placed in a brown sample bottle for testing.
Anthocyanin component detection was performed using an Agilent high performance liquid chromatography system (1100 series, VWD UV detector) and a Agilent ZORBAX SB-C18 chromatographic column (4.6 mm. Times.250 mm,5 μm). The standard substance is cyanidin-3-glucoside (Aladin, 27661-36-5), and the reagents such as methanoic acid are all of chromatographic purity grade. Methanol is analytically pure grade phosphoric acid and formic acid is HPLC grade; chromatographic conditions: mobile phase a: methanol: phosphoric acid: trifluoroacetic acid=97:2:1 (V/V); the method comprises the steps of carrying out a first treatment on the surface of the Mobile phase B: water: phosphoric acid: trifluoroacetic acid=97:2:1 (V/V); the sample injection amount is 20 mu L, and the flow rate is 1.0mL/min; detection wavelength: 515nm; column temperature: 25 ℃.
As a result, no anthocyanin was found in the wild-type control (FIG. 9A), and positive Arabidopsis leaves were peaked at 14.589min, 15.411min, 19.195min (FIG. 9B), respectively, with 15.411min peak highest, followed by 14.589min, and again 19.195min; the 14.589min peak was found to be cyanidin-3-glucoside by comparison with the standard, but the anthocyanin material represented by the 15.411min peak was unknown.
These results demonstrate that no anthocyanin was present in the Arabidopsis wild-type control, and that over-expression of PpHSP20-like1 Arabidopsis positive strain produced one of the procyanidin-3-glucosides, which mutually confirmed that red flesh of red-peach 'pheasant' was the anthocyanin substance represented by the peak 15.411min, as determined by the anthocyanin procyanidin-3-glucoside, and further qualitative analysis was required.
The leaf and the calyx of the wild type control strain and the calyx of the empty vector control strain are not red due to the overexpression of the PpHSP20-like1 gene, namely, the cyanidin-3-glucoside and another unknown anthocyanin substance in anthocyanin are generated in the leaf and the calyx of the flower due to the overexpression of the PpHSP20-like1 gene.
Example 7
Mass spectrum detection of anthocyanin component represented by 15.411min peak in arabidopsis transgenic positive strain leaves
Fresh leaves of an Arabidopsis transgenic positive line (0.2 g) are weighed, ground into powder by liquid nitrogen, added with 2.0mL of extracting solution (acetone: water: acetic acid=70:29.5:0.5), and leached for 12 hours in a refrigerator at 4 ℃ in a dark place. The supernatant was centrifuged at 10000rpm at 4℃for 10min in a fresh centrifuge tube, filtered through a microfiltration membrane (0.22 μm) and placed in a brown sample bottle for further testing.
Chromatographic conditions: shimadzu LC20+ SCIEX TripleTOF 5600+; liquid phase conditions: the column was waters BEH Xbridge 3.5.5 um,2.1 x 150mm; mobile phase a: acetonitrile, mobile phase B:0.1% formic acid water, 5uL of sample injection amount and 0.3ml/min of flow rate; LC-QTOF/MS mass spectrometry conditions: IDA method, mass range: m/z 100-1000, positive mode. High resolution mass spectrometry was performed using a tandem time of flight mass spectrometer (specification: triple tof 5600+, AB SCIEX company, usa). Formic acid, acetonitrile is HPLC grade; the experimental water was from the Milli-Q pure water system.
The peak of the transgenic positive line 15.411min represents anthocyanin cyanidin-3-rutin according to the mass spectrum secondary TOFMS chart (FIG. 10A and FIG. 10B) and the comprehensive determination of molecular weight of various anthocyanin components in the literature. Thus, over-expression of PpHSP20-like1 Arabidopsis positive strains produced cyanidin-3-glucose and cyanidin-3-rutinoside in anthocyanins.
Example 8
Instant transformation of white peach fruit by recombinant expression plasmid
Positive clone bacterial liquid containing recombinant plasmid R05-PpHSP20-like1 prepared in example 3 is respectively expanded to 50mL of YEB liquid culture medium system, shake culture is continued at 28 ℃ and 200rpm until OD600 is 0.8-1.0, and supernatant is discarded by centrifugation. With injection buffer (containing 10mM MES,10mM MgCl) 2 100 μm acetosyringone) was washed 2 times, then the pellet was resuspended to an OD600 of 0.7 with the above buffer and allowed to stand at room temperature for 4h. Subsequently, 160 fruits of white peach variety 'little safflower' provided by the academy of agricultural sciences of Jiangsu province are selected, and the design treatment is carried out: 60 fruits of empty vector plasmid (as control 1) are injected, 60 fruits of positive clone bacteria liquid of recombinant plasmid R05-PpHSP20-like1 are injected, and 40 fruits are not injected (as control 2); specifically, 1mL of the heavy suspension is sucked by a disposable sterilization syringe to inject one needle on the female side and the male side of the fruit on the tree 3 weeks before maturation. The tree body and the fruit are cultivated and managed in normal fields, and the pulp color and luster are investigated every 3 days for three times. As shown in FIG. 11, where OE1 and OE2 represent the flesh color phenotype of 2 white peaches transiently overexpressing PpHSP20-like1, respectively, CK1 represents the flesh color phenotype of an empty vector control without the PpHSP20-like1 gene, and CK2 represents the flesh color phenotype of a no-liquid control injected. The results show that: transient overexpression of PpHSP20-like1 resulted in red coloration of the white flesh at the injection site, and PpHSP20-like1 promoted the production of red flesh coloration, compared to empty vector control 1 and no injection of any control 2. The embodiment carries out the instant transformation of recombinant expression plasmid on the white peach variety 'small safflower', and has broad spectrum and similar effect on other varieties of white peaches.
Sequence listing
<110> academy of agricultural sciences in Jiangsu province
<120> Gene PpHSP20-like1 for promoting production of red color by plant organ and use thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1179
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggaagatg accctcctaa aatgtccgtc aatccatcgt ttgccgacca gctttttcta 60
ctgaacttcg taatgggaaa ttatttgggc cctgatgtca cgtttgataa cctcgaatgt 120
tcagcatttc aaaggatagc tgaaggttca cctccgtata tgtcaagtta tttggggcct 180
tcttatgtta gtgtgtctct tttggagggt ttatattact atctcttgag aaatgttcac 240
cctagtctca ttttgagacc agaaatgttg cataagtatt taaagggcag cctacctttg 300
ccaaattcag ggctgacgaa agacagttgg cagttcacaa attttttccc tttggatctt 360
catgaacaga tatggtaccc agaaagcttc agaattgtta aggggattct ttttattgat 420
gatcctgtta catcatgtat gaaagaggac gatctggaaa agttcaaatc tttatcaggt 480
ataaataaca tgaaaattga tatagatgaa gccatacgat atcaacacaa gtacctggat 540
aatggggaaa gtaagacgaa ttgcttgaat gatgattgca atttcacaaa gacagaattt 600
ttttccaatg gaaatggaaa ttggtcggaa agatttcagc aaaaatataa gagaaggtgc 660
ttctctcatt ctccatcaaa gccagcattt ccccaagttg ttactacaaa acatttaagc 720
aaatatggag cttcatggaa gacctgcaaa ccagatggac ctgtgttcat gccccttatc 780
tctgttccta atttggaaga atgtacttca gattcttctg ttgttttgtc tgggactgcc 840
aggaaagggg tagttgggcc acctgttggt gttgtggaca ttggtgttag caaggctgca 900
tattacttcc gggttgctct accaggggtc aggaaggatt tttgtcaatt caattgtgag 960
attgaatcta atggcaagat tcatctccaa ggcgtcacaa gtggtggaaa tcccataagg 1020
aaacgttcgc gtgtattcca aatgaaattg cagcaattat gcccacccgg accattcacg 1080
ctctcgttca gccttccagg acctgttgat ccaaggctct ttgcaccaaa ttttggacct 1140
gatggcattt ttgaaggagt tatcataaaa gacgagtga 1179
<210> 2
<211> 392
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Glu Asp Asp Pro Pro Lys Met Ser Val Asn Pro Ser Phe Ala Asp
1 5 10 15
Gln Leu Phe Leu Leu Asn Phe Val Met Gly Asn Tyr Leu Gly Pro Asp
20 25 30
Val Thr Phe Asp Asn Leu Glu Cys Ser Ala Phe Gln Arg Ile Ala Glu
35 40 45
Gly Ser Pro Pro Tyr Met Ser Ser Tyr Leu Gly Pro Ser Tyr Val Ser
50 55 60
Val Ser Leu Leu Glu Gly Leu Tyr Tyr Tyr Leu Leu Arg Asn Val His
65 70 75 80
Pro Ser Leu Ile Leu Arg Pro Glu Met Leu His Lys Tyr Leu Lys Gly
85 90 95
Ser Leu Pro Leu Pro Asn Ser Gly Leu Thr Lys Asp Ser Trp Gln Phe
100 105 110
Thr Asn Phe Phe Pro Leu Asp Leu His Glu Gln Ile Trp Tyr Pro Glu
115 120 125
Ser Phe Arg Ile Val Lys Gly Ile Leu Phe Ile Asp Asp Pro Val Thr
130 135 140
Ser Cys Met Lys Glu Asp Asp Leu Glu Lys Phe Lys Ser Leu Ser Gly
145 150 155 160
Ile Asn Asn Met Lys Ile Asp Ile Asp Glu Ala Ile Arg Tyr Gln His
165 170 175
Lys Tyr Leu Asp Asn Gly Glu Ser Lys Thr Asn Cys Leu Asn Asp Asp
180 185 190
Cys Asn Phe Thr Lys Thr Glu Phe Phe Ser Asn Gly Asn Gly Asn Trp
195 200 205
Ser Glu Arg Phe Gln Gln Lys Tyr Lys Arg Arg Cys Phe Ser His Ser
210 215 220
Pro Ser Lys Pro Ala Phe Pro Gln Val Val Thr Thr Lys His Leu Ser
225 230 235 240
Lys Tyr Gly Ala Ser Trp Lys Thr Cys Lys Pro Asp Gly Pro Val Phe
245 250 255
Met Pro Leu Ile Ser Val Pro Asn Leu Glu Glu Cys Thr Ser Asp Ser
260 265 270
Ser Val Val Leu Ser Gly Thr Ala Arg Lys Gly Val Val Gly Pro Pro
275 280 285
Val Gly Val Val Asp Ile Gly Val Ser Lys Ala Ala Tyr Tyr Phe Arg
290 295 300
Val Ala Leu Pro Gly Val Arg Lys Asp Phe Cys Gln Phe Asn Cys Glu
305 310 315 320
Ile Glu Ser Asn Gly Lys Ile His Leu Gln Gly Val Thr Ser Gly Gly
325 330 335
Asn Pro Ile Arg Lys Arg Ser Arg Val Phe Gln Met Lys Leu Gln Gln
340 345 350
Leu Cys Pro Pro Gly Pro Phe Thr Leu Ser Phe Ser Leu Pro Gly Pro
355 360 365
Val Asp Pro Arg Leu Phe Ala Pro Asn Phe Gly Pro Asp Gly Ile Phe
370 375 380
Glu Gly Val Ile Ile Lys Asp Glu
385 390
Claims (7)
1. GenePpHSP20-like1Application of the gene in promoting plant organs, such as arabidopsis leaves, arabidopsis calyx or peach pulp, to generate red colorPpHSP20-like1The nucleotide sequence of (2) is SEQ ID NO.1, the genePpHSP20-like1Overexpression in plants.
2. A gene comprising the gene of claim 1PpHSP20-like1Application of expression vector of (2) in promoting plant organs to generate red color, wherein the plant organs are arabidopsis leaves, arabidopsis calyx or peach pulp, and the genes arePpHSP20- like1The nucleotide sequence of (2) is SEQ ID NO.1, the genePpHSP20-like1Overexpression in plants.
3. The use according to claim 2, wherein the construction of the expression vector comprises the steps of:
the cDNA of red peach variety pheasant red is used as a template, primers are designed and amplified to obtainPpHSP20-like1The full-length fragment of the gene open reading frame is connected to pENTR/D-TOPO vector after PCR product purification, and the constructed plasmid is named pENTR/D-TOPO:PpHSP20-like1the method comprises the steps of carrying out a first treatment on the surface of the L by GATEWAY methodThe enzyme R will pENTR/D-TOPO: like1PpHSP20-like1Ligation to R05 vector, constructed plasmid was designated R05:PpHSP20-like1。
4. the use according to claim 3, wherein the primers are:
P1: 5’- CACCATGGAAGATGACCCTCCTAAAATGTCCGTC -3’，
P2: 5’- CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’。
5. a gene comprising the gene of claim 1PpHSP20-like1Or the use of the host bacterium of the expression vector of claim 2 for promoting the production of red color by plant organs, wherein the plant organs are arabidopsis leaves, arabidopsis calyx or peach pulp, and the genesPpHSP20-like1The nucleotide sequence of (2) is SEQ ID NO.1, the genePpHSP20-like1Overexpression in plants.
6. The use according to claim 5, wherein the host strain is an agrobacterium GV3101 starting strain.
7. GenePpHSP20-like1Or contains a genePpHSP20-like1Use of an expression vector of (2) in the production of transgenic peach with improved red flesh colour of peach fruit, said genePpHSP20-like1The nucleotide sequence of (2) is SEQ ID NO.1, the genePpHSP20-like1Overexpression in peach.
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