Sweet potato IbPSST2 gene and application thereof in promoting synthesis of plant anthocyanin

Abstract

The invention discloses a sweet potato IbPSST2 gene and application of the sweet potato IbPSST2 gene in promoting synthesis of plant anthocyanin, and belongs to the technical field of gene engineering. The nucleotide sequence of the IbPSST2 gene of the sweet potato disclosed by the invention is as shown in SEQ ID NO. 1. The IbPSST2 gene has a remarkable function in the aspect of regulating and controlling the synthesis of plant anthocyanin, and can be applied to the aspect of improving the quality of sweet potatoes.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically to a sweet potato IbPSST2 gene and an application thereof in promoting the synthesis of anthocyanins in plants. Background Art

[0002] Sweet potato (Ipomoea batatas L.) belongs to the genus Ipomoea of ​​the family Convolvulaceae and is the eighth largest food crop in the world. my country has always been the world's largest producer of sweet potatoes. Both the above-ground and underground parts of sweet potatoes are edible. Among them, the sweet potatoes with the above-ground stem tips as the edible part are called vegetable sweet potatoes, which are a kind of special sweet potatoes. The color variation of sweet potato stem tips is rich, including dark green, light green, yellow-green, green with purple edges, light purple, purple and other types. Generally speaking, they can be divided into two categories: purple stem tips and non-purple stem tips. The purple stem tips rich in anthocyanins have extremely high nutritional value and multiple health functions such as anti-aging and anti-tumor, and are important quality traits of vegetable sweet potatoes. At present, the stem tips of the main varieties of vegetable sweet potatoes are mostly green, and purple types are rarely seen. In addition, the genetic basis and regulatory mechanism of the formation of purple stem tips of sweet potatoes are still unclear, which seriously hinders the variety selection and genetic improvement of purple stem tips for vegetable sweet potatoes.

[0003] Anthocyanins are the material basis for the formation of purple stem tips in sweet potatoes. The anthocyanin biosynthesis pathway is highly conserved in plants, and a series of key enzyme genes in its synthesis pathway have been isolated and identified. The biosynthesis of anthocyanins requires the coordinated expression of multiple structural genes, and transcription factors are responsible for the precise spatiotemporal expression regulation of these structural genes. Studies have shown that the MBW (MYB-bHLH-WD40) ternary complex formed by transcription factors MYB, bHLH and WD40 affects the synthesis and accumulation of anthocyanins by activating or inhibiting the transcription of downstream structural genes; among them, MYB / bHLH is responsible for binding to the promoter of downstream genes, and WD40 is responsible for stabilizing the ternary complex. In particular, MYB transcription factors can be regulated at the transcriptional or post-transcriptional level by external environmental conditions and regulatory genes, affecting the accumulation of plant anthocyanins. For example, under light conditions, Arabidopsis bZIP transcription factor HY5 promotes the accumulation of anthocyanins by activating the transcription of AtMYB12 and AtMYB111 genes. Apple MdHY5 can bind to the E-box and G-box motifs of the MdMYB10 promoter to promote anthocyanin synthesis in fruits, while MdBBX20 promotes the binding of MdHY5 to the MdMYB1 promoter by forming a protein complex with MdHY5, thereby promoting the synthesis of anthocyanins in apples. Pear PpHY5 can enhance the expression of the G-box motif of the PpMYB10 promoter by binding to it, thereby promoting the synthesis of anthocyanins in pear peels; PpBBX16 and PpBBX18 can bind to the PpMYB10 promoter by interacting with PpHY5 to activate its transcription, thereby inducing the synthesis of anthocyanins in pear peels. However, in the sweet potato stem apex, the key genes regulating anthocyanin synthesis have not yet been identified.

[0004] Therefore, providing the sweet potato IbPSST2 gene and its application in promoting plant anthocyanin synthesis is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a sweet potato IbPSST2 gene and its application in promoting plant anthocyanin synthesis.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The nucleotide sequence of the sweet potato IbPSST2 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the sweet potato IbPSST2 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the sweet potato IbPSST2 gene is used to promote the synthesis of plant anthocyanins.

[0010] Furthermore, a recombinant expression vector pGreenII62-SK::IbPSST2 contains the sweet potato IbPSST2 gene.

[0011] Furthermore, a recombinant bacterium I contains the recombinant expression vector pGreenII62-SK::IbPSST2.

[0012] Furthermore, the sweet potato IbPSST2 gene, the recombinant expression vector pGreenII62-SK::IbPSST2 or the recombinant bacteria I are used to promote the accumulation of anthocyanins in tobacco leaves.

[0013] Furthermore, a recombinant expression vector pCAMV35s::DsRed-IbPSST2 contains the sweet potato IbPSST2 gene.

[0014] Furthermore, a recombinant bacterium II contains the recombinant expression vector pCAMV35s::DsRed-IbPSST2.

[0015] Furthermore, the sweet potato IbPSST2 gene, the recombinant expression vector pCAMV35s::DsRed-IbPSST2 or the recombinant bacteria II are used to promote the accumulation of anthocyanins in the sweet potato root system.

[0016] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses the sweet potato IbPSST2 gene and its application in promoting the synthesis of anthocyanins in plants, obtains the sweet potato gene IbPSST2 that promotes the synthesis of anthocyanins in sweet potato stem tips, and constructs its coding region into an overexpression vector. After the vector is transferred into tobacco and sweet potato respectively through transient expression and stem segment injection, the IbPSST2 gene in the obtained transiently expressed tobacco leaves and transgenic sweet potato roots is significantly expressed compared with the control leaves and roots, and the expression tobacco leaves and transgenic sweet potato roots are purple. The IbPSST2 gene promotes the increase of anthocyanin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 The accompanying drawing is a Manhattan plot of the IbPSST2 gene identified by genome-wide association analysis in the present invention;

[0019] Figure 2The attached drawings are scanned images of the stem tip leaves of purple stem tip and green stem tip sweet potato varieties selected by the present invention;

[0020] Among them, Figure a is a scanned image of the stem tip leaves of the sweet potato variety Yan 25 from the unexpanded leaf (NSL) to the fifth expanded leaf (SL-1 to SL-5); Figure b is a scanned image of the stem tip leaves of the sweet potato variety Xiguahong from the unexpanded leaf (NSL) to the fifth expanded leaf (SL-1 to SL-5); Figure c is a scanned image of the stem tip leaves of the sweet potato variety Australia Zibai from the unexpanded leaf (NSL) to the fifth expanded leaf (SL-1 to SL-5); Figure d is a scanned image of the stem tip leaves of the sweet potato variety Xu 32 from the unexpanded leaf (NSL) to the fifth expanded leaf (S Figure 5. Scanned images of stem apex leaves of sweet potato variety Guangcaishu No. 10 from unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 6. Scanned images of stem apex leaves of sweet potato variety Shulv No. 1 from unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 7. Scanned images of stem apex leaves of sweet potato variety Qixu 37 from unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); scale bar is 1 cm;

[0021] Figure 3 The accompanying drawings are diagrams showing anthocyanin content in the stem tips of purple and green stem tip sweet potato varieties and the expression pattern of the IbPSST2 gene in the present invention;

[0022] Among them, Figure a shows the anthocyanin content of the leaves of the sweet potato variety Yan 25 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure b shows the expression pattern of the IbPSST2 gene from the unexpanded leaves to the fifth expanded leaves of Yan 25; Figure c shows the anthocyanin content of the leaves of the sweet potato variety Xiguahong from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure d shows the expression pattern of the IbPSST2 gene from the unexpanded leaves to the fifth expanded leaves of Xiguahong; Figure e shows the anthocyanin content of the leaves of the sweet potato variety Australian Purple White from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure f shows the expression pattern of the IbPSST2 gene from the unexpanded leaves to the fifth expanded leaves of Australian Purple White; Figure g shows the anthocyanin content of the leaves of the sweet potato variety Xu 32 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5). Figure 1 shows the anthocyanin content of IbPSST2 gene in the unexpanded leaves to the fifth expanded leaves of Xu 32; Figure 2 shows the anthocyanin content of the leaves of the sweet potato variety Guangcaishu No. 10 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 3 shows the anthocyanin content of the leaves of the sweet potato variety Shulu No. 10 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 4 shows the anthocyanin content of the leaves of the sweet potato variety Qixu 37 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 5 shows the anthocyanin content of the leaves of the sweet potato variety Qixu 37 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 6 shows the anthocyanin content of the leaves of the sweet potato variety Qixu 37 from the unexpanded leaves (NSL) to the fifth expanded leaves (SL-1 to SL-5); Figure 7 shows the anthocyanin content of the leaves of the sweet potato variety Qixu 37 from the unexpanded leaves to the fifth expanded leaves;

[0023] Figure 4 The accompanying drawing is a map of the pGreenII62-SK vector of the present invention;

[0024] Figure 5 The accompanying drawing is a schematic diagram of the construction of the instantaneous infection tobacco vector of the present invention;

[0025] Figure 6 The accompanying drawings are phenotypes and detection diagrams of the effects of the IbPSST2 gene of the present invention on anthocyanin accumulation in tobacco leaves and sweet potato roots;

[0026] Figure a is a phenotypic diagram of the effect of IbPSST2 gene on anthocyanin accumulation in tobacco leaves; Figure b is the gene expression level detection results of wild type and transient overexpression of IbPSST2 gene; Figure c is the anthocyanin content determination results of wild type and transient overexpression of IbPSST2 gene in tobacco leaves; Figure d is a root system diagram of IbPSST2 gene transgenic sweet potato under natural light; Figure e is a root system diagram of IbPSST2 gene transgenic sweet potato under red excitation light, in which the shiny roots are transgenic positive roots; Figure f is a wild type root system and a wild type root system. Figure 1 is the expression level detection of IbPSST2 in the wild-type root system ck and the root system overexpressing IbPSST2; Figure g is the root longitudinal section phenotype of the wild-type root system ck (purple skin and white core); Figure h is the root longitudinal section phenotype of overexpressing IbPSST2; Figure i is the root cross-section phenotype of the wild-type root system ck (purple skin and white core); Figure j is the root cross-section phenotype of overexpressing IbPSST2; Figure k is the detection result of anthocyanin content in the wild-type root system ck and the root system overexpressing IbPSST2; the scale bars in g, h, i, and j are 100 μm;

[0027] Figure 7 The accompanying drawing is a map of the pCAMV35s::DsRed vector of the present invention;

[0028] Figure 8 The accompanying drawing is a schematic diagram of the construction of the sweet potato root genetic transformation vector of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The CDS sequence of the IbPSST2 gene is shown in SEQ ID NO.1.

[0031] ATGGTTAATTCATTATCATCTGCATGGCCTTCGCCTTCGGGATTGATGAGAAAAGGTGCATGGACCGAAGAAGAAGATAATCTTTTGAGGAAGTGCATTCAGAAGTATGGGGAAGGGAAATGGCATCTAGTTCCCCTTAGAGCTGGGTTAAACAGGTGCAGAAAAAGTTGTAGATTGAGATGGTTGAACTATCTTCGTCCTGATATCAAACGAGGAGACTTCAGTGTGGATGAAGTTGATCTCATTATGCGCCTCCATCGACTCTTAGGCAACAGGTGGTCGCTTATTGCCGGCAGAATCCCGGGAAGAACAGCAAACGATGTGAAGAATTACTGGAACACCCATATTCAGAAGAAGGTATTTGCCATGGCTGCTGCTTCATCTAGGATGCAAGACAATTGGAAGGGCAAAGCCCCAGAAATCAGTAAAAACACCGTCGTTAAGCCTCAACCTCGGAGATTCTTAAACACCTCATCATTATCTCGGACACCAATGACCGGAAAAGCCACCGCCGTTACCAATTACGCTCAAATCCAAGCACACACGTTACCGCAGCGGGAAACAACAACAACATCGGACTTGGTAATGGAAAATGTTCAAAAAAACGACACAATCGCGAGTTTTCCGTCAGAGTTAGAAACAACAACGTTTGACGACAGAGTCCAGTGGTGGGAGGAATTGCTCTTTGACAAGGAACTCAACGATGAAGGAACCGCGTGCATGCACGAAGGTCAAATCGGTTGGTCTCACTTGCCAACTGATAT TGACCTTTTGGAACTTCTAAGCTAA ; SEQ ID NO.1.

[0032] The amino acid sequence of the IbPSST2 gene is shown in SEQ ID NO.2.

[0033] MVNSLSSAWPSPSGLMRKGAWTEEEDNLLRKCIQKYGEGKWHLVPLRAGLNRCRKSCRLRWLNYLRPDIKRGDFSVDEVDLIMRLHRLLGNRWSLIAGRIPGRTANDVKNYWNTHIQKKVFAMAAASSRMQDN SEQ ID NO.2.

[0034] Example 1 Ib Localization of PSST2 Gene

[0035] To obtain relevant SNP sites, relevant resequencing data were downloaded from public databases, and Ipomoeatrifida was used as the reference genome. BWA software was used for comparison, and Samtools software was used to identify SNPs in the population. SNP sites were then filtered (MAF>5%, missing rate<30%), and 1999880 SNPs sites were finally obtained. The phenotypes were graded into purple and non-purple. After determining the color trait classification, EMMAX software was used for association analysis. The kinship matrix between individuals was automatically generated by the software. Bonferroni was then used to test the significance of the association between markers and expression traits in GWAS, and the significant threshold obtained was 5.0*10 -7 LD analysis was performed on the significantly associated SNPs to determine the candidate intervals. 2 SNPs with a correlation greater than 0.1 were merged into QTLs, and finally SNP loci with at least 3 significant associations were retained as candidate QTLs. -7 ) was used as the potential significant threshold to detect significant loci, and it was found that a QTL locus was significantly associated with stem tip color. The physical location of the QTL was (chr12:1879095-3743995) ( Figure 1 ).

[0036] After initially obtaining the candidate QTL segment, the annotated genes in the localized region were retrieved from the Ipomoea trifida genome for functional annotation, and finally 18 genes were found to be related to anthocyanin synthesis in Ipomoea trifida (Table 1). At the same time, combined with the expression patterns of these candidate genes, genes specifically expressed in the stem apex were screened, and finally the candidate gene itf12g04080 was located in Ipomoea trifida.

[0037] Table 1 Functional annotation of candidate genes

[0038]

[0039]

[0040] In order to further confirm the candidate genes controlling the color of sweet potato stem tips, the present invention selected four purple stem tip sweet potatoes, Australia Purple White, Watermelon Red, Xushu 32, and Yan 25, and three green stem tip sweet potatoes, Guangcaishu No. 10, Shulu No. 1, and Qixu 37, to analyze the expression pattern of the candidate genes. First, six leaves of these varieties were sampled from the unexpanded leaf to the fifth expanded leaf, and then anthocyanins in each leaf were extracted using an organic solvent extraction method.

[0041] The extraction of anthocyanins from sweet potato leaves adopts organic solvent extraction method, and the specific operation is as follows:

[0042] (1) Prepare a 1% hydrochloric acid methanol solution by measuring 99 mL of methanol (Xilong 67-56-1) and 1 mL of hydrochloric acid (Guoyao 7647-01-0) using a measuring cylinder, and then mix the two in a glass bottle.

[0043] (2) Three samples of fresh plant tissue (tobacco leaves) were mixed as one biological replicate. After successful mixing, the samples were quickly frozen in liquid nitrogen. Three biological replicates were required for each sample.

[0044] (3) Place the sample to be tested into a grinding machine (Shanghai Jingxin Tissuelyser-64), adjust the vibration frequency to 50 Hz, and the grinding time to 120 seconds to fully grind the tissue. Then weigh 0.2 g of the powdered sample and pour it into a 2 mL centrifuge tube.

[0045] (4) Add 1 mL of 1% hydrochloric acid-methanol to the centrifuge tube and store it at 4°C in the dark overnight. Shake the tube to ensure sufficient extraction. Set a blank control with only 1 mL of 1% hydrochloric acid-methanol and no plant tissue sample added.

[0046] (5) Place the blank control and plant samples in (4) in a centrifuge (Eppendorf SHPZ5424R), set the temperature to 4°C, the speed to 12,000 rpm, and centrifuge for 5 min. After centrifugation, pipette 500 μL of supernatant into a new centrifuge tube, add 300 μL of chloroform (Guoyao 8301-54-5) and 500 μL of ddH2O, and mix well to remove chlorophyll.

[0047] (6) Place the blank control and plant samples in (5) in a centrifuge, set the temperature to 4°C, the speed to 12000 rpm, and centrifuge for 5 min. After centrifugation, take 200 μL of the supernatant (aqueous phase) for anthocyanin content determination.

[0048] (7) The absorbance of the sample and blank control was measured at 530 nm and 657 nm using an ELISA reader (Thermo, Varioskan TMLUX).

[0049] (8) The relative content of anthocyanins was calculated using the following formula: Anthocyanin content = (OD 530 -0.25×OD 657 ) / m; where OD 530 OD is the optical density of anthocyanin at 530 nm wavelength; 657 It is the optical density of chlorophyll at a wavelength of 657 nm (Note: m: sample mass, unit: g, three groups of biological replicates were set, and each group was measured 3 times).

[0050] It was found that in the sweet potato varieties with purple stem tips, the anthocyanin content showed a gradual decrease from the unexpanded leaves to the fifth expanded leaves, and the anthocyanin content in the leaves after the third or fourth leaf decreased significantly ( Figure 2 a, b, c, d); in green shoot tip seedlings, the anthocyanin content in each part was low and there was no obvious change trend ( Figure 2 e,f,g).

[0051] The expression pattern of Itf12g04080 in the stem tips of the eight sweet potatoes was further analyzed by qRT-PCR. The specific steps are as follows:

[0052] 1) The sweet potato stem tip RNA was extracted using the polysaccharide and polyphenol plant total RNA extraction reagent (TIANGEN, DP441), and the specific extraction steps were the same as the product instructions. The concentration of the extracted RNA was measured using a Nanophotometer N60 Touch micro-spectrophotometer, and the integrity of the RNA was detected by agarose gel electrophoresis.

[0053] 2) cDNA synthesis. The qualified RNA extracted in step (1) was reverse transcribed using the reverse transcription kit HiScriptIIQRTSuperMix forqPCR (Vazyme, R222), and the obtained cDNA was used for real-time fluorescence quantitative (qRT-PCR) analysis of the IbPSST2 gene. The reverse transcription system and reaction conditions were the same as those in the product manual. All cDNAs obtained by reverse transcription were stored at -80°C.

[0054] 3) qRT-PCR experiment. Gene-specific qRT-PCR primers IbPSST2_RT-F / R were designed based on the full-length CDS sequence of IbPSST2. qRT-PCR experiments were performed using cDNA as a template.

[0055] The sequence of primer IbPSST2_RT-F / R is as follows:

[0056] IbPSST2_RT-F: 5'-CCTTCGCCTTCGGGATTGAT-3'; SEQ ID NO.3;

[0057] IbPSST2_RT-R: 5'-GATGCCATTTCCCTTCCCCA-3'; SEQ ID NO. 4.

[0058] qRT-PCR reaction system: total volume 15 μL, including 1.5 μL cDNA template, 0.75 μL forward and reverse primers, 7.5 μL 2×chamQ SYBR MasterMix (Vazyme, Q311), 4.5 μL ddH2O (primers synthesized at General Biosystems (Anhui) Co., Ltd.). qRT-PCR reaction procedure: 95°C pre-denaturation for 30 seconds, 95°C for 10 seconds, 60°C for 30 seconds for 40 cycles, and preparation of melting curve (qRT-PCR reaction instrument is BIO-RAD CFX Connect TM Real-Time System). After obtaining the data, 2 -ΔΔCt The relative expression of genes was calculated.

[0059] The results showed that the expression pattern of Itf12g04080 was close to the content changes of anthocyanins in the four varieties, especially the expression pattern of Itf12g04080 was basically consistent with the content change trend of anthocyanins in Australian Purple White and Xushu 32 ( Figure 3 ah). In green stem tip sweet potatoes, the expression pattern of Itf12g04080 was inconsistent with the change trend of anthocyanins in the stem tip of sweet potatoes ( Figure 3 Based on this, the applicant believed that Itf12g04080 was the major gene regulating the color of sweet potato stem tips and named Itf12g04080 IbPSST2 (Purple Sweetpotato Stem Tips 2).

[0060] Example 2 Ib Cloning of PSST2 gene

[0061] 1) RNA extraction and cDNA synthesis

[0062] (1) Sweet potato stem tip RNA was extracted using polysaccharide and polyphenol plant total RNA extraction reagent (TIANGEN, DP441), and the specific extraction steps were the same as the product instructions. The concentration of the extracted RNA was measured using a Nanophotometer N60 Touch micro-spectrophotometer, and the integrity of the RNA was detected by agarose gel electrophoresis.

[0063] (2) cDNA synthesis. The qualified RNA extracted in step (1) was transcribed using the reverse transcription kit HiScript III1 st Strand cDNA Synthesis Kit (+gDNAwiper) (Vazyme, R312) was used for reverse transcription, and the obtained cDNA was used for CDS amplification of IbPSST2 gene. The reverse transcription system and reaction conditions were the same as those in the product manual. All cDNAs obtained by reverse transcription were stored at -80°C.

[0064] In the Sweetpotato Genomic Source (http: / / sweepotato.plantbiology.msu.edu / ) website, the genome sequence of the wild relative of sweet potato, Ipomoea trifida, was searched with the ID Itf12g04080, and the nucleotide sequence of the IbPSST2 gene was further obtained. PCR primers IbPSST2-F / R were designed to amplify the full length of the CDS of the gene, and PCR amplification was performed using the purple stem tip sweet potato Xu 32 cDNA as a template.

[0065] The sequence of primer IbPSST2-F / R is as follows:

[0066] IbPSST2-F: 5'-ATGGTTAATTCATTATCATCTGCATGG-3'; SEQ ID NO.5;

[0067] IbPSST2-R: 5'-TTAGCTTAGAAGTTCCAAAAGGTCA-3'; SEQ ID NO. 6.

[0068] The total volume of the PCR reaction system was 20 μL: 2× PrimeSTAR Max Premix (PrimeSTAR Max DNA Polymerase, TAKARA) 12.5 μL, forward and reverse primers 0.5 μL each, cDNA template (about 100 ng / μL) 1.0 μL, ddH2O was added to 20 μL (primers were synthesized at General Biosystems (Anhui) Co., Ltd.). The PCR reaction program was: denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 60 seconds, 35 cycles, and storage at 4°C after the reaction. The PCR product was detected by gel electrophoresis: the PCR product was electrophoresed with 1% agarose with EB (ethidium bromide) at a voltage of 100-120V for 30 minutes, and the final result was imaged on a gel imaging system (Bio-RAD ChemiDOC TM XR). The results showed that the CDS of the IbPSST2 gene was 789bp. The gel block where the target fragment was located was cut off and recovered using the agarose gel DNA recovery kit (TIANgel MidiPurification Kit) (the operating steps are the same as the product manual). The recovered fragment was cloned into the full-length gold T vector (pEASY-Blunt Simple Cloning Kit) (the connection steps are the same as the product manual). The obtained clone was positively identified by conventional colony PCR (2×Taq MasterMix) detection, and the positive colony was extracted with plasmids (Tiangen Plasmid Mini-Extraction Kit). All operating steps are the same as the product manual. The positive plasmid was sent to Universal Biosystems (Anhui) Co., Ltd. for sequence determination. The CDS sequence of the IbPSST2 gene finally obtained is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0069] Example 3 IbPSST2 gene is used to promote the accumulation of anthocyanins in tobacco leaves

[0070] The diploid wild-type Nicotiana benthamiana used in the present invention is stored in Jiangsu Normal University. The specific planting method is as follows: place sterilized nutrient soil in a pot and fully absorb water, cover with plastic wrap after sowing and leave pores, place in an incubator or greenhouse, and the culture conditions are 20-24°C, relative humidity 50%, and light 16h / dark 8h. After the seedlings grow two true leaves, transplant them to separate flower pots (potted according to the ratio of nutrient soil, vermiculite, and perlite of 5:1:1) for cultivation, and the culture conditions are 20-24°C, relative humidity 50%, and light 16h / dark 8h. During this period, water and control pests and diseases from time to time, and after the tobacco grows to the six to eight leaf stage (about 4 weeks), subsequent transient infection experiments can be carried out.

[0071] 1) According to the pGreenII62-SK vector (map see Figure 4 ) sequence, designed the IbPSST2 gene primer IbPSST2_62sk-F / R containing the homologous recombination linker for vector construction, and used the T vector fusion plasmid containing the IbPSST2 gene as a template, and selected high-fidelity DNA polymerase (PrimeSTAR Max DNA Polymerase, TAKARA) for PCR amplification.

[0072] The sequence of primer IbPSST2_62sk-F / R is as follows:

[0073] IbPSST2_62sk-F:

[0074] 5'- gccgctctagaactagtggatcc ATGGTTAATTCATTATCATCTGCATGG-3'; SEQ ID NO.7;

[0075] IbPSST2_62sk-R:

[0076] 5'- ttggtaccgggccccccctcgag TTAGCTTAGAAGTTCCAAAAGGTCA-3'; SEQ ID NO.8;

[0077] The underlined lowercase letters in the primers are linker sequences.

[0078] 2) The amplified product was subjected to agarose gel electrophoresis, gel excision recovery, fragment recovery, and homologous recombination with the linearized pGreenII62-SK vector ( Figure 5). The pGreenII62-SK vector was linearized using restriction endonucleases Bam HI (NEB, R0136V) and Xho I (NEB, R0146V). The specific operation was carried out according to the product manual. The enzyme digestion system was: 3-5μg of pGreenII62-SK plasmid, 5μL of 10×NEB buffer, 2μL of each restriction endonuclease, and ddH2O was added to 50μL; the enzyme digestion reaction procedure was: 37°C, 12 hours. The obtained enzyme digestion products were detected by agarose gel electrophoresis, gel excision recovery and fragment recovery to obtain the linearized pGreenII62-SK vector for homologous recombination. Homologous recombination (ClonExpress II One Step Cloning Kit, Vazyme, C112) was carried out strictly according to the product manual, wherein the reaction system was: 70 ng of linearized vector, about 32 ng of insert fragment, 4 μL of 5×CE II buffer, 2 μL of homologous recombination enzyme, and ddH2O was added to 20 μL; the reaction procedure was: 37°C, 30 minutes. The obtained ligation product was transformed into Escherichia coli DH5α (TOLOBIO, CC96102) by heat shock transformation. After the transformation, the plate was inverted and cultured overnight at 37°C until a single colony grew. The single colony was identified by PCR using gene-specific primers IbPSST2-F / R, and the positive single colony obtained by identification was shaken and plasmid was extracted. The plasmid was sequenced, and the plasmid with correct sequencing was transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19) (TOLOBIO, CC96303) by heat shock.

[0079] 3) The Agrobacterium containing the fusion plasmid was streaked on YEP (Qingdao Haibo HB8474) solid medium (containing 50 mg / L Kan and 25 mg / L rifampicin), inverted and placed in a 28°C incubator for activation for 48 hours, and the grown single colonies were shaken in YEB liquid medium (containing 50 mg / L Kan and 25 mg / L rifampicin) (positive single clone Agrobacterium was picked and placed in 2 mL YEP liquid medium (containing 50 mg / L Kan and 25 mg / L rifampicin), and cultured overnight at 28°C and 250 r / min (OD 600 Value about 1.0)), shake vigorously (shake vigorously at a volume ratio of 1:100, i.e., take 500 μL of the small shaking bacterial solution and inoculate it into 50 mL of fresh YEP liquid medium (containing 50 mg / L Kan and 25 mg / L rifampicin), place it at 28°C, shake at 250 r / min for about 12 h, until OD 600The suspension treatment method is as follows: prepare IM suspension (10mM MgCl2 (Sinopharm 10012818), 10mM MES (Solabo M8010-10) to 500mL of sterile water, adjust the pH to 5.6, and finally add 100μM acetosyringone (Solabo M8110-1)); centrifuge the bacteria obtained by shaking at 5000r / min for 5min, discard the supernatant, resuspend the bacteria with 10mL IM suspension, centrifuge again and retain the bacterial pellet; dilute the bacteria with IM suspension to OD 600 In the range of 0.6-0.8, incubate in a 28°C incubator for 2-3 hours after suspension.

[0080] 4) Transient infection experiment: Select diploid wild-type Nicotiana benthamiana leaves at the 6-8 leaf stage and in good growth condition for injection. The infection method is: inject the bacterial solution containing pGreenII62-SK::IbPSST2 plasmid (OD 600 =0.8) was injected into one half of the leaves; the other half of the leaves were injected with pGreenII62-SK empty vector solution (OD 600 =0.8). The injection method is: first use a needle to poke a hole at the place where injection is required, and then use a syringe without a needle to inject the bacterial solution into the leaf until half of the leaf is filled. After infection, the tobacco is placed in the dark at 25°C for 12 hours, and then cultured under 16h light / 8h dark conditions for about a week to observe the phenotype.

[0081] 5) For leaves showing phenotypes (half of the tobacco leaves injected with pGreenII62-SK::IbPSST2 bacterial solution turned purple, and half of the tobacco leaves injected with pGreenII62-SK empty vector remained green) Figure 6 a), using a 1 cm diameter puncher to take samples. RNA was extracted from a portion of the samples, and the expression of IbPSST2 was detected by qRT-PCR ( Figure 6 b), and part of the samples were tested for anthocyanin content ( Figure 6 c).

[0082] Example 4 IbPSST2 gene is used to promote the accumulation of anthocyanins in sweet potato roots

[0083] 1) The transgenic sweet potato receptor material Xu Zishu No. 8 was stored in Jiangsu Normal University. The tubers of Xu Zishu No. 8 were buried in nutrient soil and placed at room temperature for germination. After one week, when the seedlings grew out, they were moved to a plant culture room at a temperature of 30° C. and a relative humidity of 50%, and cultured for about 2-4 weeks at a light of 16 hours / darkness of 8 hours, until the aboveground length was about 30 cm.

[0084] 2) pCAMV35s::DsRed vector references: Yu, Y., Xuan, Y., Bian, cellularNa(+)homeostasis and salttoleranceby activatingplasmamembrane Na(+) / H(+)antiport activity in sweetpotato roots.Hortic Res 7,131.

[0085] According to the pCAMV35s::DsRed vector (map see Figure 7 ) sequence, designed IbPSST2 gene primers IbPSST2_TNRT-F / R containing homologous recombination linkers for vector construction, and used T vector fusion plasmid containing IbPSST2 gene as template, and selected high-fidelity DNA polymerase for PCR amplification.

[0086] The sequence of primer IbPSST2_TNRT-F / R is as follows:

[0087] IbPSST2_TNRT-F:

[0088] 5'- cttcactgttgatacacgcgt ATGGTTAATTCATTATCATCTGCATGG-3'; SEQ ID NO.9;

[0089] IbPSST2_TNRT-R:

[0090] 5'- tgttgattcagaattgtcgac TTAGCTTAGAAGTTCCAAAAGGTCA-3'; SEQ ID NO. 10.

[0091] The underlined lowercase letters in the primers are linker sequences.

[0092] 3) The amplified product was subjected to agarose gel electrophoresis, gel excision recovery, fragment recovery, and homologous recombination with the linearized pCAMV35s::DsRed vector ( Figure 8). The pCAMV35s::DsRed vector was linearized using restriction endonucleases Mlu I (NEB, R3198V) and Sal I (NEB, R0138V). The specific operation was carried out according to the product manual. The enzyme digestion system was: pCAMV35s::DsRed plasmid 3-5μg, 10×NEB buffer 5μL, restriction endonuclease 2μL each, ddH2O filled to 50μL; the enzyme digestion reaction program was: 37℃, 12 hours. The above-obtained enzyme digestion products were detected by agarose gel electrophoresis, gel excision recovery and fragment recovery to obtain the linearized pCAMV35s::DsRed vector for homologous recombination. Homologous recombination was carried out strictly according to the product manual, wherein the reaction system was: linearized vector 192ng, insert fragment about 32ng, 5×CE II buffer 4μL, homologous recombination enzyme 2μL, ddH2O filled to 20μL; the reaction program was: 37℃, 30 minutes. The obtained ligation product was transformed into Escherichia coli DH5α by heat shock transformation. After the transformation, the plate was inverted and cultured overnight at 37°C until a single colony grew. The single colony was identified by PCR using gene-specific primers IbPSST2-F / R. The positive single colony obtained by identification was shaken and the plasmid pCAMV35s::DsRed-IbPSST2 was extracted. The plasmid was sequenced, and the correctly sequenced plasmid was transformed into Agrobacterium rhizogenes K599 (Weidi Biotechnology, AC1080) by heat shock.

[0093] 4) The rhizogenes containing the fusion plasmid were streaked on TY solid medium (yeast powder (Solarbio, LP0021) 3 g / L, peptone (Solarbio, LP0042) 5 g / L, CaCl2 (Shanghai Guoyao, 10005861) 10 mmol / L, agarose (Solarbio, NO.A8200) 15 g / L, containing 50 mg / L Kan), inverted and placed in a 28°C incubator for activation for 48 hours, and the grown single colonies were shaken in TY liquid medium (containing 50 mg / L Kan) (positive single clone Agrobacterium was picked and placed in 2 mL TY liquid medium (containing 50 mg / L Kan), and cultured overnight at 28°C and 250 r / min (OD 600 value of about 1.0)), shake vigorously (shake vigorously at a volume ratio of 1:100, i.e., take 500 μL of the small shaking bacterial solution and inoculate it into 50 mL of fresh TY liquid medium (containing 50 mg / L Kan), place it at 28°C, shake at 250 r / min for about 12 h, until OD 600 The value reaches 0.8 to 1.0) and then the suspension treatment is carried out to prepare for instant infection. The suspension treatment method is the same as step 3) of Example 3.

[0094] 5) Cut the seedlings about 15 cm below the stem tip of Xu Zishu No. 8 (2-3 leaves and 2-3 internodes need to be retained), and use a 1 mL syringe to absorb the incubated bacterial solution with fusion plasmid (OD 600 =0.8) and bacterial solution with empty vector (OD 600 =0.8), respectively, injected into the internodes of the seedlings. After the injection, the seedlings were left at room temperature for 1 hour, then inserted into nutrient soil, and cultured at room temperature under 16h light / 8h dark conditions for about one month.

[0095] 6) Dig the injected seedlings out of the soil, rinse the soil off the roots with tap water, and observe whether the roots emit fluorescence under red fluorescence. The roots that emit fluorescence are transgenic positive roots ( Figure 6 d, e). The root systems of wild-type root system CK (purple skin and white core) and transgenic Yangxin root system were cut longitudinally and cross-sectionally, and the accumulation of anthocyanins in the roots was observed under an optical microscope (Olympus CX43). Figure 6 gj).

[0096] 7) RNA was extracted from transgenic positive roots, and the expression level of IbPSST2 was detected by qRT-PCR ( Figure 6 f), and part of the samples were tested for anthocyanin content ( Figure 6 k).

[0097] The IbPSST2 gene was overexpressed in tobacco and sweet potato roots, respectively, and it was found that IbPSST2 could significantly promote the accumulation of anthocyanins in tobacco leaves and sweet potato roots compared with the control. This gene can be used for genetic improvement of sweet potato stem tip color.

[0098] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Sweet potato IbPSST2 gene, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the sweet potato IbPSST2 gene according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the sweet potato IbPSST2 gene according to claim 1 in promoting plant anthocyanin synthesis.

4. A recombinant expression vector pGreenII62-SK::IbPSST2, characterized in that: Contains the sweet potato IbPSST2 gene according to claim 1.

5. A recombinant bacterium I, characterized in that Contains the recombinant expression vector pGreenII62-SK::IbPSST2 according to claim 3.

6. Use of the sweet potato IbPSST2 gene according to claim 1, the recombinant expression vector pGreenII62-SK::IbPSST2 according to claim 4 or the recombinant bacteria I according to claim 5 in promoting the accumulation of anthocyanins in tobacco leaves.

7. A recombinant expression vector pCAMV35s::DsRed-IbPSST2, characterized in that: Contains the sweet potato IbPSST2 gene according to claim 1.

8. A recombinant bacterium II, characterized in that: Contains the recombinant expression vector pCAMV35s::DsRed-IbPSST2 according to claim 7.

9. Use of the sweet potato IbPSST2 gene according to claim 1, the recombinant expression vector pCAMV35s::DsRed-IbPSST2 according to claim 7 or the recombinant bacteria II according to claim 8 in promoting the accumulation of anthocyanins in sweet potato roots.

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