Psabf2 gene and snp marker related to Siberian apricot flowering period regulation and application thereof

Abstract

The application discloses a PsABF2 gene and SNP markers related to Siberian apricot flowering period regulation and application thereof. The coding sequence of the PsABF2 gene is shown in SEQ ID NO. 1; the PsABF2 gene is used for regulating the flowering period of Siberian apricot. Based on the gene, the application further provides a protein encoded by the PsABF2, a method for delaying the flowering period of plants by using the PsABF2 gene and four SNP markers linked to the PsABF2 gene, and the SNP markers can be used for identifying the flowering period trait of Siberian apricot, predicting the flowering period and molecular marker assisted breeding. The application provides important tools and technical support for the genetic breeding and flowering period management of Siberian apricot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering. Specifically, it is a PsABF2 gene and SNP marker related to Siberian apricot flowering regulation and application thereof. BACKGROUND

[0002] Siberian apricot (Prunus sibirica) is a plant of the genus Armeniaca in the subfamily Prunoideae of Rosaceae, which is an important ecological and economic tree species in China. Siberian apricot has strong ecological adaptability and characteristics such as wind and sand prevention, water and soil conservation, cold resistance, drought resistance, and poor soil tolerance. It has irreplaceable ecological value in ecologically fragile areas such as drought and semi-arid regions, and has become an important afforestation tree species for sand fixation, soil conservation, water conservation and desertification control in the three northern regions of China. Siberian apricot not only plays an important role in ecological function, but is also widely used in food, medical and industrial fields. The apricot kernel contains rich nutrients, with a protein content of about 30% and an oil content of 50%-60%. It can be used for oil extraction or processed into food or beverage, and has high economic value. Due to the short dormancy period and early flowering of Siberian apricot, it is easily affected by early spring "spring frost" and seriously restricts the yield and quality of Siberian apricot fruit.

[0003] Flower bud dormancy is one of the important links in the growth cycle of plants, which directly affects its future growth and development. It can be used as an important basis for judging plant cold resistance and predicting geographical distribution, and is also the theoretical basis for adjusting the dormancy period of woody crops and promoting the planting of deciduous woody plants. Flower bud dormancy is a complex life phenomenon regulated by multiple factors and multiple genes. Its formation and release are influenced and regulated by external environment such as temperature, light and water, as well as internal changes such as genes and hormones. In this process, the content of hormones and the expression of genes also change to adapt to the changes. Abscisic acid (ABA) is considered to be an important substance that responds to changes in the external environment to regulate the change of flower bud dormancy state, and promotes the deepening of flower bud dormancy. Its content changes in response to environmental changes, and regulates the initiation, development and termination of flower bud dormancy. During dormancy, the development of flower organs also deepens with the dormancy process, preparing for flowering. Usually the content of ABA in flower bud increases in autumn, thereby inducing flower bud to enter dormancy, and then the content of ABA decreases until the flower bud germinates. Related studies have shown that the content of ABA has an inhibitory effect on the release of flower bud dormancy. When the content of ABA is high during flower bud dormancy, the release time of flower bud dormancy will be late, thereby delaying the flowering period. Therefore, increasing the content of ABA during flower bud dormancy of Siberian apricot can delay the flowering period of Siberian apricot, thereby preventing it from being affected by frost, and promoting the healthy and stable development of Siberian apricot industry. SUMMARY

[0004] To this end, the technical problem to be solved by the present application is to provide a PsABF2 gene related to Siberian apricot flowering regulation and a SNP marker and application thereof, to provide a theoretical and methodological basis for analyzing the molecular regulation mechanism of Siberian apricot flower bud dormancy and exploring the molecular mechanism of ABA signal pathway related genes on Siberian apricot flower bud dormancy release and flowering regulation, and to provide a molecular biology tool for regulating Siberian apricot flowering.

[0005] To solve the above technical problems, the present application provides the following technical solutions.

[0006] The PsABF2 gene related to Siberian apricot flowering regulation, the coding sequence of the PsABF2 gene is shown as SEQ ID NO. 1; the PsABF2 gene is used for regulating the flowering of Siberian apricot.

[0007] The protein encoded by the PsABF2 gene, the PsABF2 gene is the PsABF2 gene related to Siberian apricot flowering regulation described above, the amino acid sequence of the protein encoded by the PsABF2 gene is shown as SEQ ID NO. 3, and the protein is involved in the regulation of Siberian apricot flowering.

[0008] Application of the PsABF2 gene in delaying plant flowering, the PsABF2 gene is the PsABF2 gene related to Siberian apricot flowering regulation described above; the plant is Siberian apricot and / or Arabidopsis thaliana; when the PsABF2 gene is used to delay plant flowering, the expression amount of the PsABF2 gene in the plant is increased.

[0009] The application of the PsABF2 gene in delaying plant flowering, the expression amount of the PsABF2 gene is detected by fluorescent quantitative PCR; the sequences of the primers used in the fluorescent quantitative PCR are shown as SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0010] The application of the PsABF2 gene in delaying plant flowering, when the PsABF2 gene is used to delay plant flowering, a recombinant vector is used to transform the plant to increase the expression amount of the PsABF2 gene in the plant; the recombinant vector contains a sequence shown as SEQ ID NO. 1.

[0011] The application of the PsABF2 gene in delaying plant flowering, the original vector of the recombinant vector is an overexpression vector pMDC32.

[0012] The SNP marker related to Siberian apricot flowering regulation, the SNP marker is SNP1-SNP4 linked to the PsABF2 gene related to Siberian apricot flowering regulation described above, wherein: The SNP marker related to Siberian apricot flowering regulation, the SNP marker is SNP1-SNP4 linked to the PsABF2 gene related to Siberian apricot flowering regulation described above, wherein:

[0013] SNP1 is located at 32680522 of chromosome 1 of Siberian apricot, the reference base of SNP1 is T, and the base after mutation is TG;

[0014] SNP2 is located at 32680714 of chromosome 1 of Siberian apricot, the reference base of SNP2 is A, and the base after mutation is G;

[0015] SNP3 is located at 32681144 of chromosome 1 of Siberian apricot, the reference base of SNP3 is A, and the base after mutation is T;

[0016] SNP4 is located at 32681347 of chromosome 1 of Siberian apricot, the reference base of SNP4 is G, and the base after mutation is A.

[0017] The application of the SNP marker related to the flowering time regulation of Siberian apricot is used for identifying the flowering time trait of Siberian apricot, or for predicting the early or late flowering of Siberian apricot, or for molecular marker assisted breeding of Siberian apricot.

[0018] In the application of the SNP marker related to the flowering time regulation of Siberian apricot for identifying the flowering time trait of Siberian apricot, the genotype of the to-be-tested Siberian apricot at the sites of the SNP1, SNP2, SNP3 and SNP4 is obtained from the genomic DNA of the to-be-tested Siberian apricot plant; wherein:

[0019] When the genotype of the SNP1 is T / T, the genotype of the SNP2 is A / A, the genotype of the SNP3 is A / A, and the genotype of the SNP4 is G / G in the genomic DNA of the to-be-tested Siberian apricot, the to-be-tested Siberian apricot is early-flowering Siberian apricot;

[0020] When the genotype of the SNP1 is TG / T or TG / TG, or the genotype of the SNP2 is G / G or G / A, or the genotype of the SNP3 is T / T or T / A, or the genotype of the SNP4 is A / A or A / G in the genomic DNA of the to-be-tested Siberian apricot, the to-be-tested Siberian apricot is late-flowering Siberian apricot.

[0021] The genotype of the to-be-tested Siberian apricot at the sites of the SNP1, SNP2, SNP3 and SNP4 is obtained from the genomic DNA of the to-be-tested Siberian apricot plant, that is, the base composition of these SNP markers in the to-be-tested Siberian apricot and a group of Siberian apricots is detected, and common SNP detection methods such as mutation amplification blocking system PCR (ARMS PCR), TaqMan probe, molecular beacon and sequencing method can be used in the detection.

[0022] The application obtains the genotype of the test Siberian apricot at the site of the SNP1, SNP2, SNP3 and SNP4 from the genomic DNA of the test Siberian apricot, takes the genomic DNA of the test Siberian apricot as a template to perform PCR, obtains a PCR product, and sequences the PCR product to obtain the genotype of the test Siberian apricot at the site of the SNP1, SNP2, SNP3 and SNP4.

[0023] The positions of the SNP1-SNP4 on the chromosome 1 are obtained according to the genomic sequence of the Siberian apricot F106. The sequence of the genome of the Siberian apricot F106 is based on Prunus sibirica F106 Whole Genome v1.0 Assembly & Annotation, and the relevant genomic sequence can be obtained from https: / / www.rosaceae.org / Analysis / 10254124.

[0024] The technical scheme of the present application achieves the following beneficial technical effects:

[0025] The present application discloses a Siberian apricot flowering period regulation related gene PsABF2 and SNP marker and application thereof. The PsABF2 gene is a Siberian apricot ABA transduction pathway related gene during flower bud dormancy, and plays an important role in the Siberian apricot flower bud dormancy process. Experiments prove that after the overexpression vector pMDC32-PsABF2 carrying the PsABF2 gene is transformed into Arabidopsis thaliana by means of Agrobacterium-mediated genetic transformation, the flowering period of Arabidopsis thaliana is changed, which confirms that the PsABF2 has the function of regulating the flowering period of Arabidopsis thaliana, and indirectly proves that the PsABF2 gene in the Siberian apricot has the function of regulating the flowering period of the Siberian apricot. Further, the present application obtains four SNP sites linked to the PsABF2 gene in 66 Siberian apricot plants by mining the SNP sites of PsABF2, and provides a method for judging whether the Siberian apricot is a late-flowering variety according to the four SNP sites. The Siberian apricot flower bud dormancy ABA transduction pathway regulation gene PsABF2 provided in the present application provides a new tool for Siberian apricot breeding and flower bud dormancy mechanism regulation. The PsABF2 gene and the SNP sites linked thereto can be used as a marker assisted tool in late-flowering breeding, and are used for Siberian apricot genetic background analysis and screening, and late-flowering Siberian apricot variety breeding. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The electrophoresis map of the Siberian apricot total RNA gel extracted in the embodiment of the present application;

[0027] Figure 2 Gel electrophoresis diagram of PCR amplification product of PsABF2 gene in the embodiment of the present application;

[0028] Figure 3 Measurement results of expression amount of PsABF2 gene at each stage during the flower bud dormancy period of Siberian apricot in the embodiment of the present application;

[0029] Figure 4 PCR identification results of the transgenic positive Arabidopsis thaliana into which the PsABF2 gene is transferred in the embodiment of the present application;

[0030] Figure 5 Comparison of growth conditions of wild-type Arabidopsis thaliana and the transgenic positive Arabidopsis thaliana into which the PsABF2 gene is transferred in the embodiment of the present application;

[0031] Figure 6 Statistical analysis of bolting time of the transgenic positive Arabidopsis thaliana into which the PsABF2 gene is transferred in the embodiment of the present application;

[0032] Figure 7 Statistical analysis of flowering period of the transgenic positive Arabidopsis thaliana into which the PsABF2 gene is transferred in the embodiment of the present application. DETAILED DESCRIPTION

[0033] The methods in the following examples are all conventional operation methods in the art unless otherwise specified. The materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. In the fluorescent quantitative experiment in the following examples, three repeated experiments are set, and the experimental results are averaged for analysis. Wild-type Arabidopsis thaliana is genetically transformed using Columbia wild-type Arabidopsis thaliana Columbia-0 (Col-0).

[0034] Example 1

[0035] In this embodiment, sequence analysis of PsABF2 gene related to ABA transduction pathway during the flower bud dormancy period of Siberian apricot is performed, and the cDNA sequence of PsABF2 gene is cloned through RNA extraction and reverse transcription.

[0036] The inventors have cloned and obtained PsABF2 gene related to ABA transduction pathway in the dormant flower bud of Siberian apricot through long-term research combined with bioinformatics means. The sequence of ORF (open reading frame) region of PsABF2 gene related to ABA transduction pathway during the flower bud dormancy period of Siberian apricot is shown as SEQ ID NO. 1, and the reverse complementary sequence of the sequence shown as SEQ ID NO. 1 is SEQ ID NO. 2. The sequence shown as SEQ ID NO. 1 is 1266 bp in length, encodes 421 amino acids, and the amino acid sequence encoded thereby is shown as SEQ ID NO. 3.

[0037] SEQ ID NO. 1 (PsABF2):

[0038]

[0039] SEQ ID NO. 2 (PsABF2 reverse complement):

[0040]

[0041] SEQ ID NO. 3 (amino acid sequence encoded by the sequence shown in SEQ ID NO. 1):

[0042] MGTNMNFKGFGNEPPGINPLARQSSIYSLTFEELQNTIGGSGKDFGSMNMDELLKSIWTAEETQIMAPSGGGAGGQNGLGLGGGSLQRQGSLTLPRTLSQKTVDEVWKNISKEGTGAGGSNMPQRQQTLGEMTLEEFLVKAGVVREEAQLAPKPANNGAGFFGDLSRFGNTGGNLDFEFQQTNRGVGVMGNRVSETNSQVPNQASNLPLNANGVRSNQQQQQLPQQQQIFPKQQPVTYNTSPLPMGPNAQLGSPGMRGGIMGIGDQGLNGTLVQSSGMGMVGLGAAGAVRVATGSPANQLSSDGIGKSNGTDTSSVSPVPYVFNGGFRGRKGGGPVEKVVERRQRRMIKNRESAARSRARKQAYTMELEAEVAKLKEENQELQKKQVCFVVVSHSCWKHELTDVINLQCLTLIYCSLVAAG*

[0043] The sequence of the ORF region shown in SEQ ID NO. 1 is identical to the CDS sequence (coding sequence) obtained by reverse transcription of the mRNA of PsABF2. That is, the sequence shown in SEQ ID NO. 1 is the coding sequence of the PsABF2 gene encoding protein.

[0044] Total RNA was extracted from the dormant flower buds of Siberian apricot using VazymE FastPure Universal Plant Total RNA Isolation Kit polysaccharide polyphenol plant total RNA extraction kit (Novozyme, Nanjing). The steps were carried out according to the instructions:

[0045] (1) Take 100 g of Siberian apricot flower bud tissue, grind it in liquid nitrogen, and immediately add 600 μL of BufferPSL, vortex vigorously for 30 sec, and mix the sample and lysis solution evenly.

[0046] (2) Centrifuge at 12000 rpm for 5 min, and immediately proceed to the next step.

[0047] (3) Take about 500 μL supernatant to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been put into the collection tube).

[0048] (4) Centrifuge at 12000 rpm (13400 x g) for 30 sec.

[0049] (5) Discard the FastPure gDNA-Filter Columns III, and keep the filtrate in the collection tube.

[0050] (6) Add 0.5 times the volume of the filtrate of absolute ethanol (about 250 μL, adjust according to the actual situation of the supernatant) to the collection tube, shake and mix for 15 sec.

[0051] (7) Transfer the mixture obtained in step (6) to FastPure PNA Columns V (FastPure PNA Columns V has been put into the collection tube).

[0052] (8) Centrifuge at 12000 rpm for 30 sec, and discard the filtrate.

[0053] (9) Add 700 μL Buffer RWA to the FastPure PNA Columns V.

[0054] (10) Centrifuge at 12000 rpm for 30 sec, and discard the filtrate.

[0055] (11) Add 500 μL Buffer RWB to the FastPure PNA Columns V.

[0056] (12) Centrifuge at 12000 rpm (13400 x g) for 30 sec, and discard the filtrate.

[0057] (13) Repeat steps (11) and (12).

[0058] (14) Put the FastPure PNA Columns V back into the collection tube.

[0059] (15) Centrifuge at 12000 rpm for 2 min.

[0060] (16) Transfer the FastPure PNA Columns V to a new RNase-free Collection Tubes 1.5 mL centrifuge tube.

[0061] (17) Drop 30-100 μL of RNase-free ddH2O to the center of the membrane of the adsorption column.

[0062] (18) 12000 rpm (13400 x g) centrifugation for 1 min.

[0063] (19) The extracted RNA was detected for quality by ultraviolet spectrophotometer, and the qualified RNA (OD260 / 280 = 1.8-2.2) was immediately stored at -80°C. Siberian apricot dormant flower bud RNA was obtained after the end of this step, and its electrophoretogram is shown in Figure 1

[0064] The extracted Siberian apricot dormant flower bud RNA was reverse transcribed to synthesize cDNA by using HiScript II QRT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novogene, Nanjing). The steps were performed according to the instructions:

[0065] (1) Genomic DNA removal. The reaction solution was prepared according to the formula shown in Table 1 in a nuclease-free centrifuge tube:

[0066] Table 1

[0067] Element volume RNase-free ddH2O Dilute to 16 μL 4×gDNA wiper mix 4μL Siberian apricot dormant flower bud RNA Total RNA: 1pg~1μg

[0068] (2) The reaction solution in the centrifuge tube was mixed and centrifuged by gently blowing with a pipette.

[0069] (3) Incubation at 42°C for 2 min.

[0070] (4) Preparation of reverse transcription reaction system. After step (3) was completed, 5 x HiScript II qRT SuperMix II was directly added to the reaction solution according to the ratio shown in Table 2.

[0071] Table 2

[0072] Element volume 5×HiScriptⅡqRT SuperMixⅡ 4μL The reaction solution after completing step (3) 16μL

[0073] (5) Gently mix by blowing with a pipette.

[0074] (6) The reverse transcription reaction was performed according to the procedure shown in Table 3:

[0075] Table 3

[0076] Reaction temperature Reaction time 50℃ 5min 85℃ 5sec

[0077] The product obtained in step (6) was Siberian apricot dormant flower bud cDNA, which could be immediately used for PCR reaction to clone PsABF2 gene, or stored at -20°C for subsequent operation.

[0078] ​Furthermore, primers were designed based on the sequence shown in SEQ ID NO. 1 to clone the coding sequence of the PsABF2 gene from the dormant flower bud cDNA of Siberian apricot. The specific method is as follows:

[0079] (1) Primer design for PsABF2 gene; as shown in Table 4:

[0080] Table 4

[0081] Primer name Sequence (5'→3') PsABF2-F1 (SEQ ID NO. 4) ATGGGTACCAACATGAACTTC PsABF2-R1 (SEQ ID NO. 5) TCAGCCTGCAGCAACCAATGA

[0082] (2) Preparation of PCR reaction system (using 2×Phusion produced by Thermo Fisher Scientific) TM The PCR reaction system was prepared according to the formula shown in Table 5. The template DNA was the cDNA of dormant flower buds of Siberian apricot obtained by reverse transcription.

[0083] Table 5

[0084] Element volume 2 x Phusion TM Plus Green PCR Master Mix 25 μL PsABF2-F1 primer 1 μL PsABF2-R1 primer 1 μL Template DNA 1 μL Nuclease-free water 22 μL total Dilute to 50 μL

[0085] (3) Perform PCR reaction according to the PCR reaction program shown in Table 6.

[0086] Table 6

[0087]

[0088] After the PCR reaction program is completed, the PCR reaction products are subjected to 1% agarose gel electrophoresis. The electrophoresis results are as follows: Figure 2 As shown, Figure 2 A clear PsABF2 gene band (target band) was found in the sample. The target band was recovered by gel excision. The Tiangen Agarose Gel DNA Recovery Kit DP209-02 was used for gel excision and recovery. Follow the steps below:

[0089] (1) Column equilibration step: Add 500 μL of equilibration solution BL to the adsorption column CA2 (the adsorption column is placed in the collection tube).

[0090] (2) Centrifuge at 12000 rpm for 1 min.

[0091] (3) Pour out the waste liquid in the collection tube and put the adsorption column back into the collection tube.

[0092] (4) Cut the target DNA band from the agarose gel (try to remove the excess part) and place it in a clean centrifuge tube, and weigh the gel block.

[0093] (5) According to the weight of the gel block, 100 μL of PN solution was added to the centrifuge tube containing the gel block, calculated as 0.1 g of gel block weight.

[0094] (6) The centrifuge tube was placed in a 50°C water bath for 10 min, and was constantly turned upside down to ensure that the gel block was fully dissolved.

[0095] (7) The solution obtained in step (6) was added to an adsorption column CA2 (the adsorption column was placed in a collection tube), and was left at room temperature for 2 min.

[0096] (8) Centrifugation was performed at 12000 rpm for 60 sec.

[0097] (9) The waste liquid in the collection tube was discarded, and the adsorption column CA2 was placed in the collection tube.

[0098] (10) 600 μL of rinse solution PW was added to the adsorption column CA2.

[0099] (11) Centrifugation was performed at 12000 rpm for 60 sec.

[0100] (12) The waste liquid in the collection tube was discarded, and the adsorption column CA2 was placed in the collection tube.

[0101] (13) Steps (10), (11) and (12) were repeated.

[0102] (14) Centrifugation was performed at 12000 rpm for 2 min.

[0103] (15) The rinse solution was removed as much as possible. The adsorption column CA2 was left at room temperature for several minutes, and was completely air-dried to prevent the residual rinse solution from affecting the next operation.

[0104] (16) The adsorption column CA2 was placed in a clean centrifuge tube, and 30 μL of ddH2O was added to the middle of the adsorption membrane, and was left at room temperature for 2 min.

[0105] (17) Centrifugation was performed at 12000 rpm for 2 min.

[0106] (18) The DNA solution was collected, and was stored at -20°C.

[0107] Example 2

[0108] In this example, the expression pattern of PsABF2, which is related to the ABA transduction pathway of Siberian apricot flower buds, during the dormancy of Siberian apricot flower buds was analyzed.

[0109] (1) RNA extraction was performed on flower buds at different dormancy times, and the operation method was the same as in Example 1.

[0110] (2) Reverse transcription of the flower bud RNA of different stages as described above, the operation method is the same as in Example 1.

[0111] (3) The cDNA of the flower bud of different stages obtained after reverse transcription is used as a template to perform fluorescent quantitative PCR on PsABF2 gene. The quantitative PCR primer design is shown in Table 7.

[0112] Table 7

[0113] Primer name Sequence (5'→3') PsABF2-F2 (SEQ ID NO. 6) TGGGTAGTCCTGGAATGAG PsABF2-R2 (SEQ ID NO. 7) CATCCCTGAACTCTGAACC 18SRNA-F (SEQ ID NO. 8) GTTACTTTTAGGACTCCGCC 18S RNA-R (SEQ ID NO. 9) TTCCTTTAAGTTTCAGCCTTG

[0114] (4) Preparation of the fluorescent quantitative PCR reaction system. The reaction system is prepared according to the formula shown in Table 8.

[0115] Table 8

[0116] Components volume 2*SYBR Green qPCR Premix 10 μL F primer 1 μL R primer 1 μL Template DNA 1 μL ddH2O 7μL total Dilute to 20 μL

[0117] (5) PCR reaction is performed according to the PCR reaction program in Table 9.

[0118] Table 9

[0119]

[0120] (6) 18sRNA is used as an internal reference gene, and the 2 -ΔΔt method is used to calculate the relative expression amount of the gene.

[0121] As Figure 3 the expression amount measurement result, the expression amount of PsABF2, the ABA transduction pathway related gene during the dormancy of Siberian apricot flower, is higher during the dormancy induction period (October 10 to November 10), and the expression amount of PsABF2 during the dormancy period (November 10 to February 15 of the next year) and the dormancy release period (February 15 of the next year to April 3 of the next year) shows a downward trend as a whole, indicating that PsABF2, the ABA transduction pathway related gene during the dormancy of Siberian apricot flower, can regulate the dormancy of Siberian apricot flower. Correspondingly, the protein encoded by the gene is involved in a series of physiological and biochemical processes in the ABA transduction pathway during the dormancy of Siberian apricot flower.

[0122] Example 3

[0123] In this embodiment, the construction of PsABF2 gene overexpression vector (recombinant vector) is carried out. Gateway recombination technology is used in the construction of the recombinant vector, and the kits used are BP Clonase II Enzyme mix (for BP reaction) and LR Clonase Enzyme mix (for LR reaction), both of which are purchased from Thermo Fisher Scientific. The original vector of the recombinant vector is pMDC32, which contains a 35S promoter and is an overexpression vector that can be expressed in plants. The specific method for constructing the recombinant vector is as follows:

[0124] (1) The coding sequence of PsABF2 gene is cloned completely using PCR method, and the gel recovery of PCR product is completed. The method is the same as that in Example 1.

[0125] (2) The DNA obtained after gel recovery in step (1) is taken as a template, and PCR is carried out using the primers shown in Table 10 to add adapters to both ends of the coding sequence of PsABF2 gene. The program for PCR is as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 54℃ annealing for 10s, 72℃ extension for 30s, cycle for 35 times; 72℃ terminal extension for 5min; 4℃ preservation.

[0126] Table 10

[0127] Primer name Sequence (5'→3') PsABF2-F3 GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGGTACCAACATGAACTTC PsABF2-R3 GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGCCTGCAGCAACCAATGA

[0128] (3) The PCR product obtained in step (2) is recovered by gel recovery to obtain the target gene containing adapters. The method for gel recovery is the same as that in Example 1.

[0129] (4) The target gene containing adapters recovered in step (3) is connected to the pDONR207 entry vector (the vector is preserved by the Institute of Economic Forests, Chinese Academy of Forestry, and the vector map can be obtained by the public from BioVector NTCC). The operation steps are carried out according to the Gateway system product manual. The reaction system and reaction conditions for connection are shown in Table 11.

[0130] Table 11

[0131]

[0132] (5) The reaction liquid after the reaction in step (4) is transformed into E. coli DH5a (Shanghai Weidi Biology) by heat shock transformation method; the reaction system during transformation is shown in Table 12, and the reaction conditions are shown in Table 13 (the reaction system is sequentially placed in reaction condition 1, reaction condition 2 and reaction condition 3 during transformation).

[0133] Table 12

[0134] Components volume The above reaction solution 5μL DH5a competent E. coli 50μL

[0135] Table 13

[0136] Reaction conditions 1 Reaction Condition 2 Reaction Condition 3 30 minutes on ice 42℃30sec 5 minutes on ice

[0137] (6) To the transformed E. coli, 1000 μL of LB medium without antibiotics was added, and the mixture was incubated at 37°C in a shaker at 200 rpm / min for 1 h;

[0138] (7) After the incubation, the bacterial solution was centrifuged at 6000 rpm / min for 1 min, 800 μL of supernatant was removed, and the remaining solution was uniformly blown and spread on LB solid medium containing gentamycin (50 mg / mL gentamycin). The medium was incubated at 37°C in an incubator for 14 h until single colonies were formed;

[0139] (8) The positive clones were picked, sequenced, and preserved;

[0140] (9) The plasmid of the positive clone with correct sequence was extracted, and the target gene (PsABF2 gene) on the plasmid was connected with the expression vector pMDC32 (the vector was preserved by the Institute of Economic Forests, Chinese Academy of Forestry, and the vector map was available to the public from BioVector NTCC). The operation steps were performed according to the instructions of the LR Clonase Enzyme mix kit. The preparation method and reaction conditions of the reaction system are shown in Table 14:

[0141] Table 14

[0142]

[0143] (10) The above reaction solution was transformed into E. coli DH5a (Shanghai Weidi Biology) by heat shock transformation. The reaction system during the transformation was the same as shown in Tables 12 and 13.

[0144] (11) To the transformed E. coli, 1000 μL of LB medium without antibiotics was added, and the mixture was incubated at 37°C in a shaker at 200 rpm / min for 1 h;

[0145] (12) After the incubation, the bacterial solution was centrifuged at 6000 rpm / min for 1 min, 800 μL of supernatant was removed, and the remaining solution was uniformly blown and spread on LB solid medium containing kanamycin (50 mg / mL kanamycin). The medium was incubated at 37°C in an incubator for 14 h until single colonies were formed;

[0146] (13) Extract the positive clones, sequence and preserve the bacteria. The plasmid extracted from the positive clone with correct sequence is the recombinant vector pMDC32-PsABF2 containing the coding sequence of PsABF2 gene.

[0147] Example 4

[0148] In this example, the function of PsABF2 gene in regulating flowering time is verified by heterologous expression experiment.

[0149] The following steps are followed:

[0150] (1) The overexpression vector pMDC32-PsABF2 is transformed into Agrobacterium GV3101 (Shanghai Weidi Biology) by heat shock method. The reaction system during transformation is shown in Table 15, and the reaction conditions are shown in Table 16 (the reaction system is placed in reaction condition 1, reaction condition 2, reaction condition 3 and reaction condition 4 in turn during transformation).

[0151] Table 15

[0152] Components volume pMDC32-PsABF2 200 μg GV3101 competent Agrobacterium 50μL

[0153] Table 16

[0154] Reaction conditions 1 Reaction Condition 2 Reaction Condition 3 Reaction Conditions 4 5 minutes on ice Liquid nitrogen 5 minutes 37℃5min 5 minutes on ice

[0155] (2) After transformation is completed, 1000 μL of non-resistant liquid LB medium is added to the Agrobacterium, which is placed in a 28°C shaking bed at 200 rpm / min, and is shaken and cultured for 1 h;

[0156] (3) The Agrobacterium bacterial solution obtained after shaking and culturing is centrifuged at 6000 rpm / min for 1 min, 800 μL of supernatant is removed, and the remaining is blown evenly and spread on the resistant LB solid medium containing kanamycin (50 mg / mL Kanamycin) and rifampicin (20 mg / mL Rifampicin), and is placed in a 28°C constant temperature incubator for inverted culture for 48 h, until single colonies are grown;

[0157] (4) The single colonies on the above-mentioned resistant LB medium are picked to the LB liquid medium containing the same resistance, and are shaken and cultured at 28°C.

[0158] The Agrobacterium-mediated inflorescence dipping method is used to transform Arabidopsis thaliana, and the steps are as follows:

[0159] (1) The fruit pods of Arabidopsis thaliana in the flowering stage are cut, and the whole plant of Arabidopsis thaliana is placed in the bacterial solution for infection;

[0160] (2) The infected Arabidopsis thaliana plant is taken out for 24 h light shielding treatment;

[0161] (3) The light-shielded grown Arabidopsis plants were cultured under normal conditions, and the T0 generation seeds were harvested after the seeds were matured;

[0162] (4) The harvested T0 generation seeds were sterilized and planted in 1 / 2MS solid medium containing hygromycin (30 mg / mL Hygromycin B), and were subjected to 3 d vernalization treatment. The plants after vernalization were cultured under normal artificial climate.

[0163] After the Arabidopsis seeds formed seedlings, the seedlings were subjected to fluorescent quantitative PCR (RT-PCR) identification to screen out transgenic positive plants.

[0164] First, RNA extraction was performed on plant tissues, and the operation steps were the same as the RNA extraction method in Example 1.

[0165] The primers PsABF2-F2 and PsABF2-R2 were subjected to fluorescent quantitative PCR (RT-PCR) using 18S RNA as an internal reference. If the fluorescent signal of the PsABF2 gene was detected, it indicated that the Arabidopsis plant was a transgenic positive Arabidopsis plant.

[0166] As shown in Figure 4 The fluorescent quantitative PCR (RT-PCR) identification results of the transgenic Arabidopsis plants into which the PsABF2 gene was introduced in the present example are shown in the figure. WT is a wild-type Arabidopsis line, and PsABF2-#1, PsABF2-#2, PsABF2-#3, PsABF2-#4 and PsABF2-#5 are five transgenic positive Arabidopsis plants into which the PsABF2 gene was successfully introduced.

[0167] As shown in Figure 5 The transgenic positive Arabidopsis plants and wild-type Arabidopsis (WT) plants that were sown at the same time and cultured under the same conditions were observed. As shown in the figure, the wild-type Arabidopsis had already bolted and was about to bloom, while the three transgenic positive Arabidopsis plants had not yet bolted. This indicates that the flowering period of the transgenic positive Arabidopsis plants was delayed.

[0168] Statistical analysis was performed on the bolting and flowering times of the transgenic positive Arabidopsis and wild-type Arabidopsis, and the results are shown in Figure 6 and Figure 7 Figure 6 In the figure, the average bolting time of the wild-type Arabidopsis under normal culture conditions was 27.7 d, while the average bolting time of the PsABF2 transgenic Arabidopsis was 29.9 d. The bolting time of the transgenic positive Arabidopsis was about 2.2 d later than that of the wild-type Arabidopsis.

[0169] Figure 7 ​In the normal culture condition, the average flowering time of wild type Arabidopsis is 30 days, while the average flowering time of PsABF2 transgenic positive Arabidopsis is 32.25 days, and the flowering time of the transgenic positive Arabidopsis is about 2.25 days later than that of the wild type Arabidopsis.

[0170] The above results show that the bolting and flowering time of the PsABF2 transgenic positive Arabidopsis is later than that of the wild type Arabidopsis.

[0171] The pMDC32-PsABF2 is a plant expression vector, which can theoretically be used to transform Siberian apricot plants. When it is expressed in Siberian apricot plants, the flower buds of the Siberian apricot will be in a deeper dormant state, which will delay the flowering time of the Siberian apricot.

[0172] Example 5

[0173] In this embodiment, the SNP of the late flowering trait of Siberian apricot is mined and determined.

[0174] Taking the F106 genome data of Siberian apricot as a template, 66 Siberian apricots in Hulunbuir region of Inner Mongolia (including 23 early flowering and 43 late flowering materials) were selected as test materials for whole genome association analysis. Combined with the early and late flowering traits of each material, the SNP sites in the PsABF2 gene and linked to the gene were mined, and a total of 4 SNP sites were mined, which were SNP1, SNP2, SNP3 and SNP4.

[0175] SNP1 is located at the 32680522th site of chromosome 1 of Siberian apricot, 952 bp away from the start codon of PsABF2, and the nearby sequence is ACATT T GGACC (the SNP1 site is indicated by underlining). The genotype of Siberian apricot at the SNP1 site has 3 types, which are TG / T, TG / TG and T / T, wherein T is the reference base (the base at this site in the reference sequence), and TG is the base after mutation. TG / T means that one of the two chromosomes 1 of Siberian apricot has TG at the SNP1 position, and the other has T at the SNP1 position; TG / TG and T / T have the same meaning. The SNP1 site genotyping and the phenotypic variation of the trait explain 88.5%.

[0176] SNP2 is located at the 32680714th site of chromosome 1 of Siberian apricot, 1144 bp away from the start codon of PsABF2, and the nearby sequence is: GTAAT ATCGTC (underlined SNP2 site). The genotype of Siberian apricot at the SNP2 site is G / A, G / G and A / A, in which A is the reference base and G is the base after mutation; the phenotypic variation explanation rate of SNP2 site genotyping and the trait is 83.1%.

[0177] SNP3 is located at the 32681144 site of chromosome 1 of Siberian apricot, 1574 bp away from the start codon of PsABF2, and the sequence nearby is: AATAA A AGTTA (underlined SNP3 site). The genotype of Siberian apricot at the SNP3 site is T / A, T / T and A / A, in which A is the reference base and T is the base after mutation; the phenotypic variation explanation rate of SNP3 site genotyping and the trait is 83.1%.

[0178] SNP4 is located at the 32681347 site of chromosome 1 of Siberian apricot, 1777 bp away from the start codon of PsABF2, and the sequence nearby is: GGTTC G AATCT (underlined SNP4 site). The genotype of Siberian apricot at the SNP4 site is A / A, A / G and G / G, in which G is the reference base and A is the base after mutation; the phenotypic variation explanation rate of SNP4 site genotyping and the trait is 81.8%.

[0179] The statistical situation of SNP site variation types is shown in Table 17. The variation types of each strain at the four SNP sites are shown in Table 18, and four SNP sites related to late flowering are mined, i.e. SNP1-SNP4.

[0180] Table 17 SNP site variation types

[0181]

[0182]

[0183] Table 18 SNP site variation types of each strain

[0184]

[0185]

[0186] In the table, some individual strains have "0 / 0" at some SNP sites, which can be regarded as incidental variation and discarded.

[0187] The above results show that according to the genotypes of a certain plant to be detected at SNP1-SNP4, the SNP sites obtained by comparing the plant with the F106 Siberian apricot reference sequence have a good correspondence with the traits, and the phenotype variation explanation rate of SNP1-SNP4 is between 81.8% and 88.5%.

[0188] Obviously, the above embodiments are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the patent application claims.

Claims

1. PsABF2 The use of a gene for delaying the flowering period of a plant is characterized in that: PsABF2 The coding sequence of the gene is shown in SEQ ID NO.1, and the plant is Siberian apricot or Arabidopsis thaliana; PsABF2 When genes are used to delay flowering, they increase PsABF2 Gene expression level.

2. according to claim 1 PsABF2 The use of a gene for delaying the flowering period of a plant is characterized in that: PsABF2 The expression levels of the genes were detected by fluorescence quantitative PCR; the sequences of the primers used in fluorescence quantitative PCR were shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively.

3. according to claim 1 PsABF2 The use of a gene for delaying the flowering period of a plant is characterized in that: Will PsABF2 When the gene is used to delay the flowering period of plants, the recombinant vector is used to transform the plants to increase the PsABF2 The recombinant vector contains the sequence shown in SEQ ID NO.

1.

4. according to claim 3 PsABF2 The use of a gene for delaying the flowering period of a plant is characterized in that: The original vector of the recombinant vector is the overexpression vector pMDC32.

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