PsABF2 gene related to flowering phase regulation of Siberia apricots, SNP marker and application of PsABF2 gene and SNP marker

Abstract

The invention discloses a PsABF2 gene related to flowering phase regulation and control of Siberia apricots, an SNP (Single Nucleotide Polymorphism) marker and application of the PsABF2 gene and the SNP marker. The coding sequence of the PsABF2 gene is as shown in SEQ ID NO. 1; the PsABF2 gene is used for regulating and controlling the flowering phase of apricot sibirica. Based on the gene, the invention further provides a protein encoded by PsABF2, a method for delaying the flowering phase of plants by using the PsABF2 gene and four SNP markers linked with the PsABF2 gene, and the SNP markers can be used for identifying the flowering phase character of apricot siberia, predicting the flowering phase and molecular marker assisted breeding. The invention provides an important tool and technical support for genetic breeding and flowering phase management of the Siberia apricot.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and specifically to a PsABF2 gene and SNP marker related to the regulation of Siberian apricot flowering period and applications thereof. Background Art

[0002] Siberian apricot (Prunus sibirica) is a plant of the genus Armeniaca in the subfamily Prunoideae of the family Rosaceae. It is an important ecological and economic tree species in my country. Siberian apricot has strong ecological adaptability and has the characteristics of windbreak and sand fixation, soil and water conservation, cold resistance, drought resistance, and barrenness resistance. It has irreplaceable ecological value in arid and semi-arid areas with fragile ecological environment. It has become an important afforestation tree species for sand fixation and soil conservation, water conservation and desertification control in the three northern regions of my country. Siberian apricot not only plays an important role in ecological functions, but is also widely used in food, medical and industrial fields. Its almonds are rich in 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 beverages, and has high economic value. Due to its short dormancy period and early flowering period, Siberian apricot is very susceptible to the frost damage of the early spring "late spring cold", which seriously restricts the yield and quality of Siberian apricot fruits.

[0003] Flower bud dormancy is one of the important links in the plant growth cycle, which directly affects its future growth and development. It can be used as an important basis for judging plant cold resistance and inferring geographical distribution, and is also a 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 affected and regulated by external environments such as temperature, light, and water, as well as changes in genes, hormones, etc. in the body. In this process, hormone content and gene expression will also change accordingly to adapt to it. Abscisic acid (ABA) is considered to be an important substance that responds to changes in the external environment and regulates changes in the dormancy state of flower buds. It promotes the deepening of flower bud dormancy. The form of its content changes in response to environmental changes, regulating the initiation, development and termination of flower bud dormancy. During dormancy, the degree of development of flower organs also deepens with the dormancy process, preparing for flowering. Usually, the content of ABA in flower buds increases in autumn, which induces flower buds to enter dormancy, and then the ABA content decreases until the flower buds sprout. Related studies have shown that ABA content has an inhibitory effect on the release of flower bud dormancy. When the ABA content level is high during the dormancy period, the dormancy release time will be later, which will lead to a delay in the flowering period. Therefore, increasing the ABA content level during the dormancy period of Siberian apricot flower buds and delaying the flowering period of Siberian apricots can prevent them from being damaged by frost, thereby promoting the healthy and stable development of the Siberian apricot industry. Summary of the invention

[0004] To this end, the technical problem to be solved by the present invention is to provide PsABF2 genes and SNP markers related to the regulation of Siberian apricot flowering period and their applications, which provide a theoretical and methodological basis for analyzing the molecular regulatory mechanism of Siberian apricot flower bud dormancy and exploring the molecular mechanism of ABA signaling pathway-related genes on the release of Siberian apricot flower bud dormancy and flowering period regulation, and provide a molecular biological tool for regulating the flowering period of Siberian apricot.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

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

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

[0008] The application of the PsABF2 gene in delaying the flowering period of plants, the PsABF2 gene is the above-mentioned PsABF2 gene related to the regulation of the flowering period of Siberian apricot; the plants are Siberian apricot and / or Arabidopsis; when the PsABF2 gene is used to delay the flowering period of plants, the expression level of the PsABF2 gene in the plant is increased.

[0009] The above-mentioned application of PsABF2 gene in delaying the flowering period of plants, the expression level of PsABF2 gene is detected by fluorescence quantitative PCR; the sequences of the primers used in fluorescence quantitative PCR are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

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

[0011] The above-mentioned application of the PsABF2 gene in delaying the flowering period of plants, the original vector of the recombinant vector is the overexpression vector pMDC32.

[0012] Siberian apricot flowering period regulation-related SNP markers, the SNP markers are SNP1 to SNP4 linked to the PsABF2 gene related to the Siberian apricot flowering period regulation, wherein:

[0013] SNP1 is located at position 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 position 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 position 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 position 32681347 on chromosome 1 of Siberian apricot. The reference base of SNP4 is G, and the base after mutation is A.

[0017] Application of SNP markers related to flowering period regulation of Siberian apricot, using the above-mentioned SNP markers related to flowering period regulation of Siberian apricot to identify the flowering period traits of Siberian apricot, or to predict the early or late flowering period of Siberian apricot, or for molecular marker-assisted breeding of Siberian apricot.

[0018] When the above-mentioned Siberian apricot flowering period regulation-related SNP markers are used to identify the flowering period traits of Siberian apricot, the genotypes of the Siberian apricot to be tested at the sites of SNP1, SNP2, SNP3 and SNP4 are obtained from the genomic DNA of the Siberian apricot plant to be tested; wherein:

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

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

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

[0022] In the above application, when obtaining the genotype of the Siberian apricot to be tested at the sites where the SNP1, SNP2, SNP3 and SNP4 are located from the genomic DNA of the Siberian apricot plant to be tested, PCR is performed using the genomic DNA of the Siberian apricot to be tested as a template to obtain PCR products, and the PCR products are sequenced to obtain the genotype of the Siberian apricot to be tested at the sites where the SNP1, SNP2, SNP3 and SNP4 are located.

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

[0024] The technical solution of the present invention achieves the following beneficial technical effects:

[0025] The present invention discloses a gene PsABF2 and a SNP marker related to the regulation of Siberian apricot flowering period and an application thereof. The PsABF2 gene is a gene related to the ABA transduction pathway during the dormancy of Siberian apricot flower buds, and plays an important role in the dormancy of Siberian apricot flower buds. Experiments have shown that after the overexpression vector pMDC32-PsABF2 carrying the PsABF2 gene is transformed into Arabidopsis thaliana by Agrobacterium-mediated genetic transformation, the flowering period of Arabidopsis thaliana changes, confirming that PsABF2 has the function of regulating the flowering period of Arabidopsis thaliana, and indirectly proving that the PsABF2 gene in Siberian apricot has a regulatory function on the flowering period of Siberian apricot. Furthermore, the present invention obtains 4 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 Siberian apricot is a late-flowering variety based on the 4 SNP sites. The ABA transduction pathway regulatory gene PsABF2 during the dormancy period of Siberian apricot flower buds provided in the present invention provides a new tool for Siberian apricot breeding and the regulation of flower bud dormancy mechanisms. The PsABF2 gene and the SNP loci linked thereto can be used as marker-assisted tools in late-flowering breeding, for genetic background analysis and screening of Siberian apricots, and for the selection of late-flowering Siberian apricot varieties. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0028] Figure 3 The expression measurement results of the PsABF2 gene at various stages of the Siberian apricot flower bud dormancy period in the embodiment of the present invention;

[0029] Figure 4 PCR identification results of transgenic positive Arabidopsis thaliana with PsABF2 gene transferred in the embodiment of the present invention;

[0030] Figure 5 Comparison of the growth of wild-type Arabidopsis and transgenic positive Arabidopsis into which the PsABF2 gene was introduced in the examples of the present invention;

[0031] Figure 6 Statistical analysis of bolting time of transgenic positive Arabidopsis thaliana into which PsABF2 gene was introduced in the embodiment of the present invention;

[0032] Figure 7 Statistical analysis of the flowering period of transgenic positive Arabidopsis thaliana into which the PsABF2 gene was introduced in the examples of the present invention. DETAILED DESCRIPTION

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

[0034] Example 1

[0035] In this example, the sequence analysis of the ABA transduction pathway-related gene PsABF2 during the dormancy period of Siberian apricot flower buds was performed, and the cDNA sequence of the PsABF2 gene was cloned by RNA extraction and reverse transcription.

[0036] After long-term research and combined with bioinformatics methods, the inventor cloned and obtained the ABA transduction pathway related gene PsABF2 in the dormant flower buds of Siberian apricot. SEQ ID NO.1 is the ORF (open reading frame) region sequence of the ABA transduction pathway related gene PsABF2 during the dormant period of Siberian apricot flower buds, and SEQ ID NO.2 is the reverse complementary sequence of the sequence shown in SEQ ID NO.1. The length of the sequence shown in SEQ ID NO.1 is 1266bp, and the encoded protein is 421 amino acids, and the encoded amino acid sequence is shown in 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] MGTNMNFKGFGNEPPGINPLARQSSIYSLTFEELQNTIGGSGKDFGSMNMDELLKSIWTAEETQIMAPSGGGAGGQNGLGLGGGSLQRQGSLTLPRTLSQKTVDE VWKNISKEGTGAGGSNMPQRQQTLGEMTLEEFLVKAGVVREEAQLAPKPANNGAGFFGDLSRFGNTGGNLDFEFQQTNRGVGVMGNRVSETNSQVPNQASNLPLNA NGVRSNQQQQQLPQQQQIFPKQQPVTYNTSLPPMGPNAQLGSPGMRGGIMGIGDQGLNGTLVQSSGMGMVGLGAAGAVRVATGSPANQLSSDGIGKSNGTDTSSV SPVPYVFNGGFRGRKGGGPVEKVVERRQRRMIKNRESAARSRARKQAYTMELEAEVAKLKEENQELQKKQVCFVVVSHSCWKHELTDVINLQCLTLIYCSLVAAG*

[0043] The ORF region sequence shown in SEQ ID NO.1 is the same as the CDS sequence (coding sequence) obtained by reverse transcription of PsABF2 mRNA, that is, the sequence shown in SEQ ID NO.1 is the coding sequence of the protein encoded by the PsABF2 gene.

[0044] Total RNA was extracted from dormant flower buds of Siberian apricot using the VazymE FastPure Universal Plant Total RNA Isolation Kit (Novagen, Nanjing). Follow the instructions:

[0045] (1) Take 100 g of Siberian almond flower bud tissue, grind it thoroughly in liquid nitrogen, and immediately add 600 μL of Buffer PSL. Vortex vigorously for 30 seconds to mix the sample and lysate thoroughly.

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

[0047] (3) Take about 500 μL of the supernatant and transfer it to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube).

[0048] (4) Centrifuge at 12000 rpm (13400×g) for 30 seconds.

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

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

[0051] (7) Transfer the mixed solution obtained in step (6) to FastPure PNA Columns V (FastPure PNA Columns V has been placed in a collection tube).

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

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

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

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

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

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

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

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

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

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

[0062] (18) Centrifuge at 12000 rpm (13400 × g) for 1 min.

[0063] (19) The extracted RNA was tested for quality using an ultraviolet spectrophotometer. If the RNA was of acceptable quality (OD260 / 280 = 1.8 to 2.2), it was immediately stored at -80°C. After this step, the Siberian apricot dormant flower bud RNA was obtained, and its electrophoresis pattern was as follows: Figure 1 shown.

[0064] The extracted RNA from dormant flower buds of Siberian apricot was reverse transcribed using HiScript IIQRT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozyme, Nanjing) to synthesize cDNA. Follow the steps in the instruction manual:

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

[0066] Table 1

[0067] Element volume <![CDATA[RNase-free ddH2O (Nuclease-free water)]]> Make up to 16 μL 4×gDNA wiper Mix 4μL Siberian Apricot Dormant Flower Bud RNA Total RNA: 1pg~1μg

[0068] (2) Use a pipette to gently mix the reaction solution in the centrifuge tube.

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

[0070] (4) Preparation of reverse transcription reaction system. After step (3) is completed, 5×HiScriptⅡqRT SuperMixⅡ is 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) Mix by gently pipetting.

[0074] (6) Perform reverse transcription reaction 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) is the Siberian apricot dormant flower bud cDNA, which can be used immediately for PCR reaction to clone the PsABF2 gene, or stored at -20°C for subsequent operations.

[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 of 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 Plus Green PCR Master Mix, catalog number F632L). The PCR reaction system was prepared according to the formula shown in Table 5. The template DNA was the Siberian apricot dormant flower bud cDNA obtained by reverse transcription.

[0083] Table 5

[0084] Element volume <![CDATA[2×Phusion TM Plus Green PCR Master Mix]]> 25μL PsABF2-F1 primer 1μL PsABF2-R1 primer 1μL Template DNA 1μL Nuclease-free water 22μL total Make up 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 was completed, the PCR reaction products were subjected to 1% agarose gel electrophoresis. The electrophoresis results were as follows: Figure 2 As shown, Figure 2 There is a clear PsABF2 gene band (target band) in the sample. The target band is excised and recovered. Tiangen Agarose Gel DNA Recovery Kit DP209-02 is 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) and place it in a clean centrifuge tube and weigh the gel block.

[0093] (5) According to the weight of the gel block, add 100 μL of PN solution according to the weight of 0.1 g of gel block, and add PN solution to the centrifuge tube containing the gel block.

[0094] (6) Place the tube in a 50°C water bath for 10 min, gently turning the tube upside down to ensure that the gelatin block is fully dissolved.

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

[0096] (8) Centrifuge at 12000 rpm for 60 seconds.

[0097] (9) Pour away the waste liquid in the collection tube and place the adsorption column CA2 into the collection tube.

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

[0099] (11) Centrifuge at 12000 rpm for 60 seconds.

[0100] (12) Pour away the waste liquid in the collection tube and place the adsorption column CA2 into the collection tube.

[0101] (13) Repeat steps (10), (11) and (12).

[0102] (14) Centrifuge at 12000 rpm for 2 min.

[0103] (15) Remove as much rinse solution as possible. Leave the adsorption column CA2 at room temperature for several minutes to dry thoroughly to prevent residual rinse solution from affecting the next step.

[0104] (16) Place the adsorption column CA2 in a clean centrifuge tube, add 30 μL of ddH2O dropwise to the middle of the adsorption membrane, and leave at room temperature for 2 min.

[0105] (17) Centrifuge at 12000 rpm for 2 min.

[0106] (18) Collect the DNA solution and store it at -20°C.

[0107] Example 2

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

[0109] (1) RNA was extracted from flower buds at different dormancy stages using the same method as in Example 1.

[0110] (2) The flower bud RNAs at different stages are reverse transcribed using the same operation method as in Example 1.

[0111] (3) Using the cDNA of flower buds at different stages obtained after reverse transcription as a template, fluorescent quantitative PCR was performed on the 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 fluorescent quantitative PCR reaction system: The reaction system was prepared according to the formula shown in Table 8.

[0115] Table 8

[0116] Components volume 2*SYBR Green qPCR Premix 10μL F Primer 1μL R Primer 1μL Template DNA 1μL <![CDATA[ddH2O]]> 7μL total Make up to 20 μL

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

[0118] Table 9

[0119]

[0120] (6) 18sRNA was used as the internal reference gene and 2 -ΔΔt The relative gene expression was calculated by the method.

[0121] like Figure 3 The results of the expression measurement showed that the expression level of PsABF2, a gene related to the ABA transduction pathway during the dormancy of Siberian apricot flower buds, was high during the dormancy induction period (October 10 to November 10), and the expression level of PsABF2 showed an overall downward trend during the dormancy period (November 10 to February 15 of the following year) and the dormancy release period (February 15 of the following year to April 3 of the following year), indicating that PsABF2, a gene related to the ABA transduction pathway during the dormancy of Siberian apricot flower buds, can regulate the dormancy of Siberian apricot flower buds. Accordingly, the protein encoded by this gene is involved in regulating a series of physiological and biochemical processes in the ABA transduction pathway during the dormancy of Siberian apricot flower buds.

[0122] Example 3

[0123] In this example, a PsABF2 gene overexpression vector (recombinant vector) was constructed. Gateway recombination technology was used to construct the recombinant vector, and the kits used were BP Clonase II Enzyme mix (for BP reaction) and LRClonase Enzyme mix (for LR reaction), both of which were purchased from Thermo Fisher Scientific. The original vector of the recombinant vector was 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 the PsABF2 gene was completely cloned using PCR, and the PCR product was recovered by gel excision. The method was the same as in Example 1.

[0125] (2) Take the DNA obtained after gel excision and recovery in step (1) as a template, and perform PCR with the primers shown in Table 10 to add adapters to both ends of the PsABF2 gene coding sequence. The PCR program is: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 54°C for 10s, extension at 72°C for 30s, 35 cycles; final extension at 72°C for 5min; storage at 4°C.

[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 excised and recovered to obtain the target gene containing the linker. The method of excising and recovering the target gene is the same as that in Example 1.

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

[0130] Table 11

[0131]

[0132] (5) The reaction solution after the reaction in step (4) was transformed into Escherichia coli DH5a (Shanghai Weidi Biotechnology) by heat shock transformation; the reaction system during transformation was as shown in Table 12, and the reaction conditions were as shown in Table 13 (during transformation, the reaction system was placed under reaction condition 1, reaction condition 2 and reaction condition 3 in sequence).

[0133] Table 12

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

[0135] Table 13

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

[0137] (6) Add 1000 μL of non-resistant liquid LB medium to the transformed E. coli, place in a shaker at 37°C and 200 rpm / min for 1 h;

[0138] (7) After the shaking culture, the bacterial solution was centrifuged at 6000 rpm / min for 1 min, 800 μL of the supernatant was removed, and the remaining was evenly pipetted and spread on LB solid medium containing gentamycin (50 mg / mL gentamycin), and incubated upside down in a 37°C constant temperature incubator for 14 h until a single colony grew;

[0139] (8) Select positive clones, sequence them and preserve them;

[0140] (9) Select the positive clones with correct sequencing to extract plasmids, and connect the target gene (PsABF2 gene) on the plasmid with the expression vector pMDC32 (the vector is preserved by the Economic Forestry Research Institute of the Chinese Academy of Forestry, and the public can obtain the vector map from BioVector NTCC). The operation steps are carried out 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 liquid was transformed into Escherichia coli DH5a (Shanghai Weidi Biotechnology) by heat shock transformation. The reaction system during transformation was the same as shown in Table 12 and Table 13.

[0144] (11) Add 1000 μL of non-resistant liquid LB medium to the transformed E. coli and place in a shaker at 37°C at 200 rpm / min for 1 h;

[0145] (12) After the shaking culture, the bacterial solution was centrifuged at 6000 rpm / min for 1 min, 800 μL of the supernatant was removed, and the remaining was evenly pipetted and spread on LB solid medium containing kanamycin (50 mg / mL Kanamycin), and incubated upside down in a 37°C constant temperature incubator for 14 h until a single colony grew;

[0146] (13) Extract the positive clones, sequence them and inoculate them. The plasmid extracted from the positive clones with correct sequencing is the constructed recombinant vector pMDC32-PsABF2 containing the PsABF2 gene coding sequence.

[0147] Example 4

[0148] In this example, the role of the PsABF2 gene in regulating flowering period was verified by heterologous expression experiments.

[0149] Follow these steps:

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

[0151] Table 15

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

[0153] Table 16

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

[0155] (2) After transformation, add 1000 μL of non-resistant liquid LB medium to Agrobacterium and place in a shaker at 28°C at 200 rpm / min for 1 h;

[0156] (3) After the shaking culture, the Agrobacterium culture liquid obtained was centrifuged at 6000 rpm / min for 1 min, 800 μL of supernatant was removed, the remaining liquid was evenly pipetted, and spread on a resistant LB solid medium containing kanamycin (50 mg / mL Kanamycin) and rifampicin (20 mg / mL Rifampicin), and inverted in a constant temperature incubator at 28°C for 48 h until a single colony grew;

[0157] (4) Pick a single colony from the above-mentioned resistance LB medium and transfer it to LB liquid medium containing the same resistance, and culture it on a shaker at 28°C.

[0158] The Agrobacterium-mediated inflorescence transformation of Arabidopsis thaliana was carried out as follows:

[0159] (1) Cut off the fruit pods of Arabidopsis thaliana in full bloom, and place the whole plant of Arabidopsis thaliana in bacterial solution for infection;

[0160] (2) taking out the infected Arabidopsis plants and subjecting them to 24-hour shading treatment;

[0161] (3) culturing the Arabidopsis plants grown under shade conditions under normal conditions, and harvesting T0 generation seeds after the seeds mature;

[0162] (4) The harvested T0 generation seeds were sterilized and planted in 1 / 2MS solid culture medium containing hygromycin (30 mg / mL Hygromycin B) and vernalized for 3 days. After vernalization, the plants were cultured under normal artificial climate.

[0163] After the Arabidopsis seeds form seedlings, they are subjected to fluorescence quantitative PCR (RT-PCR) identification to screen out transgenic positive plants.

[0164] First, RNA was extracted from plant tissues, and the operating steps were the same as the RNA extraction method in Example 1.

[0165] Primers PsABF2-F2 and PsABF2-R2 were used for fluorescence quantitative PCR (RT-PCR), and 18S RNA was used as an internal reference. If the fluorescence signal of the PsABF2 gene was detected, it indicated that the Arabidopsis thaliana strain was a transgenic positive Arabidopsis thaliana plant.

[0166] like Figure 4 The results of fluorescent quantitative PCR (RT-PCR) identification of transgenic Arabidopsis thaliana with the PsABF2 gene in this example. In the figure, WT is the wild-type Arabidopsis strain, and PsABF2-#1, PsABF2-#2, PsABF2-#3, PsABF2-#4 and PsABF2-#5 are 5 transgenic positive Arabidopsis thaliana strains successfully introduced with the PsABF2 gene.

[0167] like Figure 5 The transgenic positive Arabidopsis plants and wild-type Arabidopsis (WT) plants were sown at the same time and cultured under the same conditions. It can be observed from the figure that the wild-type Arabidopsis has bolted and is about to bloom, while the three transgenic positive Arabidopsis plants have not bolted yet. This indicates that the flowering period of the transgenic positive Arabidopsis plants has been delayed.

[0168] The bolting and flowering time of transgenic positive Arabidopsis and wild-type Arabidopsis were statistically analyzed, and the results were as follows: Figure 6 and Figure 7 shown. Figure 6 In the experiment, under normal culture conditions, the average bolting time of wild-type Arabidopsis was 27.7 days, while that of PsABF2 transgenic Arabidopsis was 29.9 days. The bolting time of transgenic positive Arabidopsis was delayed by about 2.2 days compared with that of wild-type Arabidopsis.

[0169] Figure 7In the experiment, under normal culture conditions, the average flowering time of wild-type Arabidopsis was 30 days, while the average flowering time of PsABF2 transgenic-positive Arabidopsis was 32.25 days. The flowering time of transgenic-positive Arabidopsis was delayed by about 2.25 days compared with wild-type Arabidopsis.

[0170] The above results showed that the bolting and flowering time of PsABF2 transgenic Arabidopsis were later than those of wild-type Arabidopsis.

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

[0172] Example 5

[0173] In this example, SNP mining and determination of the late flowering trait of Siberian apricot were performed.

[0174] Using the genome data of Siberian apricot F106 as a template, 66 Siberian apricots from the Helingeer area of ​​Inner Mongolia were selected as test materials (including 23 early-flowering and 43 late-flowering materials) for genome-wide association analysis. Combined with the early and late flowering traits of each material, the SNP sites in the PsABF2 gene and those linked to the gene were mined, and a total of 4 SNP sites were mined, namely SNP1, SNP2, SNP3 and SNP4.

[0175] SNP1 is located at position 32680522 on chromosome 1 of Siberian apricot, 952 bp away from the start codon of PsABF2, and the nearby sequence is ACATT T GGACC (underline indicates SNP1 site). There are three genotypes of Siberian apricot at the SNP1 site, namely TG / T, TG / TG and T / T, where T is the reference base (the base at this site in the reference sequence) and TG is the mutated base. TG / T means that one of the two chromosomes 1 of Siberian apricot has a TG base at the SNP1 position, and the other has a T base at the SNP1 position; TG / TG and T / T have the same meaning. The phenotypic variation explanation rate of SNP1 site genotyping and traits is 88.5%.

[0176] SNP2 is located at position 32680714 on chromosome 1 of Siberian apricot, 1144 bp away from the start codon of PsABF2, and the nearby sequence is: GTAAT ATCGTC (underline indicates SNP2 locus). The genotypes of Siberian apricot at SNP2 are G / A, G / G and A / A, where A is the reference base and G is the mutated base; the phenotypic variation explanation rate of SNP2 locus genotyping and traits is 83.1%.

[0177] SNP3 is located at position 32681144 on chromosome 1 of Siberian apricot, 1574 bp away from the start codon of PsABF2, and the nearby sequence is: AATAA A AGTTA (underline represents SNP3). The genotypes of Siberian apricot at SNP3 are T / A, T / T and A / A, where A is the reference base and T is the mutated base; the phenotypic variation explanation rate of SNP3 genotyping and traits is 83.1%.

[0178] SNP4 is located at position 32681347 on chromosome 1 of Siberian apricot, 1777bp away from the start codon of PsABF2, and the nearby sequence is: GGTTC G AATCT (underline represents SNP4). The genotypes of Siberian apricot at SNP4 are A / A, A / G and G / G, where G is the reference base and A is the mutated base; the phenotypic variation explanation rate of SNP4 genotyping and traits is 81.8%.

[0179] The statistics of the variation types of SNP sites are shown in Table 17. The variation types of each strain at these four SNP sites are shown in Table 18. A total of four late flowering-related SNP sites were mined, namely SNP1 to 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 strains have "0 / 0" at some SNP sites, which can be regarded as accidental mutations and discarded.

[0187] The above results show that according to the genotype of a plant to be tested at SNP1~SNP4, it is compared with the F106 Siberian apricot reference sequence, and the corresponding relationship between the genotyping of the SNP loci and the traits is good, and the phenotypic variation explanation rate of SNP1~SNP4 is between 81.8% and 88.5%.

[0188] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. The PsABF2 gene related to flowering period regulation of Siberian apricot is characterized by: The coding sequence of the PsABF2 gene is shown in SEQ ID NO.1; the PsABF2 gene is used to regulate the flowering period of Siberian apricot.

2. The protein encoded by the PsABF2 gene is characterized in that: The PsABF2 gene is the PsABF2 gene related to the regulation of Siberian apricot flowering period as claimed in claim 1. The amino acid sequence of the protein encoded by the PsABF2 gene is shown in SEQ ID NO.3, and the protein is involved in the regulation of Siberian apricot flowering period.

3. The use of PsABF2 gene in delaying the flowering period of plants, characterized in that: The PsABF2 gene is the PsABF2 gene related to Siberian apricot flowering period regulation as described in claim 1; the plant is Siberian apricot and / or Arabidopsis thaliana; when the PsABF2 gene is used to delay the flowering period of the plant, the expression level of the PsABF2 gene in the plant is increased.

4. The use of the PsABF2 gene in delaying the flowering period of plants according to claim 3, characterized in that: The expression level of PsABF2 gene was detected by fluorescence quantitative PCR; the sequences of the primers used in fluorescence quantitative PCR were shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

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

1.

6. The use of the PsABF2 gene in delaying the flowering period of plants according to claim 5, characterized in that: The original vector of the recombinant vector is the overexpression vector pMDC32.

7. A SNP marker related to flowering period regulation of Siberian apricot, characterized in that: The SNP markers are SNP1 to SNP4 linked to the PsABF2 gene related to the Siberian apricot flowering period regulation according to claim 1, wherein: SNP1 is located at position 32680522 of chromosome 1 of Siberian apricot. The reference base of SNP1 is T, and the base after mutation is TG; SNP2 is located at position 32680714 of chromosome 1 of Siberian apricot. The reference base of SNP2 is A, and the base after mutation is G; SNP3 is located at position 32681144 of chromosome 1 of Siberian apricot. The reference base of SNP3 is A, and the base after mutation is T; SNP4 is located at position 32681347 on chromosome 1 of Siberian apricot. The reference base of SNP4 is G, and the base after mutation is A.

8. Application of SNP markers related to flowering period regulation of Siberian apricot, characterized in that: The Siberian apricot flowering period regulation-related SNP markers as described in claim 7 are used to identify the flowering period traits of Siberian apricot, or to predict the early or late flowering period of Siberian apricot, or to be used for molecular marker-assisted breeding of Siberian apricot.

9. The use of the SNP markers related to flowering period regulation of Siberian apricot according to claim 8, characterized in that: When used to identify the flowering period trait of Siberian apricot, the genotype of the Siberian apricot to be tested at the sites of SNP1, SNP2, SNP3 and SNP4 is obtained from the genomic DNA of the Siberian apricot plant to be tested; wherein: When the genotype of the site where SNP1 is located in the genomic DNA of the Siberian apricot to be tested is T / T, the genotype of the site where SNP2 is located is A / A, the genotype of the site where SNP3 is located is A / A, and the genotype of the site where SNP4 is located is G / G, the Siberian apricot to be tested is an early-flowering Siberian apricot; When the genotype of the site SNP1 in the genomic DNA of the Siberian apricot to be tested is TG / T or TG / TG, or the genotype of the site SNP2 is G / G or G / A, or the genotype of the site SNP3 is T / T or T / A, or the genotype of the site SNP4 is A / A or A / G, the Siberian apricot to be tested is a late-flowering Siberian apricot.

10. The use of SNP markers related to flowering period regulation of Siberian apricot according to claim 8, characterized in that: When obtaining the genotype of the Siberian apricot to be tested at the sites of SNP1, SNP2, SNP3 and SNP4 from the genomic DNA of the Siberian apricot to be tested, PCR is performed using the genomic DNA of the Siberian apricot to be tested as a template to obtain PCR products, and the PCR products are sequenced to obtain the genotype of the Siberian apricot to be tested at the sites of SNP1, SNP2, SNP3 and SNP4.

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