Siberia apricot kernel size regulation gene and coding protein, primer pair and application thereof

Abstract

The invention discloses a Siberia apricot kernel size regulation gene as well as an encoding protein, a primer pair and application thereof. The regulation gene is a PsPYL12 gene and a PsABI5 gene; the nucleotide sequence of the PsPYL12 gene is as shown in SEQ ID NO. 1, and the nucleotide sequence of the PsABI5 gene is as shown in SEQ ID NO. 2. The amino acid sequences of the encoding proteins of the two regulatory genes are respectively shown as SEQ ID NO.3 and SEQ ID NO.4; nucleotide sequences of a forward primer and a reverse primer for amplifying the PsPYL12 gene are respectively as shown in SEQ ID NO. 5 and SEQ ID NO. 6; and the nucleotide sequences of a forward primer and a reverse primer for amplifying the PsABI5 gene are respectively as shown in SEQ ID NO.7 and SEQ ID NO.8. The invention also discloses a kit for detecting the PsABI5 gene. The regulation gene participates in the abscisic acid signal channel to positively regulate the seed size of the plant. The invention provides a theoretical basis and a technical tool for high-yield breeding of the Siberia apricot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering. Specifically, it is a Siberian apricot kernel size regulation gene, its encoding protein, primer pair and application. BACKGROUND

[0002] Siberian apricot (Prunus sibirica L.) is a deciduous shrub or small tree of Rosaceae family, with strong cold, drought and poor soil tolerance, and is the main ecological and economic forest tree species in the "three north" region of China. At the same time, Siberian apricot is an important woody oil tree species, with kernel oil content of 36-52wt% and protein content of 23-31wt%, and has great economic value. Kernel size is the core agronomic trait that determines the yield of Siberian apricot, directly affecting the unit area yield and commodity value. Kernel development is a complex dynamic process, mainly divided into two stages of morphological establishment and mature accumulation: the former is mainly cell division and morphological differentiation of seed coat, embryo and endosperm, and the latter is the synthesis and accumulation of storage substances such as starch, lipid and protein; the size and weight of kernel are co-regulated by the two stages.

[0003] Related studies have shown that abscisic acid (ABA) plays an important role in kernel development, and regulates processes such as maturation, dormancy and storage material accumulation through complex metabolic and signaling networks. The biosynthesis of ABA (such as NCEDs, AAO3, etc. Gene regulation) and signal transduction (such as PYR / PYL receptor, PP2C protein phosphatase, SnRK2 kinase and ABF / ABI5 transcription factor) together constitute a regulatory network, affecting kernel cell division, swelling and material accumulation. However, the molecular regulation mechanism of Siberian apricot kernel size has not been clearly defined, and the specific role of key genes in the ABA signaling pathway still needs to be further analyzed, which limits the process of improving kernel size and yield through molecular breeding. Therefore, it is of great significance to explore the key genes and their mechanisms regulating the size of Siberian apricot kernel to promote high-yield breeding and industrial development. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a Siberian apricot kernel size regulation gene, its encoding protein, primer pair and application. By overexpressing PsPYL12 gene and / or PsABI5 gene, the kernel / seed weight, length and width of Siberian apricot and Arabidopsis can be significantly increased, providing a theoretical basis and technical tool for high-yield breeding of Siberian apricot.

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

[0006] The size-regulating genes of Siberian apricot kernel are PsPYL12 gene and PsABI5 gene; the nucleotide sequence of PsPYL12 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of PsABI5 gene is shown as SEQ ID NO. 2.

[0007] SEQ ID NO. 1:

[0008] ATGGTGGTATTTTATCCAACCCAAAAGCAACAAACCCTAACCAAAACACAGCTCATGATCAGCAGCTACCATAGCCACACTCTGTTACCAAACCAGTGTGGCTCGAGCCTTGTCCAAACCATCGACGCGCCGCTGCCCCTGGTCTGGTCCGTCCTCCGCCAATTCGACAACCCTCAAGCCTACAAGCAGTTCATCAAGAGCTGCAGCATGCGTGCCGGCAATGGAGGCATCGGAAGCATCCGGGAAGTCGTGGTTAAAACCGGCTTGCCGGCGAAAACCAGCATGGAGAGGCTGGATGAGCTCGACGACAACATGCATGTCATGCATTATAGCATTGTTGGTGGAGATCACAGGCTTGCAAATTACAGTTCTACCACTACCGTGCACAGAGAAGAGGAAGGGAAGGCTGTTGTGATTCAGTCGTATGTGGTGGATGTGCCTGCCGGGAGCAGCGAGGAGGACACTTGTTCGTTTACTAATACGATCATAGGCTGCAATCTTAAGTCGCTGGCTAAAGTCACAGAAAAGATGGCTGCTAATAATTAA;

[0009] SEQ ID NO. 2:

[0010] ATGGGGATACAGACAATGGGTTCTCAAGGTGGGGCTGATGGAAATTGCAAACAGTCGCAGTTCCAGCCTTTGGCACGACAAAACTCAATTTACAGTCTCACACTCGATGAGGTACAAAATCAGTTAGGTGATTTGGGGAAGCCACTCAGCAGCATGAACCTTGACGAGCTTCTAAAAAATGTATGGAGTGCTGAGGCCAACCAGACCATGGGTATGGACATTGAAGGCACTACACTGGTCAATCAAGCTACACTGCAGCGTCAGGCAAGCCTGTCATTAACTAGTGCATTGAGCAAGAAGACAGTTGATGAGGTTTGGAGAGATATTCAACAAAGCAAAAATAATGAGGAAAAGAAATCTCAAGAACGACAACGTACTTTGGGAGAGATGACCTTGGAGGATTTCTTGGTAAAAGCGGGAGTTGTTGCTGAAGCTGAAGCATCTTCAGACAAAAAGTGTTCTGCTCCTCTTGCCGTGGTTGATGCGAATGTGGCATCTCAGTTTCCGCAAGGTCAGTGGCTGCAGTACCAACAACCACAATATCAACATCCACAACAAAGTATGATGGGGGTATACATGCCAAGCCAACCTATACCACCGCCACTGCACATAGGGGCTGGTGCTATAATGGAAGTGCCGTATCCTGACAACCAAGTTGCGTTGCCTTCACCCTTAATGGGTACGTTATCAGATACACAGACACCTGGGAGGAAAAGGGGCAACCCAGAGGACATAGTTGAGAAGACTGTTGAACGGAGACAGAAAAGAATGATAAAGAACCGAGAATCTGCTGCTCGTTCACGAGCACGAAAGCAGGCTTATACAAATGAACTGGAGAACAAAGTTTCACGTCTAGAGGAGGAAAATGAAAGGCTAAGGAAACAGAAGGAGCTAGAGAAGGTGTTGCCAAGTGCACCACCTCCCGAGCCAAAGTATCAGCTTCGTAGAACGACGTCAGCTCCATTCTAA.

[0011] The protein encoded by the Siberian apricot kernel size regulation gene, the PsPYL12 protein encoded by the Siberian apricot kernel size regulation gene or the PsPYL12 protein is involved in the regulation of the size of the Siberian apricot kernel. The amino acid sequence of the PsPYL12 protein is shown as SEQ ID NO. 3, and the amino acid sequence of the PsPYL12 protein is shown as SEQ ID NO. 4.

[0012] SEQ ID NO. 3 (amino acid sequence encoded by the sequence shown as SEQ ID NO. 1):

[0013] MVVFYPTQKQQTLTKTQLMISSYHSHTLLPNQCGSSLVQTIDAPLPLVWSVLRQFDNPQAYKQFIKSCSMRAGNGGIGSIREVVVKTGLPAKTSMERLDELDDNMHVMHYSIVGGDHRLANYSSTTTVHREEEGKAVVIQSYVVDVPAGSSEEDTCSFTNTIIGCNLKSLAKVTEKMAANN

[0014] SEQ ID NO. 4 (amino acid sequence encoded by the sequence shown as SEQ ID NO. 2):

[0015] MGIQTMGSQGGADGNCKQSQFQPLARQNSIYSLTLDEVQNQLGDLGKPLSSMNLDELLKNVWSAEANQTMGMDIEGTTLVNQATLQRQASLSLTSALSKKTVDEVWRDIQQSKNNEEKKSQERQRTLGEMTLEDFLVKAGVVAEAEASSDKKCSAPLAVVDANVASQFPQGQWLQYQQPQYQHPQQSMMGVYMPSQPIPPPLHIGAGAIMEVPYPDNQVALPSPLMGTLSDTQTPGRKRGNPEDIVEKTVERRQKRMIKNRESAARSRARKQAYTNELENKVSRLEEENERLRKQKELEKVLPSAPPPEPKYQLRRTTSAPF

[0016] The primer pair for amplifying the Siberian apricot kernel size regulation gene is used to amplify the Siberian apricot kernel size regulation gene. The nucleotide sequences of the forward primer and the reverse primer for amplifying the PsPYL12 gene are shown as SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The nucleotide sequences of the forward primer and the reverse primer for amplifying the PsABI5 gene are shown as SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

[0017] SEQ ID NO. 5: ATGGTGGTATTTTATCCAAC;

[0018] SEQ ID NO. 6: TTAATTATTAGCAGCCATCT;

[0019] SEQ ID NO. 7: ATGGGGATACAGACAATGGG;

[0020] SEQ ID NO. 8: TTAGAATGGAGCTGACGTCG.

[0021] The primer pair for amplifying the Siberian apricot kernel size regulating gene is used for amplification of the cDNA obtained by reverse transcription of the Siberian apricot kernel RNA.

[0022] The Siberian apricot kernel size regulating gene is used for participating in the positive regulation of the kernel size of plants by the abscisic acid signal pathway.

[0023] The Siberian apricot kernel size regulating gene is used for the following any one of the following uses:

[0024] (a) increasing the weight of the Siberian apricot kernel;

[0025] (b) increasing the length of the Siberian apricot kernel;

[0026] (c) increasing the width of the Siberian apricot kernel;

[0027] (d) improving the seed size trait of Arabidopsis thaliana by transgenic technology;

[0028] When the expression amount of the PsPYL12 gene and / or the PsABI5 gene is increased, the weight, length and width of the Arabidopsis thaliana seed are increased.

[0029] The Siberian apricot kernel size regulating gene is used for improving the seed size of Siberian apricot or Arabidopsis thaliana by the method of using the PsPYL12 gene and / or the PsABI5 gene, which comprises the following steps: recombining the CDS sequence of the PsPYL12 gene and / or the PsABI5 gene into the corresponding site of the pDMC32 plasmid through the Gateway™ system to obtain a recombinant overexpression vector; transforming the recombinant overexpression vector into Agrobacterium GV3101 and introducing it into Arabidopsis thaliana plants for overexpression to increase the expression amount of the PsPYL12 gene and / or the PsABI5 gene in the plants, and the hundred-grain weight, length and width of the seed of the Siberian apricot or Arabidopsis thaliana plant in which the PsPYL12 gene and / or the PsABI5 gene is successfully expressed are all increased.

[0030] The application of the Siberian apricot kernel size regulation gene is used for predicting the apricot kernel size by using the Siberian apricot kernel size regulation gene.

[0031] The technical scheme of the present application has the following beneficial technical effects:

[0032] 1. The Siberian apricot kernel size regulation gene PsPYL12 gene and PsABI5 gene provided by the present application regulate the Siberian apricot kernel size through the ABA signal pathway, and the overexpression of the PsPYL12 gene and the PsABI5 gene can significantly improve the weight, length and width of the Siberian apricot kernel, which further verifies the role of the PsPYL12 gene and the PsABI5 gene in regulating the kernel size.

[0033] 2. The PsPYL12 gene and the PsABI5 gene recombinant overexpression vector and the transgenic technology constructed by the present application can be directly used for genetic improvement of the Siberian apricot, and the precise improvement of the kernel size can be realized by directional regulation of gene expression, thereby shortening the breeding cycle. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figures 1A-1D respectively, the length, width, thickness and weight of the Siberian apricot kernel in different development periods (S1-S6) in the embodiment of the present application are shown in the four phenotype change graphs;

[0035] Figure 2 the content change graph of abscisic acid (ABA) in the Siberian apricot kernel in different development periods (S1-S6) in the embodiment of the present application is shown in the graph;

[0036] Figures 3A-3D respectively, the proportion distribution graph of expressed genes in the total detected genes, the gene expression level (FPKM) distribution graph, the hierarchical clustering graph based on the Pearson correlation coefficient and the principal component analysis (PCA) result graph of the Siberian apricot kernel in different development periods (S1-S6) in the embodiment of the present application are shown in the graphs;

[0037] Figures 4A-4C respectively, the DEGs (differentially expressed genes) number analysis result graph, the Venn analysis result graph of DEGs and the STEM clustering analysis result graph of DEGs of each pair comparison (S1 vs S2, S2 vs S3, S3 vs S4, S4 vs S5, S5 vs S6) in the continuous stage of the Siberian apricot kernel development in the embodiment of the present application are shown in the graphs;

[0038] Figure 5A and Figure 5B respectively, the DEGs number statistical graph involved in the plant hormone signal transduction pathway in the pair stage comparison and the expression mode graph of DEGs in the ABA biosynthesis and signal transduction pathway in the embodiment of the present application are shown in the graphs.

[0039] Figure 6A and Figure 6B Fig. 9 and Fig. 10 are respectively RT-qPCR and RNA-seq data correlation analysis results of differentially expressed genes (DEGs) in Siberian apricot kernel development in the embodiment of the present application;

[0040] Figure 7 Fig. 11 is a weight comparison chart of large kernel and small kernel mature kernels of Siberian apricot in the embodiment of the present application;

[0041] Figure 8A and Figure 8B Fig. 12 and Fig. 13 are respectively expression amount comparison charts of PsPYL12 gene and PsABI5 gene in large kernel and small kernel mature kernels in the embodiment of the present application;

[0042] Figure 9 Fig. 14 is a gel electrophoresis chart of PCR amplification products of PsPYL12 gene and PsABI5 gene in the embodiment of the present application;

[0043] Figure 10A and Figure 10B Fig. 15 and Fig. 16 are respectively fluorescence quantitative qPCR identification results of transgenic positive Arabidopsis thaliana into which PsPYL12 gene and PsABI5 gene are transferred in the embodiment of the present application;

[0044] Figure 11 Fig. 17 is a seed phenotype comparison chart of wild-type Arabidopsis thaliana WT, transgenic Arabidopsis thaliana PsPYL12#1, transgenic Arabidopsis thaliana PsPYL12#2, transgenic Arabidopsis thaliana PsPYL12#3, transgenic Arabidopsis thaliana PsABI5#1, transgenic Arabidopsis thaliana PsABI5#2 and transgenic Arabidopsis thaliana PsABI5#3 in the embodiment of the present application;

[0045] Figures 12A-12F Fig. 18, Fig. 19 and Fig. 20 are respectively seed weight, length and width comparison results charts of wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana in the embodiment of the present application. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the methods in the embodiments are all conventional operations; the materials used are purchased from conventional biochemical reagent companies; the fluorescence quantitative experiment is set with three repetitions, and the results are averaged. Wild-type Arabidopsis thaliana is selected as Columbia ecotype Columbia-0 (Col-0). The 'Zhalute' (ZLT) Siberian apricot kernel is used as the material in the embodiments.

[0047] 1. Changes of Siberian apricot kernel phenotype and abscisic acid content at different development stages

[0048] This embodiment uses the kernels of 'Zhalute' (ZLT) Siberian apricots grown at the experimental base of the Economic Forest Research Institute in Yuanyang County, Henan Province as material. Kernels were collected from late March to early June 2020 at 15 days (S1), 30 days (S2), 45 days (S3), 60 days (S4), 75 days (S5), and 90 days (S6) after flowering.

[0049] (1) Phenotypic changes

[0050] Phenotypic analysis of six developmental stages indicated that Siberian apricot kernel development can be divided into two stages: S1 to S4 is a rapid growth period, during which the length, width, thickness, and weight of the kernel ( Figures 1A-1D All three phases continued to increase, with the most significant growth occurring from S1 to S2. In S2, the length, width, thickness, and weight were 1.927 times, 2.54 times, 2.45 times, and 8.85 times that of S1, respectively. From S5 to S6, the growth period was slow, and the changes in morphology and weight tended to stabilize. In S6, all indicators were only 1.01-1.08 times that of S5. This stage was mainly focused on nutrient accumulation, and S4 was the key turning point between the two stages.

[0051] (2) Changes in abscisic acid content

[0052] The content of abscisic acid (ABA) was determined using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), with at least three biological replicates for each measurement. Figure 2 As shown, the ABA content gradually increased from 50.40 ng / g in stage S1, reaching a peak of 128.93 ng / g in stage S4, a 2.56-fold increase compared to stage S1; then it rapidly decreased, reaching a minimum of 19.77 ng / g in stage S6. The rapid increase in ABA content from stage S1 to S4 is similar to the growth pattern of Siberian apricot kernel phenotype, suggesting that ABA may be involved in regulating the kernel development process.

[0053] 2. Screening of genes regulating kernel size in Siberian apricots

[0054] (1) RNA sequencing

[0055] In this example, RNA sequencing was performed on 6 developmental stages of ZLT kernel samples, with 3 biological replicates for each stage, for a total of 18 samples. Sequencing was performed using the Illumina Novaseq 6000 platform, generating 2x150 bp paired-end reads. After filtering low-quality sequences with fastp software, 132.14 Gb of high-quality clean data was obtained, with an average of 7.34 Gb per sample. The average values of Q20 and Q30 reached 99.98% and 98.22%, respectively, and the average GC content was 47.25%. 98.50% of the clean reads could be successfully aligned to the Siberian apricot 'F106' reference genome (tfGDR1049), indicating high data reliability.

[0056] In this example, a total of 32959 genes were detected, of which 25986 genes were expressed at least at one developmental stage (FPKM>0). The proportion of genes expressed at each stage accounted for 59.86%-70.65% of the total detected genes. The proportion of genes expressed at S1-S3 was higher than that at S4-S6 (P<0.05). Figure 3A The distribution of gene expression levels showed that 49.91%-62.49% of the genes were extremely lowly expressed (0<FPKM≤1), and 2.68%-7.67% of the genes were extremely highly expressed (FPKM>60). Figure 3B Hierarchical clustering and principal component analysis (PCA) based on Pearson correlation coefficients showed that the transcriptome data was clearly divided into two groups: S1-S3 and S4-S6, and the samples within the S4-S6 group clustered more closely. Figure 3C and Figure 3D This further supports the molecular differences between the two stages of kernel development.

[0057] (2) Pairwise comparison of transcriptome data

[0058] Pairwise comparison of transcriptome data was performed on the consecutive stages of kernel development (S1 vs S2, S2 vs S3, S3 vs S4, S4 vs S5, S5 vs S6). EdgeR software was used to screen significant differentially expressed genes (DEGs) with |Log2(fold change)|>1 and corrected p-value (q-value)<0.05 as the threshold. Principal component analysis (PCA) and short-term expression mining (STEM) clustering analysis were performed on the OmicShare platform. Transcription factors were annotated using the PlantTFDB database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to determine the number of DEGs related to each pathway.

[0059] The results are as follows: Figures 4A-4CAs shown, the number of DEGs in the five pairwise comparisons ranged from 2604 to 6276, with the most DEGs in S6 vs S5 (6276, 3350 up-regulated and 2926 down-regulated) and the least in S3 vs S2 (2604, 1296 up-regulated and 1308 down-regulated) Figure 4A Venn analysis showed that 42 DEGs were common to all comparison groups, while S2 vs S1, S3 vs S2, S4 vs S3, S5 vs S4, and S6 vs S5 had 1460, 595, 731, 578, and 3161 unique DEGs, respectively, which may be involved in the regulation of kernel growth and development Figure 4B Short-term expression mining (STEM) clustering analysis of 9678 significant DEGs yielded 20 main profiles (Profiles 0-19), of which Profile 0 contained the most DEGs (4449) and showed a decreasing trend throughout the entire process; Profile 19 contained 529 DEGs and showed an increasing trend, consistent with the overall increase in kernel weight and size; the expression patterns of Profile 10 (269 DEGs) and Profile 14 (159 DEGs) were consistent with the content of abscisic acid (ABA), both reaching a peak at S4, while ABA also reached a peak at S4 of kernel development, indicating that these DEGs may be related to ABA Figure 4C .

[0060] (3) KEGG identification of DEGs in kernel development hormone pathways

[0061] KEGG analysis identified a large number of DEGs involved in plant hormone signal transduction, biosynthesis, and metabolic pathways Figure 5A and Figure 5B Among them, 80, 85, 101, 75, and 134 DEGs in S2 vs S1, S3 vs S2, S4 vs S3, S5 vs S4, and S6 vs S5 stages were enriched in the plant hormone signal transduction pathway, indicating that these genes play an important role in kernel growth and development. Further analysis of the ABA biosynthesis and signaling pathway revealed 26 DEGs (15 up-regulated and 11 down-regulated), of which PsABI5 gene (PaF106G0200007562.01) and PsPYL12 gene (PaF106G0400017615.01) reached a peak at S4, consistent with the peak of ABA content, and then down-regulated.

[0062] (4) Verification of differentially expressed genes (DEGs)

[0063] Verification of DEGs by RT-qPCR: Total RNA of each sample was reverse transcribed using the All-in-One First-Strand Synthesis MasterMix kit (Thermo Scientific), and specific primers for 2 candidate DEGs were designed using Primer Premier 5 software (Table 1). UBQ2 was used as an internal reference gene to correct the RT-qPCR results. The reaction was performed using 2x SYBR Green qPCR Master Mix (Beijing Kangwei Century), with 4 technical replicates for each reaction. The relative gene expression was calculated using 2 -ΔΔCT The Pearson correlation coefficient (r) and statistical significance (p value) of the mean expression of RNA-seq and RT-qPCR at each time point were analyzed using SPSS v27.0.1.0 software, and statistical graphs were drawn using OriginPro 2021 software.

[0064] Table 1

[0065]

[0066] The verification results showed that the RT-qPCR and RNA-seq data of DEGs were significantly correlated (p≤0.05, r>0.8), confirming the reliability of the RNA-seq data Figure 6A and Figure 6B ).

[0067] In addition, 14 seed kernels (7 large kernels and 7 small kernels) were collected from late May to mid-June 2023 for weight determination Figure 7 ), RNA extraction, reverse transcription, and comparison of the expression of PsPYL12 and PsABI5 genes in 7 large kernels and 7 small kernels using RT-qPCR. The results showed that the expression of both genes in small kernels was significantly lower than that in large kernels Figure 8A and Figure 8B ), which was consistent with the phenotypic differences, indicating that these two genes played an important role in seed kernel growth and size regulation.

[0068] (5) Coding sequences of PsPYL12 and PsABI5 genes

[0069] Based on the Siberian apricot genome database, the coding sequence (CDS) regions of PsPYL12 and PsABI5 genes were finally determined.

[0070] PsPYL12 gene is located at position 3881352-3882144 on chromosome 4, with gene ID PaF106G0400017615. The CDS sequence information is as follows:

[0071] PaF106G0400017615 (SEQ ID NO.1) :

[0072] ATGGTGGTATTTTATCCAACCCAAAAGCAACAAACCCTAACCAAAACACAGCTCATGATCAGCAGCTACCATAGCCACACTCTGTTACCAAACCAGTGTGGCTCGAGCCTTGTCCAAACCATCGACGCGCCGCTGCCCCTGGTCTGGTCCGTCCTCCGCCAATTCGACAACCCTCAAGCCTACAAGCAGTTCATCAAGAGCTGCAGCATGCGTGCCGGCAATGGAGGCATCGGAAGCATCCGGGAAGTCGTGGTTAAAACCGGCTTGCCGGCGAAAACCAGCATGGAGAGGCTGGATGAGCTCGACGACAACATGCATGTCATGCATTATAGCATTGTTGGTGGAGATCACAGGCTTGCAAATTACAGTTCTACCACTACCGTGCACAGAGAAGAGGAAGGGAAGGCTGTTGTGATTCAGTCGTATGTGGTGGATGTGCCTGCCGGGAGCAGCGAGGAGGACACTTGTTCGTTTACTAATACGATCATAGGCTGCAATCTTAAGTCGCTGGCTAAAGTCACAGAAAAGATGGCTGCTAATAATTAA;

[0073] PsABI5 is located at 6741964~6749130 of the 2nd chromosome in the genome, and the gene ID is PaF106G0200007562. The CDS sequence (i.e. coding sequence) information is as follows:

[0074] PaF106G0200007562 (SEQ ID NO.2) :

[0075] ATGGGGATACAGACAATGGGTTCTCAAGGTGGGGCTGATGGAAATTGCAAACAGTCGCAGTTCCAGCCTTTGGCACGACAAAACTCAATTTACAGTCTCACACTCGATGAGGTACAAAATCAGTTAGGTGATTTGGGGAAGCCACTCAGCAGCATGAACCTTGACGAGCTTCTAAAAAATGTATGGAGTGCTGAGGCCAACCAGACCATGGGTATGGACATTGAAGGCACTACACTGGTCAATCAAGCTACACTGCAGCGTCAGGCAAGCCTGTCATTAACTAGTGCATTGAGCAAGAAGACAGTTGATGAGGTTTGGAGAGATATTCAACAAAGCAAAAATAATGAGGAAAAGAAATCTCAAGAACGACAACGTACTTTGGGAGAGATGACCTTGGAGGATTTCTTGGTAAAAGCGGGAGTTGTTGCTGAAGCTGAAGCATCTTCAGACAAAAAGTGTTCTGCTCCTCTTGCCGTGGTTGATGCGAATGTGGCATCTCAGTTTCCGCAAGGTCAGTGGCTGCAGTACCAACAACCACAATATCAACATCCACAACAAAGTATGATGGGGGTATACATGCCAAGCCAACCTATACCACCGCCACTGCACATAGGGGCTGGTGCTATAATGGAAGTGCCGTATCCTGACAACCAAGTTGCGTTGCCTTCACCCTTAATGGGTACGTTATCAGATACACAGACACCTGGGAGGAAAAGGGGCAACCCAGAGGACATAGTTGAGAAGACTGTTGAACGGAGACAGAAAAGAATGATAAAGAACCGAGAATCTGCTGCTCGTTCACGAGCACGAAAGCAGGCTTATACAAATGAACTGGAGAACAAAGTTTCACGTCTAGAGGAGGAAAATGAAAGGCTAAGGAAACAGAAGGAGCTAGAGAAGGTGTTGCCAAGTGCACCACCTCCCGAGCCAAAGTATCAGCTTCGTAGAACGACGTCAGCTCCATTCTAA;

[0076] SEQ ID NO. 3 (amino acid sequence encoded by the sequence shown in SEQ ID NO. 1):

[0077] MVVFYPTQKQQTLTKTQLMISSYHSHTLLPNQCGSSLVQTIDAPLPLVWSVLRQFDNPQAYKQFIKSCSMRAGNGGIGSIREVVVKTGLPAKTSMERLDELDDNMHVMHYSIVGGDHRLANYSSTTTVHREEEGKAVVIQSYVVDVPAGSSEEDTCSFTNTIIGCNLKSLAKVTEKMAANN

[0078] SEQ ID NO. 4 (amino acid sequence encoded by the sequence shown in SEQ ID NO. 2):

[0079] MGIQTMGSQGGADGNCKQSQFQPLARQNSIYSLTLDEVQNQLGDLGKPLSSMNLDELLKNVWSAEANQTMGMDIEGTTLVNQATLQRQASLSLTSALSKKTVDEVWRDIQQSKNNEEKKSQERQRTLGEMTLEDFLVKAGVVAEAEASSDKKCSAPLAVVDANVASQFPQGQWLQYQQPQYQHPQQSMMGVYMPSQPIPPPLHIGAGAIMEVPYPDNQVALPSPLMGTLSDTQTPGRKRGNPEDIVEKTVERRQKRMIKNRESAARSRARKQAYTNELENKVSRLEEENERLRKQKELEKVLPSAPPPEPKYQLRRTTSAPF

[0080] 3. Function verification of Siberian apricot PsPYL12 gene and PsABI5 gene in regulating kernel size

[0081] Specific primers (Table 2) were designed to amplify the target sequences; after the PCR reaction program was completed, the PCR reaction products were subjected to 1% agarose gel electrophoresis, and the electrophoresis results are shown in Figure 1. Figure 9As shown in the figure, clear bands of the PsPYL12 and PsABI5 genes (target bands) are visible. The target bands were excised and recovered using a gel. The Tiangen agarose gel DNA recovery kit DP209-02 was used for gel excision and recovery. Vector construction was performed using the Gateway cloning system: purified PCR products containing adapter sequences were recombined into pDONR207 and sequenced. Correctly sequenced vectors were cloned and recombined into the pDMC32 target expression vector. The final construct was verified by sequencing. To investigate the effects of these two genes on seed size, the overexpression vector was constructed and the resulting plasmid was transformed into Agrobacterium GV3101. This plasmid was then heterologously transformed into Arabidopsis thaliana, and the inflorescence immersion method was used to transform the Arabidopsis ecotype Col-0. The obtained Arabidopsis plants were screened on 1 / 2 MS medium containing 50 mg / L hygromycin and detected by RT-qPCR. Figure 10A and Figure 10B ), obtaining 3 independent T1 transgenic lines for each gene ( Figure 11 , Figures 12A-12C ).

[0082] Table 2

[0083]

[0084] Phenotypic analysis of the transgenic plants showed that, compared with the wild type (WT, average 1.324 mg per 100 seeds, seed length 381.79 mm, width 226.72 mm), all tested PsPYL12 gene overexpression lines (1.78 mg, 2.062 mg, 2.14 mg per 100 seeds, lengths 406.38 mm, 450.53 mm, 454.51 mm, widths 260.94 mm, 271.82 mm, 273.24 mm, respectively) and PsABI5 gene overexpression lines (1.839 mg, 1.863 mg, 2.091 mg per 100 seeds, lengths 405.07 mm, 407.89 mm, 426.1 mm, widths 255.9 mm, 258.46 mm, 252.87 mm, respectively) had significantly increased seed weight, length, and width. The above results indicate that the PsPYL12 and PsABI5 genes play a positive regulatory role in seed size development.

[0085] In summary, this embodiment screened and identified key genes regulating the kernel size of Siberian apricot seeds and their application methods through phenotypic analysis, endogenous hormone content determination, transcriptome sequencing, and functional verification. It revealed the ABA-mediated growth and development pathway of Siberian apricot kernels, identified core regulatory genes for kernel size, and verified their functions, providing a theoretical basis and molecular biological tools for targeted improvement of kernel size.

[0086] Obviously, the above-described embodiments are merely exemplary but not restrictive. Based on the above description, one of ordinary skill in the art can make other different forms of changes or variations. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the patent application claims.

Claims

1. A Siberian apricot kernel size regulating gene, characterized in that, PsPYL12 gene and PsABI5 gene; the nucleotide sequence of the PsPYL12 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the PsABI5 gene is shown as SEQ ID NO.

2.

2. A protein encoded by a Siberian apricot kernel size regulatory gene, characterized in that, PsPYL12 protein or PsPYL12 protein encoded by the Siberian apricot kernel size regulatory gene according to claim 1; the amino acid sequence of the PsPYL12 protein is shown as SEQ ID NO. 3, and the amino acid sequence of the PsPYL12 protein is shown as SEQ ID NO.

4.

3. A primer pair for amplifying a Siberian apricot kernel size regulatory gene, characterized in that, The Siberian apricot kernel size regulatory gene according to claim 1; the nucleotide sequences of the forward primer and the reverse primer for amplifying the PsPYL12 gene are shown as SEQ ID NO. 5 and SEQ ID NO. 6, respectively; and the nucleotide sequences of the forward primer and the reverse primer for amplifying the PsABI5 gene are shown as SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

4. The primer pair for amplifying the Siberian apricot kernel size regulatory gene according to claim 3, characterized in that, The template used in amplification is cDNA obtained by reverse transcription of Siberian apricot kernel RNA.

5. Use of a Siberian apricot kernel size regulation gene, characterized in that, The Siberian apricot kernel size regulatory gene according to claim 1 is used to positively regulate plant kernel size in the abscisic acid signal pathway.

6. Use of the Siberian apricot kernel size regulating gene according to claim 5, characterized in that, The PsPYL12 gene and / or the PsABI5 gene are used for any of the following purposes: (a) increasing the weight of Siberian apricot kernels; (b) increasing the length of Siberian apricot kernels; (c) increasing the width of Siberian apricot kernels; (d) improving the seed size trait of Arabidopsis thaliana by transgenic technology; When the expression amount of the PsPYL12 gene and / or the PsABI5 gene is increased, the weight, length and width of Arabidopsis thaliana seeds are increased.

7. Use of the Siberian apricot kernel size regulating gene according to claim 6, characterized in that, The method for improving the size of Siberian apricot kernels or Arabidopsis thaliana seeds by using the PsPYL12 gene and / or the PsABI5 gene is as follows: the CDS sequence of the PsPYL12 gene and / or the PsABI5 gene is recombined into the corresponding site of the pDMC32 plasmid through the Gateway™ system to obtain a recombinant overexpression vector; the recombinant overexpression vector is transformed into Agrobacterium GV3101 and introduced into Arabidopsis thaliana plants for overexpression to increase the expression amount of the PsPYL12 gene and / or the PsABI5 gene in the plants.

8. Use of a Siberian apricot kernel size regulation gene, characterized in that, The Siberian apricot kernel size regulatory gene according to claim 1 is used to predict apricot kernel size.

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