Key gene AhPDS1 of plant pods and application thereof

Abstract

The invention discloses a key gene AhPDS1 of plant pods, which is characterized in that the nucleotide sequence of the key gene AhPDS1 is shown as SEQ ID NO.1. The invention also provides the key gene AhPDS1 of the plant pods, the amino acid sequence of the key gene AhPDS1 is shown as SEQ ID NO.2, the key gene AhPDS1 is used for regulating and controlling the sizes of the plant pods and seeds, and the plant is peanut. Compared with the prior art, the invention has the advantages that a site or a target gene which can be used for peanut molecular breeding is provided, and the key gene AhPDS1 of the plant legume for illustrating a biological mechanism for controlling the sizes of the legume and seeds and the application of the key gene AhPDS1 are provided.

Description

Technical Field

[0001] The invention relates to the technical fields of plant molecular biology and genetic engineering, and in particular to a key gene AhPDS1 of plant pods and an application thereof. Background Art

[0002] Cultivated peanuts belong to the leguminous plant family and have become an important oil crop because they are rich in edible vegetable oils, proteins and vitamins. Peanuts are widely grown in more than 100 countries around the world. In 2020, the global peanut production was about 54 million tons, and the planting area was about 32 million hectares.

[0003] Compared with the continuous increase in peanut consumption, the global peanut production has been sluggish and difficult to meet human demand. Therefore, it has become an urgent need to increase peanut production through molecular breeding.

[0004] Cultivated peanut is an allotetraploid species (AABB, 2n=4x=40), which is presumed to be the result of natural hybridization between two diploid ancestors, A.duranensis (AA, 2n=2x=20) and A.ipaensis (BB, 2n=2x=20) in southwestern South America. Yield improvement is the focus of breeding, and pod and seed size traits have a direct impact on it. Therefore, it is necessary to understand the genetic basis of pod and seed related traits in order to facilitate molecular marker-assisted selection and cultivate new high-yield varieties. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above technical defects, provide a site or target gene that can be used for peanut molecular breeding, and clarify a key gene AhPDS1 of plant pods that controls the biological mechanism of pod and seed size and its application.

[0006] In order to solve the above technical problems, the present invention provides a technical solution: a key gene AhPDS1 of plant pods, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] Preferably, its amino acid sequence is shown as SEQ ID NO.2.

[0008] Another aspect of the present invention provides a recombinant vector, comprising a key gene AhPDS1 and an original vector.

[0009] Preferably, the original vector is pEGOEP35S-H-GFP.

[0010] Another aspect of the present invention provides the application of the key gene AhPDS1 in regulating the size of plant pods and seeds.

[0011] Preferably, the key gene AhPDS1 regulates the biosynthesis of plant IAA through the IPA pathway.

[0012] Another method of the present invention provides the use of the recombinant vector in regulating the size of plant pods and seeds.

[0013] Preferably, the recombinant vector of the key gene AhPDS1 regulates the biosynthesis of plant IAA through the IPA pathway.

[0014] Preferably, the plant is peanut.

[0015] The advantages of the present invention over the prior art are as follows: in the present invention, the key gene AhPDS1 is extracted by studying peanut genes and can regulate the size of plant pods and seeds, providing a comprehensive understanding of the genetic and molecular mechanisms of peanut seed size regulation, and the discovery that AhPDS1 regulates the biosynthesis of IAA through the IPA pathway and localizes it to specific cellular locations further confirms its core role in determining seed size, which is of great significance to peanut breeding programs for improving seed and pod traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of research data of an embodiment of the present invention.

[0017] Figure 2 This is the chromosome analysis map of peanut B06.

[0018] Figure 3 This is a comparative analysis chart of peanut varieties.

[0019] Figure 4 is a schematic diagram of the analysis of AhPDS1.

[0020] Figure 5 This is a schematic diagram of the function of AhPDS1.

[0021] Figure 6 This is a schematic diagram of AhPDS1 localization analysis. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0023] Figure 1 :B: Visual comparison between small- and large-fruited peanut varieties is shown; C: Pearson correlation analysis was used to evaluate the phenotypic correlations between different combinations of pod- and seed-related traits in four different experimental environments.

[0024] Figure 2Middle: A, B: Manhattan plots showing SNPs (single nucleotide polymorphisms) associated with pod and seed size traits on peanut chromosome B06. The horizontal dashed line indicates the genome-wide significance threshold (P = 1 × 10 -6 ), determined by Bonferroni correction to reduce false positives; C: Selective sweep analysis of chromosome B06, indicating regions of reduced genetic diversity that may have been subject to strong positive selection during domestication; D, E, F: Linkage disequilibrium (LD) heat map of the top SNPs associated with pod length identified in this study. The heat map shows the LD strength between SNPs, with the most significant SNP highlighted in red. This SNP is associated with the candidate gene AhPDS1; G: Gene structure of AhPDS1 highlighting a non-synonymous SNP (C / a substitution) located in the fourth exon; this SNP results in an amino acid change that may affect the trait; H: Box plot of pod and seed size in different varieties. The pod and seed size of materials with CC genotype were significantly increased compared to those with AA genotype, indicating that this genotype has a strong correlation with the size trait.

[0025] Figure 3 Middle: A: Phenotypic comparison of large- and small-fruited peanut varieties. This image shows the physical differences between large-fruited and small-fruited peanuts, highlighting the variation in pod size.

[0026] B: Expression dynamics of AhPDS1 in peanut varieties with different pod sizes (Size). The figure shows the change in the expression level of AhPDS1 with pod size, indicating its potential role in pod development.

[0027] Figure 4 Middle: A: Overexpression of AhPDS1 in Arabidopsis. This panel shows the phenotypic differences between the overexpression lines (OE-SP and OE-LP) and the wild-type (Col) plants. Specifically, overexpression of AhPDS1 appears to affect the size of pods and seeds.

[0028] B: Relative expression levels of AhPDS1 in OE-SP, OE-LP and wild-type Arabidopsis. Quantitative results showed that AhPDS1 was significantly overexpressed in transgenic lines compared with the wild type.

[0029] C: Comparative analysis of phenotypic parameters between overexpressing AhPDS1 and wild-type Arabidopsis, including leaf length (n=9), leaf width (n=9), pod length (n=9), pod width (n=9), seed length (n=30) and seed width (n=30). Compared with the wild type, the overexpression lines showed significant differences in seed and pod size. The scale bar is provided in the figure.

[0030] Figure 5A: Heat map of indole-3-acetic acid (IAA) and related conjugates in AhPDS1 overexpressing and wild-type lines. The heat map provides a comparative view of auxin levels, showing an increase in IAA and conjugates in the overexpressing cell lines. B: IAA biosynthetic pathway schematic and bar graph comparing the content of each biosynthetic intermediate in overexpressing and wild-type plants. The higher levels of metabolites in the overexpressing lines indicate that AhPDS1 affects IAA biosynthesis and contributes to the observed phenotypic changes.

[0031] Figure 6 is: Subcellular localization of AhPDS1; (A) illustrates the subcellular localization of AhPDS1 protein in peanut leaf cell protoplasts. The construct pEGOEP35S-H-AhPDS1-GFP was transiently expressed in protoplasts, and GFP signals (green) were detected throughout the cytoplasm. The bright field image provides a visual reference for the structure of the protoplasts. The merged image shows the overlap of the GFP signal with the bright field image. A control construct (pEGOEP35S-H-GFP) expressing only GFP protein was used to demonstrate the specific localization pattern of AhPDS1. The red signal corresponds to a nuclear marker, highlighting the absence of AhPDS1 in the nucleus. Scale bar: 10 μm. (B) Histochemical analysis of AhPDS1 promoter activity in different tissues of Arabidopsis;

[0032] Transgenic Arabidopsis expressing pAhPDS1-GUS showed GUS activity in roots, stems, leaves, trichomes, flowers, and pods, stained blue. This broad expression pattern highlights the potential importance of AhPDS1 in different developmental stages and tissue types. Scale bar: 1 mm.

[0033] The present invention adopts the genome-wide association analysis (GWAS) method to identify new loci associated with pod and seed size, clarify the candidate genes behind these potential associations, and provide loci or target genes that can be used for peanut molecular breeding.

[0034] In specific implementation:

[0035] The association mapping population of the present invention includes 390 peanut materials from 15 countries or regions around the world. The detailed information of the origin, population structure and genetic diversity of the materials has been previously documented (Lu et al., 2024). All materials were planted in Guangzhou, China (113.28°E, 23.12°N) in spring and autumn every year in 2017 and 2018. In each season, the pod length (PL), pod width (PW), pod thickness (PT), 100 pod weight (HPW), seed length (SL), seed width (SW), seed thickness (ST) and 100 seed weight (HSW) were measured for each material after harvest.

[0036] The analysis showed that these traits had continuous distributions in different environments and were approximately normally distributed ( Table 1 ; Figure 1 C), the genetic background of different materials is diverse, which provides a basis for GWAS analysis. The broad heritability of traits related to pod and seed size (HB 2 %) ranged from 53.6% to 85.4% (Table 1).

[0037] Genotyping and association mapping:

[0038] Illumina's HiSeqX-ten sequencing platform was used to resequence the genome of each peanut material at a depth of approximately 10X, and the minor allele frequency (MAF) ≥ 0.05 was used as a threshold for screening. A total of 2,564,993 single nucleotide polymorphisms (SNPs) were detected for genome-wide association positioning. EMMAX software was used for association analysis, and the first three principal components (PCs) and the relative kinship matrix were used as covariates to eliminate the effects of population structure and kinship. The p = 1.0E-06 under Bonferroni correction was set as the significance threshold of the association, and the R software LDheatmap package was used to identify linkage disequilibrium associated with significant SNPs.

[0039] The candidate genes were identified within a linkage disequilibrium (LD) region using the reference genome "Fuhuasheng" using real-time quantitative PCR (qRT-PCR) method.

[0040] Total RNA was extracted from plant samples using a plant RNA extraction kit (Tiangen, Beijing, China) and reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. qRT-PCR was performed on an Applied Biosystems Viia7 Real-Time PCR System using SYBR Green (YEASEN, Shanghai, China) reagents.

[0041] Each sample was analyzed in triplicate to ensure accuracy and reliability. The cycling conditions for qRT-PCR were as follows: first at 95°C for 5 minutes, followed by 40 amplification cycles (95°C for 10 seconds, 60°C for 30 seconds). To normalize the expression data, ACT2 and 18S were used as endogenous controls for Arabidopsis and peanut, respectively. -ΔΔCt Method to compare the relative expression levels of candidate genes.

[0042] Overexpression analysis of AhPDS1 in Arabidopsis:

[0043] Total RNA was extracted from two-week-old peanut seedlings and then reverse transcribed into cDNA. The full-length coding sequence of the target gene was amplified by PCR using this cDNA as a template. The resulting PCR fragment was then inserted into the pGEOEP35S-H-GFP vector and overexpression analysis was performed under the drive of the cauliflower mosaic virus 35S promoter. All plasmids were introduced into the Agrobacterium strain GV3101 and transformed into Arabidopsis thaliana by the floral dip method. T4 generation transgenic plants were used for phenotypic analysis. These Arabidopsis seeds were photographed and measured using a dissecting microscope equipped with a 1× objective and a 10× eyepiece. The detailed sequences of all primers used in this study are shown in Supplementary Table 6. This method ensures the reliable generation of overexpression transgenic lines, allowing them to be used to examine the phenotype of target gene overexpression.

[0044] Promoter activity analysis:

[0045] The natural promoter of AhPDS1 was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and cloned into the pCAMBIA1391-GUS vector. To evaluate the activity of the promoter, AhPDS1-promoter transgenic plants were constructed. Plant tissues were initially immersed in phosphate buffer (50mM Na2HPO4, 50mM NaH2pO4) containing 0.5mM K3Fe(CN)6 and 0.5mM K4Fe(CN)6·3H2O5 for 5 minutes, and then the tissues were transferred to GUS staining solution, which consisted of the same phosphate buffer supplemented with 10mM EDTA Na2, 1% Triton-100 and 2mM X-Gluc, and incubated at 37°C for 12 hours. After incubation, the tissues were destained by washing with 95% ethanol at 65°C for 12 hours. The stained tissues were then examined under a stereomicroscope to observe the expression pattern of GUS.

[0046] Subcellular localization of AhPDS1-GFP fusion protein

[0047] The leaf cell protoplast transient expression system provided by Wuhan Edgegene Biotechnology (Wuhan) Co., Ltd. was used to study the subcellular localization of the AhPDS1-GFP fusion protein.

[0048] Arabidopsis leaves grown for 3 weeks were treated with a mixed solution containing 1.5% Cellulase R10, 0.75% Metase R10, 600 mM mannitol, 10 mM MES (pH 5.7) and 0.04% 2-hydroxy-1-ethanethiol at 23°C under vacuum for 3 hours.

[0049] The protoplasts were filtered through a 40 μm nylon mesh and centrifuged at 400 rpm for 5 minutes at 4°C. After discarding the supernatant, the protoplasts were washed with cold W5 solution (154 mM NaCl, 125 mM CaCl2, 2 mM KH2PO4, 2 mM MES, pH 5.7) and then resuspended in MMG solution (400 mM mannitol, 15 mM MgCl2·6H2O, 4 mM MES, pH 5.7). At a magnification of 40×, about 20-40 protoplasts were observed in each field of view.

[0050] The CDS sequence of AhPDS1 was cloned into the pEGOEP35S-H-GFP vector and transformed into the protoplasts of Col-0 and AhPDS1 overexpressing Arabidopsis plants. During transformation, 10 μl of plasmid DNA was mixed with 100 μl of protoplasts and 110 μl of 40% PEG4000 solution, and then incubated in a 22.5°C water bath for 15-20 minutes. W5 solution was added to stop the reaction, and the protoplasts were centrifuged at 400 rpm for 5 minutes at 4°C. After the protoplasts were washed twice with cold W5 solution, they were incubated overnight under weak light conditions at 23°C. The subcellular localization of AhPDS1-GFP was then observed using a laser confocal microscope (Olymbus FV3000) at an excitation wavelength of 488 nm and an emission wavelength of 510-530 nm.

[0051] Transgenic plants were grown for 10 days under different photoperiods and treated with 300 mM mannitol, and GFP signals were captured using the same confocal microscope.

[0052] result:

[0053] Table 1: Phenotypic changes of pod and seed size traits under four environments

[0054]

[0055]

[0056] In the four test environments, there were highly significant phenotypic correlations between different combinations of pods and seed-related traits. Seed pod thickness was strongly correlated with pod width (r=0.912, P<0.001). In addition, seed weight and 100-fruit weight also showed a strong correlation (r=0.857, P<0.001) ( Figure 1 C).

[0057] GWAS analysis results:

[0058] A genome-wide association study was conducted using a mixed linear model method using the EMMAX tool. In four environments, 883 significant associations related to eight pod and seed sizes were identified. The associations were mainly located on chromosome A05 (165 significant associations) and chromosome B06 (175 significant associations). A total of 584 significant associations were found on the A subgenome (At).

[0059] Some significantly enriched association regions (association hotspots) on chromosomes A02, A03, A05, A06, A07, A09 and B06 were also included.

[0060] A hotspot on chromosome B06, called B06.1, ranged from 142.8 to 149.2 Mb and contained 158 associations with six pod and seed size traits, including pod width (PW), pod thickness (PT), 100 pod weight (HPW), seed length (SL), seed width (SW), and 100 seed weight (HSW).

[0061] In addition, 26 significant associations were replicated in multiple settings (Tables 2-3; Figure 3 , 4 ). These stable associations include the association between chromosome A09 (8042653-8613448 bp) and pod length (PL), the association between chromosome B06 (143440440-145366781 bp) and pod width and pod thickness (PW and PT), and the association between chromosome A05 (9822089 bp) and seed length. In addition, a locus associated with pod length (PL) was repeatedly detected on chromosome Scaffold6 (211359 bp) in two environments (2017E and 2018E) (Table 2-3).

[0062] Genetic analysis of chromosome B06 and pleiotropic hotspots of pod and seed traits:

[0063] Chromosome B06 contains a pleiotropic hotspot (B06.1) with a significant gene, AhPDS1, which is expressed 7 times more in large-fruited peanut varieties than in small-fruited peanut varieties and has a consistent expression pattern in the husks of large-fruited and small-fruited peanut varieties at different developmental stages ( Figure 5 A, B), indicating that AhPDS1 may play a role in the regulation of pod size at different developmental stages (Table 4).

[0064] Real-time fluorescence quantitative PCR (qRT-PCR) analysis showed that the expression of AhPDS1 was significantly upregulated in large-fruit peanut materials (LP) carrying the CC genotype compared with small-fruit peanut (SP) materials. The increase in expression was particularly significant during the critical pod expansion stage, such as Figure 3As shown in A and 3B, this differential expression pattern suggests that AhPDS1 is actively involved in regulating pod growth, and the expression level of AhPDS1 is increased in large-fruited varieties, indicating that it is a key genetic factor in pod development, affecting the rate and extent of pod expansion, and AhPDS1 can be used to improve pod size traits through molecular breeding or genetic manipulation strategies.

[0065] In one embodiment:

[0066] Transgenic Arabidopsis lines were obtained by overexpressing the full-length CDS sequences of large-fruited (LP) and small-fruited (SP) peanut genotypes using the constitutive CaMV-35S promoter ( Figure 4 A). Quantitative analysis of relative expression levels showed that the overexpression line from the SP genotype (OE-SP) had higher expression levels than the wild-type (WT) and OE-LP lines ( Figure 4 B). Phenotypic analysis showed that the high-expressing LP genotype resulted in plants with significantly larger rosette, leaf, pod, and seed sizes compared to wild-type plants, while OE-SP did not show any significant differences in these characteristics compared to WT ( Figure 4 ).

[0067] result:

[0068] CDS variation between LP and SP genotypes may have different functional effects on plant morphology and development. Overexpression of the LP allele appears to promote growth and pod / seed size, whereas the SP allele does not induce similar phenotypic changes despite higher expression levels.

[0069] This suggests that the differences in CDS between LP and SP genotypes may play different functions in plant morphology and development, among which the LP allele of AhPDS1 plays a more prominent role in promoting plant growth and yield traits.

[0070] AhPDS1 overexpression promotes the biosynthesis of indole-3-acetic acid through the IPA pathway, specifically:

[0071] There are two biosynthetic pathways for indole-3-acetic acid (IAA) biosynthesis: a tryptophan-dependent pathway and a tryptophan-independent pathway using indole as a precursor.

[0072] In order to verify the expression in the present invention, the auxin profiles of overexpression (OE) plants (carrying the LP genotype) and wild-type plants were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results showed that compared with wild-type plants, the levels of IAA and most IAA amino acid conjugates in OE plants were significantly increased (Table 5), while the levels of Trp, IPA and IAA involved in the IPA pathway in OE plants were significantly higher, while other pathways such as IAOx-IAM-IAN (IAOx pathway) and TAM-IAAld (TAM pathway) were not significantly different between OE and wild-type plants, indicating that AhPDS1 participates in the biosynthesis of IAA through the IPA pathway, converting YUC-3-pyruvate (IPA) into IAA, and regulating various traits such as plant height, flower size, fruit size and seed size in different species.

[0073] Analysis of subcellular localization and expression pattern of AhPDS1: The present invention used peanut (Arachis hypogaea) leaf cell protoplasts to study the subcellular localization of AhPDS1 protein;

[0074] Protoplasts were transiently transformed with GFP-tagged AhPDS1 (pEGOEP35S-H-AhPDS1-GFP) to visualize localization. The fluorescent signal was widely present in the cytoplasm of peanut cells, indicating that AhPDS1 was mainly localized in the cytoplasm of peanut cells. This localization was further confirmed by the absence of GFP signal in the nucleus, indicating its lack of colocalization with nuclear markers (e.g. Figure 6 These observations suggest that AhPDS1 is not localized to a specific organelle but rather remains in the cytoplasm:

[0075] The invention also obtains transgenic Arabidopsis plants expressing AhPDS1 promoter fused with beta-glucuronidase (GUS) reporter gene (pAhPDS1-GUS).

[0076] Histochemical staining was used to observe the expression pattern of AhPDS1 in different tissues. Fifteen independent transgenic lines were analyzed in the T3 generation. Seeds of these lines were germinated and grown in 1x MS liquid medium, and subsequent GUS staining showed that AhPDS1 was expressed in multiple tissues, including roots, stems, leaves, trichomes, flowers, and pods, indicating that AhPDS1 plays a potential wide range of roles in plant growth and development.

[0077] Table 2: GWAS-identified association hotspots associated with pod and seed size

[0078]

[0079]

[0080] Table 3: Significantly associated SNPs identified in multiple planting seasons

[0081]

[0082] Table 4: Expression levels (FPKM values) of the AhPDS1 gene at different developmental stages in large-fruit and small-fruit varieties

[0083]

[0084]

[0085] Table 5: Auxin analysis of wild-type and LP genotype overexpressing plants (content: ng / g)

[0086]

[0087] Table 6: Primers used in the study

[0088]

[0089] In the present invention, significant association sites associated with pod and seed size traits were identified through genome-wide association studies (GWAS), and the analysis revealed known and new association sites, providing a solid foundation for improving peanut yield through selective breeding in the future. AhPDS1, as a key candidate gene for regulating seed pod and seed size, has been supported by expression and functional studies. Real-time fluorescence quantitative PCR (qRT-PCR) analysis showed that the expression of AhPDS1 (LP genotype) in large-fruit materials was significantly higher than that in small-fruit materials (SP genotype), especially in the critical pod enlargement stage, indicating that it plays an important role in pod growth. Overexpression studies in Arabidopsis thaliana confirmed that AhPDS1 plays a key role in pod and seed development. In addition, subcellular localization experiments showed that the AhPDS1 protein is mainly located in the cytoplasm, which is consistent with its role in auxin biosynthesis.

[0090] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0091] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A key gene AhPDS1 for plant pods, characterized by: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The key gene AhPDS1 of a plant pod according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: It comprises an original vector and the key gene AhPDS1 as claimed in claim 1 or 2.

4. A recombinant vector according to claim 3, characterized in that: The original vector is pEGOEP35S-H-GFP.

5. Use of the key gene AhPDS1 according to claim 1 or 2 in regulating the size of plant pods and seeds.

6. Use of the recombinant vector according to claim 3 or 4 in regulating the size of plant pods and seeds.

7. The use according to claim 5, characterized in that: The key gene AhPDS1 regulates the biosynthesis of plant IAA through the IPA pathway.

8. The use according to claim 6, characterized in that: The recombinant vector of the key gene AhPDS1 regulates the biosynthesis of plant IAA through the IPA pathway.

9. The use according to claim 7 or 8, characterized in that: The plant is peanut.

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