Gene ahwl1 for regulating peanut whitening and chloroplast development and application thereof

Abstract

The application discloses a gene Ahwl1 for regulating peanut whitening and chloroplast development and application thereof, and belongs to the technical field of plant genetic engineering, crop genetic breeding and peanut molecular biology. The application obtains the gene Ahwl1 closely related to peanut whitening and chloroplast development through phenotype identification, chlorophyll content determination, chloroplast ultrastructure observation, gene analysis and virus-induced gene silencing verification on a peanut whitening mutant, and the CDS sequence is shown as SEQ ID NO. 1. Compared with a wild type, the peanut whitening mutant shows obvious whitening of seedling stage or young leaf, and the chlorophyll content is significantly reduced. Transmission electron microscopy observation shows that the chloroplast structure development of the peanut whitening mutant is abnormal, and the grana lamella and thylakoid structure are damaged. After silencing the Ahwl1 gene by using a VIGS (Virus Induced Gene Silencing) technology, the peanut plant shows a whitening phenotype similar to the mutant, and is accompanied by a decrease in the expression amount of the target gene and a decrease in the chlorophyll content.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering, crop genetics and breeding, and peanut molecular biology, specifically relating to a gene Ahwl1 that regulates peanut albinoization and chloroplast development and its applications. Background Technology

[0002] Peanuts are an important oilseed and cash crop in my country, and their yield and quality are closely related to the photosynthetic capacity of their leaves. Leaves are the main organs for photosynthesis in plants, and chloroplasts are the core organelles for photosynthesis. The normal development of chloroplasts and the normal synthesis and accumulation of chlorophyll are essential for maintaining normal plant growth and development and yield formation.

[0003] Leaf color mutants are important materials for studying chloroplast development, chlorophyll biosynthesis, photosynthetic regulation, and plant development mechanisms. Albinism mutants typically exhibit significantly reduced chlorophyll content, abnormal chloroplast structure development, and decreased photosynthetic capacity, which can lead to seedling death in severe cases. Therefore, albino lethal mutants are not only important genetic materials for studying the molecular mechanisms of chloroplast development, but also provide new gene resources for crop leaf color trait regulation, germplasm creation, and molecular breeding.

[0004] Currently, several genes related to leaf albinism, chloroplast development, and chlorophyll synthesis have been identified in plants such as rice, maize, and Arabidopsis. However, in peanuts, the number of functional genes related to albinism and chloroplast development regulation remains relatively limited, and relevant genetic resources and functional validation evidence are insufficient. Therefore, discovering and identifying new genes controlling peanut albinism and chloroplast development is of great significance for improving the peanut chloroplast development regulatory network, creating new peanut germplasm, and conducting peanut molecular breeding. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a gene Ahwl1 that regulates peanut albinism and chloroplast development and its application, so as to fill the gap in the cloning and functional verification of albinism-related lethal genes in peanuts, and provide new gene resources for the study of peanut chloroplast development mechanism and molecular breeding.

[0006] This invention is based on a stable genetic peanut albino mutant. Through phenotypic observation, chlorophyll content measurement, transmission electron microscopy, gene analysis, and VIGS function verification, a gene Ahwl1 was identified that is involved in regulating peanut albino and chloroplast development. It was also demonstrated that the inhibition of the expression of this gene can lead to leaf albino, decreased chlorophyll content, and abnormal chloroplast development.

[0007] The technical solution adopted in this invention is as follows: The present invention first provides a gene Ahwl1 that regulates peanut albinoization and chloroplast development, the CDS sequence of the gene Ahwl1 is shown in SEQ ID NO.1; and the protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector containing the Ahwl1 gene, a recombinant microorganism or Agrobacterium strain containing the recombinant vector, and a virus-induced gene silencing vector (VIGS) for silencing the peanut Ahwl1 gene, wherein the VIGS vector contains the specific silencing fragment shown in SEQ ID NO.3.

[0009] Based on this, the present invention further provides: Applications of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1, recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1, and VIGS vectors containing specific silencing fragments as shown in SEQ ID NO.3 in regulating peanut chloroplast development, chlorophyll accumulation, and / or leaf color traits.

[0010] Specifically, by inhibiting or reducing the expression of the Ahwl1 gene, peanut plants can exhibit phenotypes such as leaf whitening, reduced chlorophyll content, and / or abnormal chloroplast development.

[0011] The present invention also provides a method for obtaining peanut albino phenotype plants, comprising the step of silencing the peanut Ahwl1 gene using VIGS technology, wherein the method uses a VIGS-TRV2 vector containing the fragment shown in SEQ ID NO.3.

[0012] This invention also provides the application of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1 in the creation of peanut germplasm resources, identification of peanut leaf color traits, research on peanut chloroplast development, or peanut molecular breeding.

[0013] This invention also provides a method for identifying peanut albino-related materials, the method comprising the following steps: detecting the expression level of the Ahwl1 gene in peanut materials, and determining whether the material is an albino-related material by combining the leaf albino phenotype, chlorophyll content and / or changes in chloroplast ultrastructure.

[0014] Preferably, the method includes the following steps: (1) Design a specific silencing fragment (SEQ ID NO.3) based on the Ahwl1 gene sequence (SEQ ID NO.1); (2) The silenced fragment is ligated to the TRV2 vector to obtain the recombinant vector TRV2-Ahwl1; (3) Transform the recombinant TRV2 vector into Agrobacterium GV3101 competent cells; (4) Infect peanut plants with Agrobacterium containing the recombinant TRV2 vector; (5) Cultivate infected peanut plants and observe their leaf phenotypes; (6) Detect the expression level of the Ahwl1 gene and / or chlorophyll content; (7) Based on the changes in leaf phenotype, Ahwl1 gene expression level and / or chlorophyll content, determine whether Ahwl1 gene silencing leads to leaf whitening, decreased chlorophyll content and / or abnormal chloroplast development.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention clones and functionally verifies the gene Ahwl1, which regulates albinism and chloroplast development, from peanut. The CDS sequence of this gene is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2. There are no reports in the prior art on the cloning and functional verification of peanut albinism-related functional genes; this invention fills a gap in gene resources in this field.

[0016] 2. This invention found that the leaves of peanut albino mutants showed obvious whitening and a significant decrease in chlorophyll content, indicating that the Ahwl1 gene is closely related to peanut chlorophyll accumulation and leaf color formation.

[0017] 3. Through transmission electron microscopy, this invention has revealed that the albino mutant exhibits abnormal chloroplast development, with damaged grana lamellae and thylakoid structures, demonstrating at the cellular level that the Ahwl1 gene is involved in normal chloroplast development.

[0018] 4. This invention utilizes VIGS technology to silence the Ahwl1 gene, resulting in peanut plants exhibiting a mottled yellowing phenotype in their leaves, accompanied by a decrease in the expression level of the target gene and / or a reduction in chlorophyll content. This demonstrates, from a functional verification perspective, that the Ahwl1 gene is involved in regulating peanut albinism and chloroplast development.

[0019] 5. The VIGS silencing system established in this invention is simple to operate and has a short cycle (phenotypes are visible in 14-21 days), which can be used for high-throughput screening of peanut gene function and provides technical support for peanut functional genomics research.

[0020] 6. The Ahwl1 gene and its VIGS silencing vector described in this invention can be used for: research on the regulatory mechanism of peanut chloroplast development, molecular marker-assisted identification and screening of peanut leaf color traits, early identification of peanut albino / yellow mutants, and creation of leaf color marker varieties through gene editing technology, providing valuable gene resources and technical reserves for peanut molecular breeding. Attached Figure Description

[0021] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 The images show the field phenotypes of the peanut albino mutant (Mutant) and wild-type (WT). Wild-type plants have normal green leaves, while albino mutant plants exhibit a distinct albino phenotype, weak growth, and show lethal or semi-lethal characteristics due to albino growth.

[0023] Figure 2 This is a comparison of chlorophyll content in leaves of peanut albino mutants (Mutant) and wild-type (WT). The albino mutant leaves showed significantly lower contents of chlorophyll a (Chla), chlorophyll b (Chlb), and / or total chlorophyll (Total) compared to the wild type.

[0024] Figure 3 Transmission electron microscopy (TEM) images of chloroplasts in leaves of peanut albino mutants and wild-type plants. In A: wild-type chloroplasts are structurally intact, with normal development of grana lamellae and thylakoid structures; in B: albino mutants exhibit abnormal chloroplast structure, with reduced, disordered, or incompletely developed grana lamellae, indicating impaired chloroplast development.

[0025] Figure 4 This image shows the albino phenotype of peanut plants after VIGS gene Ahwl1 silencing. The negative control plants had normal leaf color, while the Ahwl1-silencing plants exhibited varying degrees of leaf mottling or yellowing.

[0026] Figure 5 To detect the Ahwl1 chlorophyll gene and its expression level in VIGS plants. A: Comparison of chlorophyll a, chlorophyll b, and total chlorophyll content in leaves of wild-type control (CK) and VIGS silent plants (VIGS-APO); B: qRT-PCR detection of the relative expression level of the Ahwl1 gene in wild-type control (CK) and VIGS silent plants (VIGS-APO). Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] Biological material sources The materials were offspring obtained by our laboratory through hybridization of two varieties, SA263 and ZY. SA263 was obtained from the USDA National Plant Germplasm System (NPGS), with accession number PI 288146, introduced in 2016. ZY was obtained from the USDA National Plant Germplasm System (NPGS), with accession number PI 362130, introduced in 2016.

[0030] Example 1: Obtaining and phenotypic identifying peanut albino mutants 1.1 Experimental Materials The test material was peanut ( Arachis hypogaea L.). SA263 was used as the female parent and ZY as the male parent for hybridization. After self-pollination of F1, continuous self-pollination was carried out until F3. Homozygous mutants with stable inheritance of the albino phenotype were selected and named Mutant. The wild-type parent SA263 was used as the normal control (WT).

[0031] 1.2 Analysis of the genetic patterns of mutants Parental lines SA263, ZY, F1, and F2 segregating populations were planted in the field to investigate leaf color phenotypes and segregation ratios in each generation. Results showed that the leaves of parents SA263 and ZY were all normal green, as were the leaves of F1 plants, indicating that the albino trait was recessive. Fourteen albino phenotype plants appeared in the F2 generation. All F2 plants were harvested individually and sown for F2 propagation. 2：3 Group. F 2：3 A total of 52 mutant lines exhibiting leaf color segregation (i.e., the simultaneous presence of green-leaved and albino plants within the same lineage) emerged in the population. These segregating lines comprised a total of 448 individual plants, including 340 normal green-leaved plants and 108 albino plants, with a segregation ratio of approximately 3.15:1. This ratio conformed to Mendelian segregation ratio of 3:1 as determined by chi-square test. 2 =0.19, P >0.05 (Table 1). This result indicates that the albino trait is controlled by a single recessive nuclear gene.

[0032] Table 1. Leaf color separation ratio in the population

[0033] 1.3 Phenotypic identification of albino mutants Wild-type WT and the mutant Mutant seeds were sown in the field, with three replicate plots for each type, each containing 50 plants, and managed under the same cultivation conditions. Phenotypic observation and photography were conducted at the seedling stage (15 days after sowing).

[0034] (1) Observation of leaf color phenotype The results show that ( Figure 1 Wild-type plants grew normally with green leaves; the mutant Mutant exhibited a distinct albino phenotype even in the seedling stage, with new leaves being white or yellowish-white, the degree of albinoization decreasing from the outside in. Field phenotypic observation results are as follows: Figure 1 As shown.

[0035] (2) Determination of leaf color quantitative index The relative chlorophyll content (SPAD value) of leaves was measured using a SPAD-502 portable chlorophyll meter. Second true leaves were collected at the seedling stage (counting from bottom to top), and measurements were taken at three sites per plant, with the average value calculated. Ten plants were randomly selected from each plot for measurement. The results (Table 2) showed that the SPAD value of the mutant Mutant leaves was only 2.2 ± 1.5, a decrease of approximately 94.5% compared to the wild-type WT (39.7 ± 3.9), with a highly significant difference. P <0.01).

[0036] Table 2 Comparison of SPAD values ​​of leaves at the seedling stage between wild type and mutant WT 39.7 ± 3.9 — Mutant 2.2 ± 1.5** 94.5% Note: ** indicates a highly significant difference compared to WT. P <0.01, t-test).

[0037] The results of this embodiment indicate that the albinism trait in the Mutant mutant is controlled by a single recessive nuclear gene. This mutant exhibits a stable albinism phenotype at the seedling stage, accompanied by a significant decrease in chlorophyll content (SPAD value reduced by approximately 94.5%) and inhibited plant growth. This mutant can serve as an ideal material for studying peanut chloroplast development and also provides a reliable genetic material basis for subsequent gene localization and functional verification.

[0038] Example 2: Determination of chlorophyll content in peanut albino mutants 2.1 Experimental Materials Wild-type WT (SA263) and albino mutant Mutant plants described in Example 1 were used. At the seedling stage (15 days after sowing), leaves from the same developmental stage and leaf position (the second true leaf, counted from bottom to top) were selected for chlorophyll content determination. Each material was prepared in 3 biological replicates, and each replicate was taken from a mixed leaf sample of 3 different plants.

[0039] 2.2 Experimental Methods Fresh leaf samples were weighed, chopped, and chlorophyll was extracted using 80% acetone in the dark (Lichtenthaler, 1987). After the leaf tissue had completely faded, the chlorophyll content (A) was measured using a UV-Vis spectrophotometer. 663 and A 645The contents of chlorophyll a, chlorophyll b and total chlorophyll were calculated with reference to Arnon (1949).

[0040] 2.3 Experimental Results The results are as follows Figure 2 As shown, compared with wild-type WT, the albino mutant Mutant had significantly lower levels of chlorophyll a, chlorophyll b, and total chlorophyll. P <0.001). Among them, the total chlorophyll content decreased from 0.3214 ± 0.0007 mg / g FW in WT to 0.00035 ± 0.00005 mg / g FW in Mutant, a decrease of 99.89%.

[0041] These results indicate that the whitening phenotype of the mutant Mutant is closely related to inhibited chlorophyll accumulation.

[0042] Example 3: Observation of the ultrastructure of chloroplasts in peanut albino mutants 3.1 Experimental Materials Wild-type WT (SA263) and albino mutant Mutant plants described in Example 1 were used. Leaves from the same developmental stage and leaf position (the second true leaf, counted from bottom to top) were selected at the seedling stage (15 days after sowing) for transmission electron microscopy observation. Leaf samples from 3 different plants were taken from each material and prepared for independent slide observation.

[0043] 3.2 Experimental Methods Following standard transmission electron microscopy (TEM) sample preparation methods, the central portion of the leaf (avoiding the midrib) was cut into small sections of approximately 1 mm × 2 mm and quickly placed in 2.5% glutaraldehyde fixative, fixed overnight at 4°C. The sections were then rinsed three times with phosphate-buffered saline (PBS, pH 7.2) for 15 min each time; post-fixed with 1% osmium tetroxide for 2 h; dehydrated using a series of gradient ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, 100%); embedded in epoxy resin (Epon 812); polymerized; and sectioned using an ultramicrotome (Leica UC7) (approximately 70 nm thick). The sections were double-stained with uranium acetate and lead citrate, and observed and photographed under a TEM (HT7800, Hitachi, Japan).

[0044] 3.3 Experimental Results Transmission electron microscopy observation results as follows Figure 3 As shown.

[0045] In the mesophyll cells of wild-type WT leaves, the chloroplasts exhibit typical elliptical or fusiform morphology, with a complete and clear chloroplast membrane structure. The internal grana lamellae are tightly stacked and orderly arranged, the thylakoid structure is clear, and the stroma lamellae are evenly distributed, indicating that the wild-type chloroplasts are developing normally and structurally intact. Figure 3 (A)

[0046] In contrast, the chloroplast structure in the mesophyll cells of the albino mutant Mutant leaves exhibits significant developmental abnormalities. These abnormalities are mainly manifested as: irregular chloroplast morphology, nearly circular or irregular in shape; blurred or partially absent chloroplast membrane structure; significantly reduced and loosely stacked grana lamellae, with some areas showing almost complete absence of grana lamellae; disordered and disorganized thylakoid structure; and numerous vacuolated regions visible in some chloroplasts, indicating a marked state of degeneration. Figure 3 (B)

[0047] The results of this embodiment confirm, through transmission electron microscopy at the ultrastructural level, that the chloroplast development of the albino mutant Mutant is significantly abnormal, mainly manifested as reduced grana lamellae, disordered thylakoid structure, and chloroplast degeneration. This result corroborates the physiological data of significantly reduced chlorophyll content in Example 2, further indicating that the albino phenotype of the mutant Mutant is closely related to abnormal chloroplast development, suggesting that the Ahwl1 gene is involved in regulating normal peanut chloroplast development.

[0048] Example 4: Screening, cloning, and sequence analysis of the Ahwl1 gene sequence 4.1 Screening of candidate genes for albino mutants Based on the stable genetic phenotypic differences between the albino mutant Mutant in Example 1 and the wild-type WT, and the chloroplast ultrastructural abnormalities in Example 3, transcriptome sequencing analysis was performed on seedling leaves of wild-type WT and the albino mutant Mutant to identify candidate genes causing the albino phenotype. Total RNA was extracted from WT and Mutant leaves, and cDNA libraries were constructed followed by transcriptome sequencing. Differentially expressed genes were screened using a threshold of |log2FC|≥1 and P<0.05. Combined with GO and KEGG functional annotation, five candidate differentially expressed genes related to chloroplast development, chlorophyll biosynthesis, and photosynthesis were selected. The expression levels of the candidate genes in WT and Mutant were further verified by RT-PCR, and one gene with significantly downregulated expression in the mutant was identified. Sequence analysis showed that Ahwl1 encodes a chloroplast localization protein containing an APO RNA-binding domain (APO domain). This domain belongs to the plant-specific zinc-dependent RNA-binding domain and has high homology with APO family proteins in Arabidopsis thaliana that are involved in chloroplast RNA processing and chloroplast development. Therefore, this gene was named Ahwl1, and its encoded protein was labeled as an APO protein 2-like protein.

[0049] 4.2 Cloning of the full-length CDS of the Ahwl1 gene Based on the Ahwl1 gene sequence information obtained in section 4.1, specific amplification primers were designed. PCR amplification was performed using wild-type WT root cDNA as a template, and sequencing confirmed the full-length CDS sequence of the Ahwl1 gene. The CDS sequence of the Ahwl1 gene is shown in SEQ ID NO.1, and the amino acid sequence encoding the protein is shown in SEQ ID NO.2.

[0050] For subsequent functional validation, a specific VIGS silencing fragment was designed based on the Ahwl1 gene CDS sequence, and its nucleotide sequence is shown in SEQ ID NO.3. This fragment was used to construct the VIGS silencing vector.

[0051] Example 5: VIGS Function Verification of the Ahwl1 Gene To verify the biological function of the Ahwl1 gene, this embodiment used VIGS technology to silence the expression of the Ahwl1 gene in peanut plants and observed its effects on leaf phenotype, chlorophyll content and chloroplast development.

[0052] 5.1 Construction of VIGS silencing vector Based on the Ahwl1 gene CDS sequence obtained in Example 4, a specific silencing fragment (SEQ ID NO. 3) was selected, and amplification primers containing suitable restriction enzyme sites were designed (F: aaggttaccgaattctctagaCCCATAATTCGGATTGGGAGA, SEQ ID NO. 4; R: gagacgcgtgagctcggtaccATTTCCGGGCAATATCCACA, SEQ ID NO. 5, where lowercase letters are adapter sequences and uppercase letters are gene-specific sequences). PCR amplification was performed using a plasmid containing the full-length Ahwl1 CDS as a template. After purification, the amplified product was ligated into a TRV2 vector (pTRV2) linearized with restriction endonucleases (such as XbaI / KpnI) using homologous recombination. This vector was then transformed into *E. coli* DH5α, and positive clones were screened and sequenced for verification. The correctly sequenced recombinant plasmid was named TRV2-Ahwl1. The empty vector pTRV2 served as a negative control.

[0053] 5.2 Agrobacterium transformation and infection Recombinant plasmid TRV2-Ahwl1 and helper plasmid pTRV1 were transformed into Agrobacterium GV3101 competent cells, respectively. Positive clones were selected by kanamycin and rifampin. Single colonies of Agrobacterium positive containing TRV2-Ahwl1 and pTRV1 were picked and inoculated into LB broth containing the corresponding antibiotics, and cultured at 28°C with shaking at 220 rpm until OD. 600 =0.8~1.0. Collect bacterial cells by centrifugation at 5000 rpm for 5 min, resuspend in infection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsylgenone, pH 5.6) and adjust OD. 600 To 1.0. Let stand at room temperature for 2-4 hours.

[0054] Peanut seedlings of uniform growth, at the cotyledon stage to the first pair of true leaves unfolding stage, were selected. pTRV1 was then mixed with pTRV2 or empty vector pTRV2 in equal volumes, serving as Ahwl1 silencing treatment and empty vector control, respectively. The mixed bacterial solution was used to infect peanut seedlings via vacuum permeation at −0.08 MPa for 3.5 min. Infected seedlings were cultured in darkness for 24 h, then transferred to an artificial climate chamber at 22 ± 2°C, with 16 h light / 8 h darkness. Newly infected axillary buds or leaves were used for phenotypic observation, chlorophyll content determination, and qRT-PCR analysis.

[0055] 5.3 Phenotypic observation of gene-silenced plants Leaf phenotypes were observed from the seedling stage to the fruiting stage after infection. The results showed that ( Figure 4The empty vector control plants maintained normal green leaf color and good growth. The Ahwl1-silenced plants exhibited varying degrees of leaf mottled yellowing or whitening phenotypes, similar to the natural albino mutant phenotype. In subsequent tests, Ahwl1 expression levels were not significantly downregulated in plants that did not show a silent phenotype.

[0056] 5.4 Detection of gene silencing efficiency and chlorophyll content Total RNA was extracted from leaves of empty vector control plants and Ahwl1-silenced positive plants (showing a mottled yellowing phenotype), respectively, and reverse transcribed into cDNA. The expression level of the Ahwl1 gene was detected using real-time quantitative PCR. Peanut Actin gene was used as an internal control, and a 2... -ΔΔCt The relative expression level was calculated using this method. The results showed ( Figure 5 Compared with the empty vector control, the relative expression levels of the target gene in Ahwl1-silenced plants were significantly reduced, with decreases of 85.05% and 88.03% (A and B, respectively). P <0.01), indicating that VIGS effectively silences endogenous Ahwl1 expression.

[0057] Leaves from the same batch of plants were taken for chlorophyll content determination (method as in Example 2). The results showed ( Figure 5 In the control group (B), the chlorophyll a, chlorophyll b, and total chlorophyll content of the Ahwl1 silent plants were significantly lower than those of the empty vector control. P <0.05), the decreasing trend is consistent with that of the naturally albino mutant.

[0058] In this embodiment, the Ahwl1 gene was silenced using VIGS technology, resulting in peanut plants with mottled yellowing leaves. This was accompanied by a significant decrease in the expression level of the target gene and a significant reduction in chlorophyll content, with a phenotype consistent with the naturally occurring albino mutant. These results demonstrate that suppression of Ahwl1 gene expression can lead to albino peanut leaves and inhibited chlorophyll accumulation, functionally proving that the Ahwl1 gene is a key gene regulating albino peanut leaves and chloroplast development.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1, recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1, and VIGS vectors containing specific silencing fragments as shown in SEQ ID NO.3 in regulating peanut chloroplast development, chlorophyll accumulation, and / or leaf color traits.

2. The application according to claim 1, characterized in that, By inhibiting or reducing the expression of the Ahwl1 gene, peanut plants exhibit phenotypes such as leaf whitening, reduced chlorophyll content, and / or abnormal chloroplast development. The CDS sequence of the Ahwl1 gene is shown in SEQ ID NO.

1.

3. Application of proteins with amino acid sequences as shown in SEQ ID NO.2 in regulating peanut chloroplast development, chlorophyll accumulation, and / or leaf color traits.

4. Application of recombinant microorganisms or Agrobacterium strains containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1 in regulating peanut chloroplast development, chlorophyll accumulation and / or leaf color traits.

5. A method for obtaining peanut plants with an albino phenotype, characterized in that, The method includes the step of silencing the peanut Ahwl1 gene using VIGS technology, wherein the method uses a VIGS-TRV2 vector containing the fragment shown in SEQ ID NO.

3.

6. Application of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1 in peanut germplasm resource creation, peanut leaf color trait identification, peanut chloroplast development research, or peanut molecular breeding.

7. A method for identifying peanut albino-related materials, characterized in that, The expression level of the Ahwl1 gene in peanut materials was detected, and the albino phenotype, chlorophyll content and / or changes in chloroplast ultrastructure were combined to determine whether the material was albino-related. The CDS sequence of the Ahwl1 gene is shown in SEQ ID NO.1.

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