Rice leaf color regulation gene osprda1 and application thereof

Abstract

This invention relates to a rice leaf color regulating gene in the field of rice molecular genetic breeding. OsPRDA1 and its applications, the gene OsPRDA1 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2. This invention clones the target gene from a rice albino lethal mutant using Mutmap+ technology. OsPRDA1 The function of the gene was verified using CRISPR / Cas9 gene editing technology and complementation experiments. OsPRDA1 The protein encoded by this gene is located in chloroplasts and participates in rice chloroplast development by regulating the expression of chloroplast genes. This invention can be applied to molecular genetic breeding of rice leaf color traits and is of great significance for further understanding the mechanism of rice chloroplast development and improving rice yield.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and particularly to rice leaf color regulation genes in the field of rice molecular genetic breeding. OsPRDA1 And its applications. Background Technology

[0002] Chloroplasts are the site of photosynthesis in plants. Studying the molecular mechanisms of chloroplast development is fundamental to regulating photosynthesis and increasing crop yield. Chloroplasts are semi-autonomous organelles containing genetic material, and their genes are transcribed by plasmid-encoded RNA polymerases (PEPs) and nuclear-encoded RNA polymerases (NEPs).

[0003] PEP-associated proteins (PAPs) interact with PEP subunits and regulate chloroplast gene expression. In Arabidopsis thaliana, the gene encoding PAP (… PAP1 - PAP12 All mutations manifest as chlorosis or albinism. PRDA1 (PEP-Related Development Arrested 1) regulates chloroplast gene expression and chloroplast development through interaction with PAP9 / FSD2. In rice, PRDA1 No functional studies of this gene have been reported yet. (Regarding rice) PRDA1 Functional studies of genes can help us gain a deeper understanding of the regulatory mechanisms of chloroplast development, laying the foundation for breeding high-yield rice. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a rice leaf color regulation gene. OsPRDA1 And its applications.

[0005] The objective of this invention is achieved as follows: a rice leaf color regulating gene. OsPRDA1 The gene OsPRDA1 The nucleotide sequence is shown in SEQ ID No. 1.

[0006] The present invention OsPRDA1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

[0007] Furthermore, it also includes the above. OsPRDA1 One or more amino acids are substituted, inserted, or deleted in the amino acid sequence of the protein encoded by a gene.

[0008] This invention also provides the above-mentioned rice leaf color regulating gene. OsPRDA1Application in regulating rice leaf color.

[0009] Furthermore, the above applications reduce or destroy genes through gene interference or knockout methods. OsPRDA1 The activity of the encoded protein impairs the development of rice chloroplasts, resulting in an albinism-induced lethal phenotype.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0011] This invention reveals for the first time the rice OsPRDA1 The biological function of genes. Through 60 Co-γ ray irradiation of the rice variety Yandao 8 resulted in an albino lethal mutant. wsl1 ( white seedling and lethal 1 ). wsl1 The photosynthetic pigment content was significantly lower than that of the wild type, and chloroplast development was defective. CRISPR / Cas9 technology was used to analyze the photosynthetic pigment content of the wild type. OsPRDA1 Gene knockout resulted in a mutant exhibiting albinism and lethality. The wild-type... OsPRDA1 Gene transfer wsl1 Complementary mutant phenotypes can be observed. The OsPRDA1 protein is located in chloroplasts. qRT-PCR results indicate... wsl1 The expression of PEP-dependent chloroplast genes was significantly reduced. OsPRDA1, as a regulator of rice leaf color, can be used as a marker in rice molecular breeding. (The above information is from...) OsPRDA1 The functional analysis is of great importance for further understanding the molecular regulatory mechanism of rice chloroplast development and also provides a reference for the breeding of high-yield rice varieties. Attached Figure Description

[0012] Figure 1 Wild type and wsl1 Seedling phenotypes. A represents 4 days after germination; B represents 14 days after germination; and C represents 20 days after germination. The scale bar is 5 cm.

[0013] Figure 2 Wild type and wsl1 Photosynthetic pigment content.

[0014] Figure 3 Wild type and wsl1 The ultrastructure of chloroplasts.

[0015] Figure 4 for OsPRDA1 Schematic diagram of gene structure and mutation sites.

[0016] Figure 5 For Os PRDA1 Genotype and phenotype of knockout mutants.

[0017] Figure 6 for wsl1 Complementary plants wsl1-com Phenotype.

[0018] Figure 7 Subcellular localization of the OsPRDA1 protein.

[0019] Figure 8 for wsl1 Transcriptional analysis of mesochloroplast and nuclear genes. Detailed Implementation

[0020] The present invention will be further described below through specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0021] Example 1: wsl1 Phenotypic identification

[0022] pass 60 Co-γ ray irradiation of salt-tolerant rice variety 8 yielded an albino lethal mutant. wsl1 ( Figure 1 Compared to the wild type, wsl1 The contents of chlorophyll a, chlorophyll b and carotenoids in the middle were significantly reduced ( Figure 2 ). wsl1 The number of chloroplasts is extremely low, there is almost no accumulation of thylakoids, and no spindle-shaped starch granules are present. There are numerous oval vesicles and densely stained, spherical structures resembling plastid globules. This indicates a mutant. wsl1 The chloroplast development of the leaves is abnormal. Figure 3 ).

[0023] Example 2: OsPRDA1 Cloning of genes

[0024] In this embodiment, genetic analysis was performed using a phenotypic segregating family. The segregating family contained 74 normal plants and 20 albino fatal plants, conforming to a 3:1 (χ²) ratio. 2 = 0.70, P >0.05), indicating wsl1 It is a recessive mutant controlled by a single gene.

[0025] The target gene was cloned using the Mutmap+ method. wsl1Mutant M3 was used to replace 38 wild-type single plants and 38 albino phenotype single plants. The DNA was mixed in equal proportions into two pools, resequencing was performed and compared with the whole genome sequence of the parents to obtain the mutation index value of all mutations in the genome. According to the data analysis principle of MutMap+, the mutation was screened using the constraint rules. (1) SNP / Indel-index=1 in the mutant pool, because the genotypes in the pool are all homozygous mutations; (2) SNP / Indel-index<0.5 in the wild-type pool, because the wild-type pool contains not only wild-type but also heterozygous mutations, wild-type SNP / Indel-index=0; heterozygous mutation SNP / Indel-index=0.5. The genome was sequenced, and after screening and filtering, only one Indel in the whole genome met the conditions. This Indel is located in LOC_Os11g23790 The fourth exon contains a 12-base deletion, resulting in the loss of four amino acids. Figure 4 ), LOC_Os11g23790 The homologous gene in Arabidopsis thaliana is called PRDA1, OsPRDA1 The gene sequence length is 6282. OsPRDA1 The gene sequence is shown in SEQ ID No. 1. This gene is named... OsPRDA1 and initially LOC_Os11g23790 Identified as the target gene, OsPRDA1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

[0026] Example 3: Verification using gene editing and complementation experiments OsPRDA1 function of genes

[0027] To verify OsPRDA1 To determine the function of the gene, we used CRISPR / Cas9 technology to knock it out in the wild type. OsPRDA1 Gene. The resulting mutant. OsPRDA1-cr There is a single base deletion, such as Figure 5 As shown in A, OsPRDA1-cr Phenotype and wsl1 Phenotypic consistency, exhibiting an albino lethal phenotype, such as Figure 5 As shown in B. It will be by actin Full length of the starter driver OsPRDA1 CDS transfer wsl1 Complementation experiments were performed on callus tissue from heterozygous progeny to obtain successfully complemented tissue with a homozygous mutant background. wsl1-Com Plants, such as Figure 6 As shown. This indicates... OsPRDA1 The lack of functionality led to wsl1 The albino lethal phenotype.

[0028] Build OsPRDA1 - GFPThe fusion expression vector was transformed into rice protoplasts. The results showed that the green GFP signal co-localized with chloroplast autofluorescence (red), indicating... PRDA1 The protein is located in the cell nucleus ( Figure 7 ).

[0029] In order to investigate OsPRDA1 The effect of mutations on gene expression in rice, affecting wild-type and... wsl1 Transcriptome sequencing was performed on seedlings. Differentially expressed genes were screened according to specific rules, resulting in 3004 downregulated genes and 2902 upregulated genes. TopGO enrichment analysis was performed on the differentially expressed genes. The results showed significant enrichment of differentially expressed genes in photosynthesis, photosynthetic system, plastids, and chloroplasts. In the transcriptome data, a total of 56 chloroplast genes were identified, of which 41 were differentially expressed. 15 genes were upregulated and 26 were downregulated.

[0030] To validate the transcriptome data, eight PEP-dependent genes were selected. psbA , psbB etc.), 2 NEP-dependent genes ( rpoA , rpoB ), 2 PEP & NEP co-dependent genes ( ATP , ATP ) and two chloroplast-related nuclear coding genes ( YGL1 , DGP1 qRT-PCR experiments were performed. The results showed that the expression levels of all PEP-dependent genes were significantly reduced. Among NEP-dependent genes... rpoB The expression level of was significantly increased. rpoA The expression levels of these genes were significantly reduced. The expression levels of both PEP and NEP co-dependent genes were significantly reduced. Among chloroplast-related nuclear coding genes, YGL1 The expression level did not change significantly. DGP1 Expression levels were significantly reduced. These results indicate that OsPRDA1 plays a regulatory role in PEP-dependent plastid gene expression. Figure 8 ).

[0031] The above examples are merely some specific embodiments of the present invention, but the present invention is not limited to these embodiments. Modifications and improvements made by those skilled in the art based on the present invention should all be included within the scope of protection of the present invention.

Claims

1. The application of the rice leaf color regulating gene OsPRDA1 as a marker in rice molecular breeding, characterized in that, The nucleotide sequence of the OsPRDA1 gene is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the OsPRDA1 gene is shown in SEQ ID No. 2.

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