Application of rice chloroplast development gene OsSecY1

Abstract

The invention discloses an application of a rice chloroplast development gene OsSecY1. A gene OsSecY1 for regulating and controlling development of rice chloroplast is found in a rice development mutant library, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the amino acid sequence of encoded protein is as shown in SEQ ID NO.2. Researches show that the expression quantity of the OsSecY1 gene in leaves is relatively high, and the encoded protein is positioned in chloroplast. The OsSecY1 gene is knocked out from wild type rice, so that rice chloroplast develops abnormally, leaves are yellowed, and apoptosis begins at the later stage of a two-leaf stage. Therefore, the OsSecY1 gene provided by the invention can be used as a seedling stage marker character for assisting rice molecular breeding, is beneficial to understanding a regulation mechanism of rice chloroplast development, and provides a reference for improving the leaf photosynthetic efficiency through a genetic engineering means so as to improve the rice yield.

Description

Technical Field

[0001] This invention relates to the fields of molecular breeding and genetic engineering technology, and more specifically, to a rice chloroplast development gene. OsSecY1 Applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's three major food crops, providing staple food for nearly half of the global population. Rice yield primarily comes from photosynthesis in its leaves, and photosynthesis depends on the normal development of chloroplasts. Therefore, the development of rice chloroplasts has a direct impact on rice yield. Investigating the functional genes and regulatory pathways involved in regulating rice chloroplast development is of significant theoretical importance.

[0003] As a semi-autonomous organelle, the function of chloroplasts depends on the coordinated regulation of the nuclear genome and the chloroplast genome. During chloroplast biogenesis, approximately 3,000 nuclear-encoded proteins are synthesized in the cytosol and transported to the chloroplasts to complete the assembly of the photosynthetic apparatus. Mutations in chloroplast genes often lead to abnormal chloroplast development, resulting in leaf color mutants, such as pale green leaves, albinism, yellowing, and zebra-like leaves. Rice leaf color mutants can not only provide ideal materials for chloroplast development research but also serve as morphological markers to aid in genetic breeding.

[0004] The creation of etiolated rice materials can be used to analyze the mechanisms of chloroplast development and photosynthesis, elucidate gene function (a classic method of reverse genetics), and study the mechanisms of nucleus-chloroplast interactions. In production practice, etiolated materials can be used as marker traits in hybridization breeding, easily, accurately, and cost-effectively identifying and eliminating false hybrids and self-pollinated seedlings from the maternal parent at the seedling stage, ensuring the purity of hybrid seeds, increasing yield, and avoiding yield losses caused by planting impure seeds. Simultaneously, they can also be used to screen and create superior germplasm, providing potential genetic resources and intermediate breeding materials for cultivating new varieties with special adaptability (such as low-temperature tolerance), and also providing clues for cultivating stress-resistant varieties.

[0005] Thylakoid membrane is the place of photosynthesis light reaction. Thylakoid membrane mainly contains PSI (photosystem I), PSII (photosystem II), cytochrome and ATP synthase complex. At present, five thylakoid protein transport pathways have been found: chloroplast double arginine translocation pathway (cpTAT), chloroplast secretion pathway (cpSec), chloroplast signal recognition particle pathway (cpSRP), chloroplast guide tail anchoring protein into (cpGET) pathway and spontaneous insertion pathway. The chloroplast secretion (Sec) pathway is evolutionarily conserved, which is composed of translocation ATPase SecA and translocation channel, and the translocation channel is formed by SecY and SecE. However, the function of SecY gene and the protein encoded by the gene has not been reported in rice. Therefore, in order to reveal the mechanism of the gene in the development of rice chloroplast, and to explore more useful gene resources, it is necessary to study the function of the gene and provide a reference for improving photosynthetic efficiency through genetic engineering in the future. SecY The application relates to the application of a rice chloroplast development gene. SecY The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene.

[0006] The application relates to the application of a rice chloroplast development gene. SecY The application relates to the application of a rice chloroplast development gene. OsSecY1 The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene.

[0007] The application relates to the application of a rice chloroplast development gene. OsSecY1 The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene.

[0008] The application relates to the application of a rice chloroplast development gene. OsSecY1 The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene.

[0009] The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene.

[0010] The application relates to the application of a rice chloroplast development gene. OsSecY1 The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene.

[0011] The application relates to the application of a rice chloroplast development gene. The application relates to the application of a rice chloroplast development gene. OsSecY1 The application relates to the application of a rice chloroplast development gene. OsSecY1 The application relates to the application of a rice chloroplast development gene. OsSecY1Genes can be used as seedling marker traits to assist in molecular breeding of rice, which helps to understand the regulatory mechanism of rice chloroplast development and provides a reference for improving leaf photosynthetic efficiency and thus rice yield through genetic engineering. This is of great significance to the study of rice chloroplast development and also provides the possibility for improving photosynthetic efficiency through genetic engineering in the future.

[0012] Therefore, the present invention provides rice OsSecY1 Application of genes in regulating chloroplast development in rice.

[0013] Preferably, the OsSecY1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; OsSecY1 The sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0014] Preferably, the regulation is knockout. OsSecY1 Gene expression.

[0015] More preferably, the chloroplast development refers to abnormal chloroplast development, the absence of thylakoid stacking, and a decrease in chlorophyll content.

[0016] This invention provides rice OsSecY1 The application of genes in assisted genetic breeding, including gene knockout in rice. OsSecY1 Gene.

[0017] Preferably, the assisted genetic breeding refers to OsSecY1 Genes are used as morphological markers in genetic breeding, or in the screening of true hybrid seeds and the removal of yellowing plant seeds produced by self-pollination or false hybridization in hybrid rice seed production.

[0018] This invention provides rice OsSecY1 Application of genes in the creation of etiolated rice materials.

[0019] This invention also provides knockout rice OsSecY1 The following applications of gene expression formulations: Application in regulating chloroplast development in rice.

[0020] Application in the creation of etiolated rice materials.

[0021] Application in rice breeding.

[0022] Preferably, in rice breeding, knockout rice... OsSecY1 Gene expression agents are introduced into the male or female parent for hybridization. After hybridization, the true hybrid seeds will show a normal green color, while the seeds produced by self-pollination or false hybridization will show yellowing.

[0023] More preferably, the formulation contains knockout OsSecY1Plasmid, vector, host cell, recombinant bacteria of the gene.

[0024] Further preferably, the preparation is rice OsSecY1 CRISPR / Cas9 knockout vector of the gene, or recombinant bacteria containing the knockout vector.

[0025] Further preferably, the preparation is rice OsSecY1 The primer set of the CRISPR / Cas9 knockout vector of the gene is Primer1 (SEQ ID NO. 3) / Primer2 (SEQ ID NO. 4).

[0026] The application provides a method for creating a yellow rice material, knocking out the gene OsSecY1 of the rice, obtaining a chloroplast development abnormality, yellow leaf plant.

[0027] Preferably, the gene editing technology is used to knockout mutation of the gene OsSecY1 of the rice, obtaining a yellow leaf plant.

[0028] More preferably, a recombinant gene CRISPR / Cas9 knockout vector containing the target sequence of the gene OsSecY1 is constructed, and wild-type rice is transformed to obtain a chloroplast development abnormality plant.

[0029] In addition, the application also provides a method for assisting rice genetic breeding by using the gene OsSecY1 of the rice, knocking out the gene OsSecY1 in the rice; the assisting genetic breeding means that OsSecY1 the gene is used as a morphological marker for genetic breeding; or in hybrid rice seed production, the knockout OsSecY1 gene rice is used for hybridization, and the true hybrid seeds after hybridization will show normal green color, and the seeds produced by selfing or false hybridization will show yellowing, so as to identify and eliminate false hybrids and maternal selfing seedlings.

[0030] The application has the following beneficial effects: The application provides a new application of a rice chloroplast development gene OsSecY1 . The application identifies and analyzes a gene OsSecY1 that regulates the development of rice chloroplasts through a yellow leaf mutant in a rice development mutant library. The gene has a high expression level in leaves, and the encoded protein is located in chloroplasts. Knocking out the gene in wild-type rice can cause abnormal development of rice chloroplasts and yellow leaves, and can be used for research on the formation mechanism of rice chloroplasts and the creation of leaf color mutants. The application provides a new application of a gene Figure 1Genes can be used as seedling marker traits to assist in molecular breeding of rice, and also help to understand the regulatory mechanism of rice chloroplast development. They can also provide a reference for improving leaf photosynthetic efficiency and thus rice yield through genetic engineering. Research on rice chloroplast development is of great significance and also provides the possibility for improving photosynthetic efficiency through genetic engineering in the future. Attached Figure Description

[0031] Figure 2 The results show the map-based cloning and candidate gene sequencing of the yellow leaf mutant (A in the figure is the mutant phenotype; B is the map-based cloning map; C is the sequencing results of the mutation site).

[0032] OsSecY1 for OsSecY1 CRISPR / Cas9 knockout target sites and target editing sequencing results (Figure A shows the phenotype of CRISPR / Cas9 knockout edited materials; B shows the phenotype of the material). Figure 3 A schematic diagram of the gene structure, with black boxes representing exons and solid lines representing introns; T1 represents the editing target site.

[0033] ossecy1 for ossecy1 Chloroplast microstructure of mutant materials (A) and Figure 4 Chlorophyll content determination of mutant materials (B).

[0034] OsSecY1 for OsSecY1 Expression patterns and subcellular localization of encoded proteins (A in the figure) Figure 1 The expression pattern; B represents subcellular localization. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Example 1: Obtaining genes related to rice chloroplast development and constructing knockout vectors 1. Obtaining the target gene in rice Based on previous work in rice 60 A yellowing leaf mutant was found in the Co-induced variant library, such as Figure 1 As shown in Figure A, the segregation ratio of the heterozygous progeny indicates that this phenotype is regulated by a pair of recessive genes. Map-based cloning located the candidate genes within a 67 kb range on chromosome 8, between molecular markers 8-09386 and 8-09453. SecYAs shown in B. Sequencing analysis revealed that within the interval... Figure 1 A single base C deletion in the gene leads to a frameshift mutation, causing premature translation termination at amino acid 484. OsSecY1 As shown in C; name this gene... OsSecY1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1 (LOC_Os08g15460), and whose encoded protein amino acid sequence is shown in SEQ ID NO.2.

[0038] 2. Construction of the knockout vector exist Pubi A knockout target T was designed on the fourth exon of the gene. The target has NGG at the 3' end (N is any base of A, T, C, or G). The sequence of the knockout target T is (SEQ ID NO.5): 5'-TATACAAGATACGCATCTGTTGG-3'.

[0039] Primers were designed and synthesized targeting the knockout site T: Primer1 (SEQ ID NO.3): 5'-TATACAGATACGCATCTGTgttttagagctagaaat-3' and Primer2 (SEQ ID NO.4): 5'-ACAGATGCGTATCTTGTATACggcagccaagccagca-3'. The CRISPR / Cas9 vector pYLCRISPR / Cas9 containing the target site was then constructed. OsSecY1 - et al. (The method for constructing the vector is described in the works of Ma X, Zhang Q, and Zhu Q.) Pubi- OsSecY1 A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 2015, 8 (8): 1274-1284. The recombinant plasmid pYLCRISPR / Cas9 was processed by electroporation. Pubi- OsSecY1 Agrobacterium EHA105 strain was transformed to obtain recombinant strains. Plasmids were extracted and subjected to PCR and enzyme digestion identification to ensure the integrity and sequence correctness of the plasmids.

[0040] Using the genetic transformation method of Agrobacterium-mediated transformation in mature rice embryos (the method is based on Nishimura A, Aichi I, Matsuoka M. A protocol for Agrobacterium-mediated transformation in rice. Nature Protocols. 2006, 1(6):2796-2802), pYLCRISPR / Cas9 was transformed. OsSecY1 Transformed into the japonica rice variety Nipponbare (RBQ, germplasm preserved in our laboratory), and obtained Pubi- Gene knockout transgenic T0 generation plants.

[0041] Example 2: Identification and Decoding Analysis of Editing Sites in Transgenic Plants Genomic DNA was extracted from the T0 generation plants obtained in Example 1 and used as a template in pYLCRISPR / Cas9. OsSecY1 Hpt Carrier Hygromycin ( OsSecY1 For the protein's CDS region, upstream detection primer Primer 3 (SEQ ID NO. 6): 5'-ATTTGTGTACGCCCGACAGT -3' and downstream detection primer Primer 4 (SEQ ID NO. 7): 5'-GTGCTTGACATTGGGGAGTT -3' were designed, and PCR amplification was performed on T0 generation plants to detect whether the T0 generation plants carried the transgene.

[0042] PCR reaction system: DNA (20 ng / μL) 1 μL, Primer 3 (10 pmol / μL) 0.5 μL, Primer 4 (10 pmol / μL) 0.5 μL, 2×Buffer 10 μL, dNTP (10 mM) 0.5 μL, Taq (5 U / μL) 0.3 μL, add ddH2O to 25 μL. PCR amplification program: 94℃ 5 min; 94℃ 30 sec, 58℃ 30 sec, 72℃ 1 min, 30 cycles; 72℃ 5 min.

[0043] To detect the target site editing status in T0 generation plants, Figure 2 Primers were designed upstream and downstream of the target site in the genomic sequence corresponding to the gene. The upstream primer was Primer5 (SEQ ID NO.8) 5'-GCTGCAATTGAAGATAGTTCC -3' and the downstream primer was Primer6 (SEQ ID NO.9) 5'-CAGCCAGGAAGGTACTAGAAC -3'.

[0044] PCR reaction system: DNA (20 ng / μL) 1 μL, Primer3 (10 pmol / μL) 0.5 μL, Primer4 (10 pmol / μL) 0.5 μL, 2×Buffer 10 μL, dNTP (10 mM) 0.5 μL, Taq (5 U / μL) 0.3 μL, add ddH2O to 25 μL. PCR amplification procedure: 94℃ 5 min; 94℃ 30 sec, 58℃ 30 sec, 72℃ 1 min, 30 cycles; 72℃ 5 min.

[0045] The sequencing results were decoded by using the CRISPR-GE online analysis tool, and the results are shown in OsSecY1 B, it was found that different target points in different transgenic lines had frame shift mutations caused by deletion / insertion, and the transgenic lines of gene knockout were successfully constructed. OsSecY1

[0046] Example 3: Phenotype analysis of transgenic plants The transgenic T0 plants of gene knockout, wild type Nipponbare were planted in the rice test station of South China Agricultural University, and the water and fertilizer conditions were consistent during planting, and the plants were observed for phenotype. Pubi- OsSecY1 The results show that the chloroplasts of the transgenic plants of pYLCRISPR / Cas9

[0047] are abnormal, and the knockout of OsSecY1 the gene causes the rice to turn yellow, and the plants start to die after the late two-leaf stage, as shown in Figure 2 A, which can be used to create yellowing materials. Figure 3

[0048] Further observation and determination of the chloroplast microstructure and chlorophyll content of the mutant material show that OsSecY1 , OsSecY1 after the knockout of the gene, the chloroplasts cannot develop normally, and almost no complete thylakoid lamella can be seen. Compared with the wild type, the chlorophyll content of the knockout mutant decreases sharply.

[0049] At the same time, OsSecY1 the expression pattern experiment results of the gene show that Figure 4 the gene is expressed in the roots, stems, leaves, spikelets, anthers and leaf sheaths of rice, and the expression in the leaves is the highest, and the results are shown in OsSecY1 , the subcellular localization of the encoded protein shows that the OsSecY1 protein is located in the chloroplast.

[0050] In summary, the present application identifies and analyzes a gene OsSecY1 ​​The gene has high expression in leaves, and the coded protein is located in chloroplast. OsSecY1 Knocking out the gene will cause abnormal development of rice chloroplast, yellowing of leaves, and apoptosis starting at the late stage of the two-leaf stage, and can be used for creating materials with abnormal leaf color. OsSecY1 The gene can be used as a seedling marker trait for assisting in rice molecular breeding, and is also helpful for understanding the regulation mechanism of rice chloroplast development, and provides a reference for improving leaf photosynthetic efficiency and then improving rice yield through genetic engineering means, has important significance for the study of rice chloroplast development, and provides a possibility for improving photosynthetic efficiency through genetic engineering means in the future.

[0051] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Oryza sativa OsSecY1 application of the gene in regulating chloroplast development in rice, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the regulation is to knock out the expression of the gene. OsSecY1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the regulation is to knock out the expression of the gene. OsSecY1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the regulation is to knock out the expression of 2. Oryza sativa OsSecY1 The application of the gene in assisting the genetic breeding of rice is characterized in that, The OsSecY1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the assisted rice genetic breeding refers to... OsSecY1 Genes are used as morphological markers in genetic breeding, or in the production of hybrid rice seeds to screen for true hybrid seeds, improve seed purity, and eliminate yellowed plant seeds produced by self-pollination or false hybridization.

3. Oryza sativa OsSecY1 The use of the gene in creating a yellowing rice material is characterized in that, The OsSecY1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

4. A preparation for knocking out the expression of a gene in rice OsSecY1 application of a preparation for knocking out the expression of a gene in rice in regulating chloroplast development in rice, characterized in that, The OsSecY1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

5. A preparation for knocking out the expression of a gene in rice OsSecY1 application of a preparation for knocking out the expression of a gene in creating a yellowing rice material, characterized in that, The OsSecY1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

6. A preparation for knocking out the expression of a gene in rice OsSecY1 application in rice breeding, characterized in that, The preparation for knocking out the expression of rice OsSecY1 gene is introduced into the male parent or female parent for cross, and the true hybrid seeds after cross will show normal green color, while the seeds produced by selfing or false cross will be yellow.

7. Use according to any one of claims 4 to 6, characterized in that, The formulations are plasmids, plant expression vectors, host cells, recombinant bacteria containing knock-out OsSecY1 genes.

8. A method of creating a yellowed rice material, comprising: Knocking out rice OsSecY1 gene, obtaining chloroplast development abnormal, leaf color yellowing plant; the OsSecY1 nucleotide sequence of the gene is shown as SEQ ID NO.

1.

9. The method of claim 8, wherein, Using gene editing technology, a knockout mutation was made to the rice OsSecY1 gene, resulting in yellowing of the leaves.

10. A method of using the rice OsSecY1 A method of using the rice Knocking out the gene in rice OsSecY1 The application relates to a method for breeding rice by using the gene as a morphological marker; or a method for producing hybrid rice seeds by using the rice with the knocked-out gene to cross with another rice. OsSecY1 The application relates to a method for breeding rice by using the gene as a morphological marker; or a method for producing hybrid rice seeds by using the rice with the knocked-out gene to cross with another rice. OsSecY1 The application relates to a method for breeding rice by using the gene as a morphological marker; or a method for producing hybrid rice seeds by using the rice with the knocked-out gene to cross with another rice. OsSecY1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

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