A rice leaf albinism gene ALS and its encoding protein and application

Abstract

This invention belongs to the field of genetic engineering, specifically relating to a gene for rice leaf albinoization. ALS Its encoded protein and applications. This invention discloses a gene regulating chloroplast development. ALS ( Albino Seedling The gDNA sequence of this gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence encoding the protein is shown in SEQ ID NO.3. Experiments show that the... ALS The loss of gene function leads to abnormal chloroplast development in rice mutants, a sharp decrease in chlorophyll content, and ultimately, albinism and death during the seedling stage. This invention addresses... ALS The study of gene function provides new germplasm resources for the study of the regulatory mechanism of rice chloroplast development, and can also be used as a leaf color marker in the seedling stage for the breeding of new rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a gene for rice leaf albinoization. ALS Its encoded proteins and applications. Background Technology

[0002] Chloroplasts play a central role in plant life activities such as photosynthesis, growth and development, and responses to abiotic stresses. Chloroplasts are the most active organelles in plant aerobic metabolism and a major source of reactive oxygen species (ROS). The accumulation of ROS damages cellular macromolecules and affects crop yield. Plants require an antioxidant defense system to scavenge ROS, with superoxide dismutase (SOD) being crucial. Currently, the mechanisms of ROS scavenging in the model plant Arabidopsis thaliana have been extensively studied.

[0003] Rice, as one of the world's most important food crops, has its growth and yield directly impacting global food security. Leaf chlorosis in rice is a common physiological anomaly, usually closely related to abnormal chloroplast development and function. Leaf chlorosis leads to damage to the structure and function of chloroplasts, affecting normal photosynthesis and thus severely impacting rice growth and yield. Although rice and Arabidopsis thaliana are evolutionarily related, they differ significantly at the physiological and molecular levels. Currently, the fine-grained regulatory mechanisms of superoxide dismutase (SOD) on chloroplast stress in rice remain unclear; therefore, further gene discovery is needed to refine the chloroplast development regulatory network. Summary of the Invention

[0004] To address the above problems, this invention provides a rice leaf albino gene. ALS Its encoded proteins and applications, genes ALS The gDNA sequence is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence encoding the protein is shown in SEQ ID NO.3; gene ALS The mutation leads to abnormal chloroplast development and leaf color changes in rice; through... ALS Gene regulation can cultivate rice varieties with better chloroplast development and leaf color traits, thereby improving the photosynthetic efficiency of rice and increasing crop yield, which has important practical value in agricultural production.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] The first aspect of this invention provides a rice leaf albino gene. ALS The application of the rice leaf albino gene ALS Used for rice breeding, regulating rice chloroplast development, or regulating rice leaf color; the rice leaf albino gene ALSThe gDNA sequence is shown in SEQ ID NO.1; the rice leaf albino gene ALS The CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence encoding the protein is shown in SEQ ID NO.3.

[0007] A second aspect of the present invention provides a method for cultivating albino mutant plants, wherein the albino mutant plants are cultivated by silencing or knocking out the aforementioned... ALS Obtained after genetic modification.

[0008] Preferably, the albino mutant plant is produced by knocking out the above-mentioned... ALS The process, obtained after gene editing, specifically includes the following steps:

[0009] exist ALS Knockout targets are designed on the gDNA sequence of the gene;

[0010] Based on the knockout target sequence, a knockout primer set was synthesized. The dimer formed after annealing the knockout primers was linked to the CRISPR / Cas9 gene editing vector pC1300-Cas9-1gRNA to obtain the gene editing vector pCas9-ALS.

[0011] The gene editing vector pCas9-ALS was transferred into Agrobacterium, and the albino mutant plants were obtained by Agrobacterium-mediated transformation.

[0012] Preferably, the knockout target sequence is from 1094bp to 1113bp of the sequence shown in SEQ ID NO.1.

[0013] Preferably, the knockout primer set comprises a forward primer and a reverse primer; the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively;

[0014] SEQ ID NO.4: 5'-ggcaTATTGGCGATCTATGCAACC-3';

[0015] SEQ ID NO. 5: 5'-aaacGGTTGCATAGATCGCCAATA-3'.

[0016] Preferably, after obtaining the albino mutant plants, the plants also need to be identified, specifically:

[0017] DNA was extracted from albino mutant plants;

[0018] The DNA was amplified using sequencing primers, and then sequenced to screen for... ALS Albino mutant plants with gene mutations;

[0019] Preferably, the sequencing primer set comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO. 6; the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 7;

[0020] SEQ ID NO.6: 5'-TGGCATGATTGGTGGCAGTG-3';

[0021] SEQ ID NO.7: 5'-GAGGCAACTTGCTTGTTTTGTCT-3'.

[0022] Preferably, the Agrobacterium is Agrobacterium. EH105 strains.

[0023] Preferably, the rice variety is Zhonghua 11 or Nipponbare.

[0024] Preferably, the rice variety is Zhonghua 11.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) This invention is the first to discover a gene with a nucleotide sequence as shown in SEQ ID NO.1. ALS It is related to the regulation of chloroplast development in rice. ALS The gene encodes a Fe-superoxide dismutase located in chloroplasts, and its efficacy has been experimentally confirmed. ALS Genes play a crucial role in the biogenesis of rice chloroplasts. ALS Genes can be used to regulate rice leaf color, rice chloroplast development, or to breed new varieties. Through regulation... ALS Gene expression can be targeted to optimize chloroplast development quality, thereby improving the photosynthetic efficiency of rice. This technology provides gene editing targets for breeding high-efficiency rice varieties, and can overcome the linkage barrier between leaf color traits and yield traits in traditional breeding. It has important application value for increasing yield per unit area and meets the urgent needs of modern agriculture for "green super rice".

[0027] (2) The present invention also discloses a method for knocking out CRISPR / Cas9 technology. ALS A method for preparing albino mutant plants using genetic engineering was developed. The resulting albino mutant plants exhibit a significant phenotypic characteristic of lethal albino seedlings. This albino trait can serve as a direct marker of leaf color, enabling rapid seedling screening in hybrid rice seed production, simplifying the impurity removal process, improving breeding efficiency, and providing a novel genetic tool directly applicable to marker-assisted breeding. Furthermore, the obtained albino mutant plants can also be used as ornamental plants in agritourism, expanding the application potential of rice in both agricultural production and tourism. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 for ALS The results of mutant phenotypic identification at different mutation sites; among which, Figure 1 Figure A shows the CRISPR / Cas9 system. ALS A schematic diagram of the target site. ALS The target sequence in the fourth exon is marked in green, and the PAM sequence is marked in red. Figure 1 Figure B shows the mutant. als Two types of gene mutations in [the organism]. als-1 The mutant involves the insertion of a single base A into the target sequence. als-2 The mutant is characterized by the deletion of one base A within the target sequence; Figure 1 Figure C shows the wild-type plant (WT) and the mutant plant. als-1 and als-2 Phenotype; Figure 1 Figure D shows wild-type plants and mutant plants. als-1 and als-2 Results of chlorophyll content determination in Chl a Indicates chlorophyll a Chl b Indicates chlorophyll b ,**express p <0.01.

[0030] Figure 2 Transmission electron micrographs of chloroplasts in wild-type and mutant rice plants; among which Figure 2 Figure A and Figure 2 Figure B is a transmission electron microscope image of chloroplasts in the leaves of a wild-type rice plant. Figure 2 Figure B is Figure 2 Enlarged view within the red box in Figure A, scale bar: 1μm; Figure 2 The C diagram and Figure 2 The D diagram represents the mutant. als-1 Transmission electron microscope image of chloroplasts in rice plant leaves. Figure 2 The D diagram is Figure 2 Enlarged view within the red box in Figure C, scale bar: 1μm; Figure 2 E diagram and Figure 2 The F-plot represents the mutant. als-2 Transmission electron microscope image of chloroplasts in rice plant leaves. Figure 2 The F-graph is Figure 2 Enlarged view within the red box in Figure E, scale bar: 1μm.

[0031] Figure 3 for ALS Statistical graph showing the expression levels of gene patterns in different parts of wild-type rice.

[0032] Figure 4 Confocal laser scanning microscopy image showing subcellular localization of ALS proteins; in which Figure 4 Figure A shows the distribution of GFP signal in rice protoplasts. Figure 4 Figure B shows the autofluorescence of chloroplasts. Figure 4 Figure C shows rice protoplasts under open field conditions. Figure 4 The D diagram is Figure 4 Figure A Figure 4 Figure B and Figure 4 Overlapping graph of C-graph, Figure 4 The E-plot shows the distribution of ALS-GFP signal in rice protoplasts. Figure 4 The F-plot represents the autofluorescence of chloroplasts. Figure 4 The G diagram shows rice protoplasts under bright field conditions. Figure 4 The H diagram is Figure 4 E-graph Figure 4 F-chart and Figure 4 Overlay of the G-plot; Scale bar: 5 μm. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0034] Chloroplasts are the most active organelles in plant aerobic metabolism and a major source of reactive oxygen species (ROS). The accumulation of ROS damages cellular macromolecules and affects crop yield. Plants require an antioxidant defense system to scavenge ROS, with superoxide dismutase (SOD) being a key component. While some ROS scavenging mechanisms have been reported in Arabidopsis thaliana, the fine-grained regulatory mechanism of SOD on chloroplast stress in rice remains unclear. Therefore, further gene discovery is needed to refine the chloroplast development regulatory network.

[0035] This invention provides a rice leaf albino gene. ALS Its encoded protein and applications. Genes regulating rice leaf albinism. ALSThe gDNA sequence is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence encoding the protein is shown in SEQ ID NO.3; this invention cultivates [protein] using CRISPR / Cas9 technology. ALS Albino mutant plants with gene loss of function were obtained, and experiments revealed that the gene... ALS The mutation leads to abnormal development of chloroplast morphology and structure in rice, a significant reduction in chlorophyll content, and rice plants exhibiting a seedling-stage albinism and death phenotype.

[0036] Example 1: Obtaining rice albino mutant plants using CRISPR / Cas9 technology

[0037] ALS The gDNA nucleotide sequence of the gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence encoding the protein is shown in SEQ ID NO.3. 。

[0038] SEQ ID NO.1:

[0039]

[0040] SEQ ID NO.2:

[0041]

[0042] SEQ ID NO.3:

[0043] MAFATLVGVGGLSPALFSPSRPLSCSSSTSVSAPFILRAGGGGDARRHGLRRLVTPLRGSACRGESTNSRVLQCANEANVVTEDDIVNDGIDDETASDAEMDEDAEANGDESSGTDEDASVSWIEQQPLPYPSDALEPYISKETVEQHWGVHQNIHVERLNGMIGGSEWEGMSLGQMMLSSFNEGREAPHPPFFHAAQIWN HDFYWRSMQPGGGGKPPERLLKFINRDFGSYDGMIRQFMDAASTQFGSGWVWLCYKTSKLPHVKSRSPIPSDNYGRLVISKSPNAINPLVWGHSPLLAID LWEHAYYLDYEDRRSDYVSTFLEKLVSWETVESRLKKAVQRAVERDEYVSTKHIRKQLLARAKSQIRAMPQQVNGDAREQTSGQEKSLGV*, * represents the stop codon.

[0044] This invention firstly ALS ( LOC_Os06g02500 The target site was selected at the fourth penetrance of the gene, and the sequence of the target site is from 1094bp to 1113bp of the sequence shown in SEQ ID NO.1. Based on the target site sequence, the knockout forward primer ALS-cas9-F and the knockout reverse primer ALS-cas9-R were designed. The nucleotide sequences of ALS-cas9-F and ALS-cas9-R are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0045] SEQ ID NO.4: 5'-ggcaTATTGGCGATCTATGCAACC-3';

[0046] SEQ ID NO. 5: 5'-aaacGGTTGCATAGATCGCCAATA-3'.

[0047] Mix 20 µL and 100 µM of ALS-cas9-F and ALS-cas9-R separately at 100 °C for 5 minutes, then cool to room temperature; ligate the mixed primers into the restriction endonuclease. AarI The pC1300-Cas9-1gRNA vector was digested with enzymes to obtain the pCas9-ALS recombinant plasmid vector; the pCas9-ALS recombinant plasmid vector was transformed into Agrobacterium strain by electroporation. EHA105 ;

[0048] The Agrobacterium was then genetically transformed into the rice variety Zhonghua 11. The specific steps are as follows:

[0049] (1) Pre-culture: The rice variety Zhonghua 11 was dehulled and disinfected, rinsed several times with sterile water, dried and spread on N6D medium, and cultured under continuous light at 32℃ for 15 days.

[0050] (2) Agrobacterium infection: Agrobacterium strains transformed into the pCas9-ALS recombinant plasmid vector were infected. EHA105 (Purchased from Beijing Coollab Technology Co., Ltd.) Activated and cultured, and the activated Agrobacterium was cultured overnight in AAM+AS medium until OD. 600 The value was 0.1. The callus tissue of Zhonghua 11 was immersed in Agrobacterium bacterial solution and gently shaken for 1.5 minutes. Excess bacterial solution was absorbed with sterile filter paper. Finally, the callus tissue was placed in N6D-AS medium with sterile filter paper containing AAM and co-cultured in the dark at 25°C for 3 days.

[0051] (3) Callus redifferentiation: The callus tissue obtained in step (2) was rinsed multiple times with sterile water containing 400 mg / L carbenicillin. Then, the callus tissue was placed in sterile water containing 400 mg / L carbenicillin and shaken on a shaker for 25 minutes. The callus tissue was rinsed 5 times with sterile water containing 400 mg / L carbenicillin (to remove residual Agrobacterium). The callus tissue was then placed in sterile water containing 400 mg / L carbenicillin and shaken on a shaker for 25 minutes. The callus tissue was rinsed 5 times with sterile water containing 400 mg / L carbenicillin. The callus tissue was then placed on sterile filter paper and dried. Finally, it was inoculated on N6DS medium containing 500 mg / L hygromycin B and 400 mg / L carbenicillin and cultured under continuous light at 32°C for two weeks.

[0052] (4) Induction of germination: The vigorous callus was transferred to RE-III medium containing 50 mg / L hygromycin B and 250 mg / L carbenicillin and continuously irradiated at 32°C for 2 weeks to induce differentiation.

[0053] (5) Rooting induction: The differentiated seedlings were transferred to HF medium containing 50 mg / L hygromycin B and 200 mg / L carboxybenzyl to induce rooting.

[0054] (6) Genomic DNA was extracted from the transgenic plants using the CTAB method and used as a template. The target sequence and the sequences on both sides were amplified by PCR using sequencing primers ALS-F and ALS-R and then sequenced. The DNA of the transgenic plants was amplified by sequencing primers and identified by PCR sequencing to obtain homozygous mutant lines.

[0055] The nucleotide sequence of ALS-F is shown in SEQ ID NO.6; the nucleotide sequence of ALS-R is shown in SEQ ID NO.7;

[0056] SEQ ID NO.6: 5'-TGGCATGATTGGTGGCAGTG-3';

[0057] SEQ ID NO.7: 5'-GAGGCAACTTGCTTGTTTTGTCT-3'.

[0058] The results are as follows Figure 1 Image A~ Figure 1 As shown in Figure C, two mutant strains were obtained through gene knockout. CAS-1 and CAS-2 Both mutations were nonsense mutations, and both mutant plants exhibited albino lethal phenotypes during the seedling stage.

[0059] Example 2: Chlorophyll Content Determination

[0060] Weigh out 0.1 g of wild-type plant and mutant plant respectively. als-1 , als-2 Leaves were chopped and placed in 10 ml of 95% anhydrous ethanol. The mixture was left to stand in the dark at room temperature until the leaves turned white and all chlorophyll was extracted. Each sample was tested three times. Using 95% anhydrous ethanol as a control, the absorption peaks of the samples at 645 nm and 663 nm were measured using a UV spectrophotometer.

[0061] Chlorophyll can be calculated using the following formula. a chlorophyll b Total chlorophyll content:

[0062] chlorophyll a Content = (12.7 × D) 663 -2.69×D 645 ) × V / W;

[0063] chlorophyll b Content = (22.9 × D) 645 -4.68×D 663 )×V / W,

[0064] Total chlorophyll content = chlorophyll a Content + Chlorophyll b content;

[0065] Where V represents the volume of the extract; W represents the weight of the leaf; D 645 and D 663 These represent the light absorbance values ​​read from the ultraviolet spectrophotometer; the final calculated chlorophyll content is expressed in mg / g.

[0066] The results are as follows Figure 1 As shown in Figure D, the mutant plant als-1 and als-2 The chlorophyll content in the sample was significantly reduced compared to the wild type.

[0067] Example 3: Transmission Electron Microscopy Observation

[0068] Transmission results of wild-type and mutant leaves at the three-leaf stage showed that the mutant... als-1 and als-2 The chloroplasts in the cells exhibit abnormal morphological and structural development, lacking thylakoid membranes and stacked grana. Figure 2 ).

[0069] Example 4: Pattern Representation Analysis

[0070] Different tissues from wild-type plants were cooled and ground into powder in liquid nitrogen. Total RNA was extracted from these tissues using a total RNA extraction kit (Axygene, China), and cDNA was synthesized using a ReverTra Ace qPCR-RT kit (TOYOBO, Japan). Real-time quantitative PCR (qRT-PCR) experiments were performed using a SYBR Green real-time PCR master mix (TOYOBO, Japan) to detect cDNA in the roots, stems, leaves, leaf sheaths, and spike tissues of wild-type plants. ALS The expression level of , the results of which indicate ALS It was expressed in different parts of the wild type, but the expression level was highest in the leaves. Figure 3 ).

[0071] Example 5: Subcellular localization

[0072] To clarify the subcellular localization of ALS, the ALS coding sequence with the stop codon removed was amplified using subcellular localization primers ALS-GFP-F (SEQ ID NO. 8) and ALS-GFP-R (SEQ ID NO. 9), and cloned into the N-terminus of the green fluorescent protein (GFP) tag of the GFP vector, thus constructing the ALS-GFP recombinant vector. This recombinant vector was transformed into rice protoplasts extracted from 3-week-old rice seedlings, and subcellular localization was detected after incubation at 28°C for 16 hours in the dark.

[0073] SEQ ID NO.8: 5'-caggagctcggtaccggatccATGGCGTTCGCCACACTG-3';

[0074] SEQ ID NO.9:

[0075] 5'-gcccttgctcaccatggatccCACCCCTAGGGACTTCTCTTGA-3'.

[0076] Using a confocal laser scanning microscope (LSM 700; Zeiss), the green fluorescence of ALS-GFP overlapped with the autofluorescence of chloroplasts, indicating that the ALS protein is localized in chloroplasts. Figure 4 ).

[0077] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A gene for rice leaf albinism ALS The application, characterized in that, By silencing or knocking out the rice leaf albino gene. ALS This gene is used to regulate chloroplast development or leaf color in rice; the rice leaf albino gene... ALS The gDNA sequence is shown in SEQ ID NO.1; The rice leaf albino gene ALS The CDS sequence is shown in SEQ ID NO.2, which describes the rice leaf albino gene. ALS The amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

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