Rice leaf color regulation gene ALS8 as well as encoding protein and application thereof

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a rice leaf color regulation gene ALS8 as well as an encoding protein and application thereof. The genome gDNA sequence of the ALS8 gene is shown as SEQ ID NO.1, the coding region CDS sequence of the ALS8 gene is shown as SEQ ID NO.2, and the amino acid sequence of the coded protein of the ALS8 gene is shown as SEQ ID NO.3. An ALS8 gene knockout vector is constructed through a CRISPR / Cas9 technology, the rice mutant with ALS8 function deletion is obtained, and the mutant shows the phenotype that chloroplast develops abnormally, the chlorophyll content is remarkably reduced, and the rice is lethal after yellowing in the seedling stage. Experiments prove that the ALS8 gene plays a key role in regulation and control of rice chloroplast development and leaf color formation. The ALS8 gene knockout mutant provided by the invention can be used as a leaf color marker to be applied to seedling stage screening in a hybrid rice seed production process, and has important breeding application value and agricultural production potential.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a rice leaf color regulation gene ALS8, its encoded protein, and its applications. Background Technology

[0002] Photosynthesis is the energy source for plant growth and development. Chloroplasts, as the main site of photosynthesis, directly affect the plant's growth process and yield formation. The developmental level of the thylakoid membrane system and the stacking structure of the grana within the chloroplast determine the efficiency of light energy absorption, transmission, and conversion, thereby affecting the accumulation of photosynthetic products.

[0003] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, and its yield stability is directly related to global food security. Leaf color is an important agronomical trait during rice growth and development, and also an outward indicator of chloroplast development. In production practice, abnormal leaf color in the seedling stage, especially chlorosis (whitening or yellowing), is usually closely related to chloroplast development disorders. These mutants often exhibit decreased chlorophyll content, incomplete development of the thylakoid membrane system, and abnormal grana stacking structure, leading to reduced photosynthetic efficiency, and in severe cases, even causing plant growth stagnation or death. However, leaf color mutants also have important application value: on the one hand, they can serve as ideal materials for studying the molecular mechanisms of chloroplast development; on the other hand, they can serve as intuitive seedling stage marker traits for purity identification and rapid screening in hybrid rice seed production.

[0004] Aminomethyltransferases are key enzymes in nitrogen metabolism pathways, primarily involved in the glycine cleavage system, catalyzing tetrahydrofolate-dependent reactions, and playing a crucial role in photorespiration metabolism and the nitrogen cycle. Previous studies have shown that mutations in aminomethyltransferase genes can affect plant nitrogen balance, thereby interfering with chloroplast development and photosynthetic function. However, the specific regulatory mechanisms of aminomethyltransferase family genes in rice chloroplast development remain unclear. Related functional genes still need to be discovered and identified to further refine the molecular regulatory network of rice chloroplast development and provide new gene resources and theoretical basis for breeding rice with high photosynthetic efficiency. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies, such as the unclear regulatory mechanisms of carbamoyltransferase genes in rice chloroplast development and the scarcity of related gene resources. It aims to provide a rice leaf color regulating gene, ALS8, its encoded protein, and its applications. The specific technical problem this invention aims to solve is to reveal the key functions of the ALS8 gene in rice chloroplast development and leaf color formation, and to provide a method for regulating rice chloroplast development and creating leaf color-altering mutants by knocking out the ALS8 gene. This provides new gene resources and technical means for rice genetic breeding and seedling marker screening.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides the application of knocking out the ALS8 gene in inhibiting chloroplast development in rice, wherein the genomic gDNA sequence of the ALS8 gene is shown in SEQ ID NO.1, the coding region CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0007] Experiments have shown that knocking out the ALS8 gene can specifically inhibit the development of rice chloroplasts. This inhibitory effect is manifested in three aspects: first, it significantly reduces the chlorophyll content of rice leaves; second, it causes rice to exhibit a yellowing phenotype in the seedling stage; and third, it causes abnormal ultrastructure of rice leaf chloroplasts, with the lack of thylakoid membranes and stacked grana in the chloroplasts, resulting in disordered chloroplast morphology and developmental obstruction.

[0008] This invention is the first to clearly demonstrate the specific inhibitory effect of ALS8 gene knockout on rice chloroplast development and its specific phenotypic characteristics, providing a novel gene target and experimental basis for the study of the regulatory mechanism of rice chloroplast development. At the same time, the resulting chloroplast development inhibition-related phenotypes can serve as specific markers in rice genetic breeding and have important application value.

[0009] The second aspect of this invention provides a method for cultivating rice leaf color mutant plants. The core of this method is to directionally knock out the ALS8 gene in rice to ultimately obtain rice leaf color mutant plants with yellowing leaves.

[0010] Preferably, the present invention uses the CRISPR / Cas9 gene editing system to perform targeted knockout of the ALS8 gene. This system can accurately knock out the ALS8 gene, causing the expression level of the ALS8 gene to decrease and lose its normal function, thereby stably obtaining rice leaf color mutants with leaf yellowing phenotype. This knockout method has strong targeting and high editing efficiency, and the resulting mutant phenotype is stable and heritable.

[0011] Specifically, the method for cultivating rice leaf color mutant plants by knocking out the ALS8 gene using the CRISPR / Cas9 system includes the following steps: Step 1: Design specific knockout target sites on the gDNA sequence of the ALS8 gene to achieve precise targeting of the ALS8 gene. Step 2: Based on the sequence information of the knockout target site designed in Step 1, knockout primers are synthesized. The knockout primers include a forward primer and a reverse primer, and their nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. The dimer formed after annealing the knockout primers is ligated with the CRISPR / Cas9 gene editing vector pC1300-Cas9-1gRNA to construct the recombinant gene editing vector pCas9-ALS8 that targets the knockout of the ALS8 gene. Step 3: The successfully constructed recombinant gene editing vector pCas9-ALS8 was transferred into Agrobacterium tumefaciens. The Agrobacterium tumefaciens was used to transform rice callus tissue. After screening and differentiation culture, rice leaf color mutant plants were obtained. Due to the knockout of the ALS8 gene, the plants showed the typical phenotype of yellowing leaves, accompanied by the characteristic of inhibited chloroplast development.

[0012] The beneficial effects of this invention are: This invention reveals for the first time the specific inhibitory effect of ALS8 gene knockout on rice chloroplast development and clarifies the specific phenotypic characteristics of this inhibitory effect, filling the research gap in the regulatory mechanism of carbamoyltransferase-related genes in rice chloroplast development and providing new gene resources and experimental data for the study of molecular regulatory network of rice chloroplast development.

[0013] The application of ALS8 gene knockout in suppressing rice chloroplast development provided by this invention achieves targeted regulation of rice chloroplast development. The resulting characteristic phenotypes, such as reduced chlorophyll content, leaf yellowing, and lack of thylakoid membranes and stacked grana in chloroplasts, provide ideal experimental materials for functional studies related to rice chloroplast development.

[0014] Meanwhile, the rice leaf color mutant breeding method provided by this invention achieves precise knockout of the ALS8 gene by relying on the CRISPR / Cas9 system. The breeding method is highly targeted, efficient in editing, and reproducible, and can stably obtain rice leaf color mutant plants with yellowing leaves. Due to the significant yellowing phenotypic characteristics of the obtained leaf color mutant plants in the seedling stage, they can be used as efficient seedling leaf color screening markers and applied to scenarios such as rapid screening of hybrid rice seed production and molecular marker-assisted breeding of rice. This can greatly improve the efficiency of rice breeding, simplify the impurity removal steps, and improve the purity of seed production. It can also be used as germplasm resources for the breeding of colored rice and rice for agritourism, thus expanding the application scenarios of rice breeding. Attached Figure Description

[0015] Figure 1 The results of mutation type and phenotypic identification for ALS8 knockout mutants; among which, Figure 1 Figure A is a schematic diagram of the ALS8 target site in the CRISPR / Cas9 knockout vector. The target sequence in the first exon of ALS8 is marked in green, and the PAM sequence is marked in red. Figure 1 Figure B shows the mutant. ALS8 Two types of gene mutations, als8-1 The mutant inserts a T base into the target sequence. als8-2 The mutant is a C-base deletion within the target sequence; Figure 1 Figure C shows the wild-type plant (WT) and the mutant plant. als8-1 and als8-2 Phenotype during the seedling stage; Figure 1 Figure D shows the wild-type plant (WT) and the mutant plant. als8-1 and als8-2 Results of chlorophyll content measurement in leaves ** This means p < 0.01.

[0016] Figure 2 Wild-type WT plants and mutant rice plants als8-1, als8-2 Transmission electron micrograph of leaf chloroplasts; among which Figure 2 Figure A and Figure 2 Figure B is a transmission electron microscope image of chloroplasts in a wild-type WT rice leaf. Figure 2 Figure B is Figure 2 A magnified view of a portion of Figure A, scale bar: 1μm; Figure 2 The C diagram and Figure 2 The D diagram represents the mutant. als8-1 Transmission electron microscope image of chloroplasts in rice plant leaves. Figure 2 The D diagram is Figure 2 A magnified view of a portion of Figure C, scale bar: 1μm; Figure 2 E diagram and Figure 2 The F-plot represents the mutant. als8-2 Transmission electron microscope image of chloroplasts in rice plant leaves. Figure 2 The F-graph is Figure 2 A magnified view of a portion of the E-plot, scale bar: 1μm.

[0017] Figure 3 A statistical graph showing the expression levels of the ALS8 gene pattern in different parts of wild-type rice. Detailed Implementation

[0018] 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.

[0019] Explanation of the sequence list: SEQ ID NO. 1: SEQ ID NO .2: SEQ ID NO. 3: MRGLLACATLARRAAGATSTARRHLAGAAEAAEAELKKTALYDFHVAHGGKMVPFAGWSMPIQYKDTIMDSTLNCRANGSLFDVSHMCGLSLHGRQAIPFLESLVVADVAALKDGTGTLTVFTNDRGGAIDDSVVTKVTDQHIYLVVNAGCRDKDLAHIGEHMEAFNKKGGDVKWHVHDERSLLALQGPLAAPTLQLLTKEDLS KMYFSDFKMIDINGYACFLTRTGYTGEDGFEISVPSENAVDLAKALLEKSEGKVRLTGLGARDSLRLEAGLCLYGNDMEQHITPVEAGLSWAIGKRRKAEGG FLGADVILKQLQEGPKIRRVGLLSQGPPPRSHSEIVSNSGENIGEVTSGGFSPCLKKNIAMGYVKSGLHKAGTEFKVVVRGKSYDAVVTKMPFVPTKYYKPS SEQ ID NO. 4: GGCAACCCTCAACTGCCGCGCCAA SEQ ID NO. 5: AAACTTGGCGCGGCAGTTGAGGGT SEQ ID NO. 6: ACGACGGTGGTGAAAGAAGATG SEQ ID NO. 7: GGCGTTGACGACGAGGTAGA Example 1: Obtaining rice leaf color-altering mutant plants using CRISPR / Cas9 technology This invention first selects a target site at the first penetrance of the ALS8 (LOC_Os04g53230) gene. Based on the target site sequence, a knockout forward primer ALS8-cas9-F and a knockout reverse primer ALS8-cas9-R are designed and synthesized, wherein the nucleotide sequences of ALS8-cas9-F and ALS8-cas9-R are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively; SEQ ID NO.4: 5'-ggca ACCCTCAACTGCCGCGCCAA-3' SEQ ID NO.5: 5'-aaacTTGGCGCGGCAGTTGAGGGT-3' 10 µL and 100 µM of upstream and downstream primers ALS8-cas9-F and ALS8-cas9-R were mixed in a metal bath at 100 °C for 10 minutes, and then cooled to room temperature. The mixed primers were ligated into the pC1300-Cas9-1gRNA vector digested with restriction endonuclease AarI to obtain the pCas9-ALS8 recombinant plasmid vector. The obtained pCas9-ALS8 recombinant plasmid vector was transformed into Agrobacterium strain EHA105 by electroporation. The Agrobacterium was then genetically transformed into the rice variety Zhonghua 11. The specific steps are as follows: Callus tissue from mature rice embryos cultured for one month was mixed with diluted bacterial solution and inoculated for 5 minutes. The bacterial solution was removed using sterile filter paper, and the mixture was dried in a sterile fume hood before being transferred to N6 solid medium and co-cultured at 28°C for 15 days to obtain co-cultured callus tissue. The co-cultured callus tissue was then inoculated into N6 solid selection medium containing 100 mg / L hygromycin. Hygromycin was added to the N6 solid medium to obtain N6 solid selection medium with a hygromycin concentration of 100 mg / L for the first selection. A second selection was then performed with hygromycin at a concentration of 150 mg / L. Finally, the mixture was cultured on differentiation medium containing 150 mg / L hygromycin to obtain ALS8 gene knockout transgenic lines.

[0020] Genomic DNA was extracted from transgenic plants using the CTAB method and used as a template. Identification primers ALS8-F and ALS8-R were used to amplify the target site sequence and the sequences flanking it by PCR, followed by sequencing using ALS8-F. The transgenic plant DNA was then amplified using identification primers and identified by PCR sequencing to obtain homozygous mutant lines.

[0021] The upstream sequence of the ALS8 knockout identification primer is shown in SEQ ID NO. 6, and the downstream sequence of the identification primer is shown in SEQ ID NO. 7. SEQ ID NO.6: 5'-ACGACGGTGGTGAAAGAAGATG-3' SEQ ID NO.7: 5'-GGCGTTGACGACGAGGTAGA-3' The results are as follows Figure 1 Figure A to Figure 1 As shown in Figure C, two mutant strains were obtained through gene knockout. ALS8-1 and ALS8-2 Both of these mutation methods will lead to ALS8 The protein encoding the mutant terminated prematurely, and both mutant plants exhibited a seedling-stage yellowing and death phenotype.

[0022] Example 2: Determination of chlorophyll content in leaves Wild-type and knockout mutant plants were weighed separately. als8-1 , als8-2 50 mg of leaf samples were chopped and placed in 10 ml of 95% ethanol. The samples were then left to stand in the dark at room temperature for 72 hours until all chlorophyll was extracted from the leaves. Each sample was tested in triplicate. 95% ethanol was used as a blank control. The absorbance of the samples at 645 nm and 663 nm was measured using a spectrophotometer.

[0023] Calculate the chlorophyll a, chlorophyll b, and total chlorophyll content using the following formulas: Chlorophyll a content = (12.7 × D) 663 -2.69×D 645 ) × V / W; Chlorophyll b content = (22.9 × D) 645 -4.68×D 663 )×V / W, Total chlorophyll content = chlorophyll a content + chlorophyll b content; Where V represents the volume of the sample extract; W represents the weight of the sample; D 645 and D 663 These represent the absorbance read from the spectrophotometer; the final chlorophyll content is expressed in mg / g.

[0024] The results are as follows Figure 1 As shown in Figure D, compared with the wild type, the mutant plants als8-1 and als8-2 The chlorophyll content in the leaves decreased significantly.

[0025] Example 3: Observation of Chloroplast Ultrastructure Wild-type and knockout mutant plants were collected during the seedling stage. als8-1 , als8-2 The leaves were chopped and fixed in 3% glutaraldehyde solution and 1% osmium tetroxide. They were then dehydrated stepwise with ethanol and acetone, embedded in epoxy resin, and sectioned. The samples were stained with uranyl acetate and Reynolds lead citrate, and finally observed using a Talos L120C TEM (Thermo Fisher Scientific). The results showed that the mutant... als8-1 and als8-2 The chloroplasts in the cells exhibit disordered morphology and abnormal development, lacking thylakoid membranes and stacked grana. Figure 2 ) Example 4: Gene Expression Pattern Analysis Different tissues from the wild-type plant were collected during the heading stage, frozen in liquid nitrogen, and ground into powder. Total RNA was extracted from these tissues using a plant total RNA extraction kit (Axygene, China) according to the manufacturer's instructions. The RNA was then reverse transcribed into cDNA using a ReverTra Ace qPCR-RT kit (TOYOBO, Japan). Real-time quantitative PCR (qRT-PCR) was performed using a SYBR Greenreal-time PCR master mix (TOYOBO, Japan) to detect the expression levels of ALS8 in different tissues of the wild-type plant, including roots, stems, leaves, leaf sheaths, and spikes. The results showed that ALS8 is constitutively expressed and is expressed in all different tissues of the wild-type plant, but the relative expression level is highest in leaves. Figure 3 ).

Claims

1. The application of ALS8 gene knockout in inhibiting chloroplast development in rice, characterized in that, The gDNA sequence of the ALS8 gene is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The inhibition of rice chloroplast development is manifested in reducing the chlorophyll content of rice, causing leaf yellowing, and / or causing the absence of thylakoid membranes and stacked grana in the chloroplasts of rice leaves.

3. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the ALS8 gene is shown in SEQ ID NO.

3.

4. A method for cultivating a rice leaf color mutant plant, characterized in that, The method involves knocking out the ALS8 gene in rice to obtain rice plants with yellowing leaves. The gDNA sequence of the ALS8 gene is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that, The ALS8 gene knockout in rice is described as a targeted knockout of the ALS8 gene using the CRISPR / Cas9 system, which reduces the expression level of the ALS8 gene.

6. The method according to claim 5, characterized in that, The knockout of the ALS8 gene includes the following steps: Step 1: Design a knockout target site on the gDNA sequence of the ALS8 gene; Step 2: Synthesize knockout primers based on the sequence information of the knockout target site, and ligate the dimer formed after annealing the knockout primers to the CRISPR / Cas9 gene editing vector pC1300-Cas9-1gRNA to obtain the gene editing vector pCas9-ALS8. Step 3: The gene editing vector pCas9-ALS8 is transferred into Agrobacterium, and the transformation of rice callus tissue is mediated by Agrobacterium infection to obtain leaf color mutant rice plants. The knockout primers comprise a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

7. The cultivation method according to claim 6, characterized in that, The amino acid sequence of the protein encoded by the ALS8 gene is shown in SEQ ID NO.3.

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