Rice leaf whitening gene ALS 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 whitening gene ALS as well as an encoding protein and application thereof. The invention discloses a chloroplast development regulation gene ALS (Albino Seedling), the gDNA sequence of the gene is as shown in SEQ ID NO.1, the CDS sequence of the gene is as shown in SEQ ID NO.2, and the amino acid sequence of encoded protein is as shown in SEQ ID NO.3. The invention also discloses a chloroplast development regulation gene ALS. Experiments show that after function deletion of the ALS gene, chloroplast in a rice mutant develops abnormally, the chlorophyll content is sharply reduced, and the rice mutant is albino and lethal in the seedling stage. The study on the ALS gene function provides a new germplasm resource for the study on the rice chloroplast development regulation mechanism, and the ALS gene can also be used as a leaf color marker in the seedling stage to be applied to the breeding of a new rice variety.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a rice leaf albino gene ALS Its encoded protein and applications. Background Art

[0002] Plant chloroplasts play a core role in life activities such as photosynthesis, growth and development, and abiotic stress response. Chloroplasts are the most active organelles in plant aerobic metabolism and the main source of reactive oxygen in the body. The accumulation of reactive oxygen can damage cellular macromolecules and affect crop yields. Plants need an antioxidant defense system to remove reactive oxygen, among which superoxide dismutase (SOD) is the key. At present, in the model plant Arabidopsis, the mechanism of reactive oxygen scavenging has been studied in depth.

[0003] As one of the most important food crops in the world, the growth and yield of rice are directly related to global food security. Rice leaf albinism is a common physiological abnormality, which is usually closely related to the development and functional abnormalities of chloroplasts. Leaf albinism can cause damage to the structure and function of chloroplasts, affect the normal photosynthesis, and thus seriously affect the growth and yield of rice. Although rice and Arabidopsis have a certain evolutionary relationship, there are many differences between the two at the physiological and molecular levels. At present, the fine regulatory mechanism of SOD in rice on chloroplast stress is still unclear, so it is still necessary to explore genes to improve the chloroplast development regulatory network. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a rice leaf whitening gene ALS Its encoded protein and application, gene ALS The gDNA 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 of the encoded protein is shown in SEQ ID NO.3; ALS After the mutation, the rice chloroplasts will develop abnormally and the leaf color will change; ALS Gene regulation can cultivate rice varieties with better chloroplast development and leaf color traits, thereby improving the photosynthetic efficiency of rice and achieving the goal of increasing crop yields. It has important practical value in agricultural production practice.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows: The first aspect of the present invention provides a rice leaf albino gene ALS 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 of the encoded protein is shown in SEQ ID NO.3.

[0006] The second aspect of the present invention provides a method for cultivating an albino mutant plant, wherein the albino mutant plant is cultivated by silencing or knocking out the above-mentioned ALS Obtained after the gene.

[0007] Preferably, the albino mutant plant is produced by knocking out the above-mentioned ALS The specific steps include: exist ALS Design knockout targets on the gDNA sequence of the gene; A knockout primer set was synthesized according to the knockout target sequence, and the dimer formed after annealing of the knockout primer was connected to the CRISPR / Cas9 gene editing vector pC1300-Cas9-1gRNA to obtain the gene editing vector pCas9-ALS; The gene editing vector pCas9-ALS is introduced into Agrobacterium, and the albino mutant plant is obtained by Agrobacterium-mediated transformation of the plant.

[0008] Preferably, the knockout target sequence is bp 1094 to bp 1113 of the sequence shown in SEQ ID NO.1.

[0009] 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 as SEQ ID NO.4 and SEQ ID NO.5, respectively; SEQ ID NO.4: 5'-ggcaTATTGGCGATCTATGCAACC-3'; SEQ ID NO. 5: 5'-aaacGGTTGCATAGATCGCCAATA-3'.

[0010] Preferably, after obtaining the albino mutant plants, the plants need to be identified, specifically: Extract DNA from albino mutant plants; The DNA was amplified with a sequencing primer set and the ALS Albino mutant plants with gene mutations; 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; SEQ ID NO.6: 5'-TGGCATGATTGGTGGCAGTG-3'; SEQ ID NO.7: 5'-GAGGCAACTTGCTTGTTTTGTCT-3'.

[0011] Preferably, the Agrobacterium is Agrobacterium EH105 strains.

[0012] Preferably, the rice variety is Zhonghua 11 or Nipponbare.

[0013] Preferably, the rice variety is Zhonghua 11.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention first discovered the gene with the nucleotide sequence shown in SEQ ID NO.1 ALS Related to the regulation of chloroplast development in rice, ALS The gene encodes a chloroplast-localized Fe-superoxide dismutase, and experimentally confirmed ALS Gene plays a key role in rice chloroplast biogenesis. ALS The gene can be used to regulate rice leaf color, rice chloroplast development or breed new varieties. ALS Gene expression can optimize the quality of chloroplast development, thereby improving the photosynthesis efficiency of rice. This technology provides gene editing targets for breeding high-light-efficiency rice varieties, which can break through the linkage barrier between leaf color traits and yield traits in traditional breeding, and has important application value for increasing yield per unit area, meeting the urgent needs of modern agriculture for "green super rice".

[0015] (2) The present invention also discloses a method for knocking out a gene using CRISPR / Cas9 technology. ALS A method for preparing albino mutant plants obtained by gene acquisition, wherein the albino mutant plants obtained by this method exhibit significant phenotypic characteristics of albinism and lethality in the seedling stage. The albino trait can be used as an intuitive leaf color marker to achieve rapid screening in the seedling stage during hybrid rice seed production, simplify the impurity removal process, improve breeding efficiency, and provide a new type of genetic tool that can be directly applied for molecular marker-assisted breeding. At the same time, the albino mutant plants obtained can also be used as plant ornamental resources in sightseeing agriculture, expanding the application potential of rice in agricultural production and sightseeing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 for ALS The results of mutant phenotype identification at different mutation sites; Figure 1 Figure A shows the CRISPR / Cas9 system ALS Schematic diagram of the target site. ALS The target sequence within 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 als-1 The mutant has a base A inserted into the target sequence. als-2 The mutant was a deletion of a base A in 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 the wild-type plant and the mutant plant als-1 and als-2 The results of chlorophyll content determination in a Chlorophyll a , Chl b Chlorophyll b ,**express p <0.01.

[0018] Figure 2 The transmission electron micrographs of chloroplasts of wild-type and mutant rice plants are shown in Figure 1. Figure 2 Figure A and Figure 2 Figure B is a transmission electron micrograph of chloroplasts in leaves of wild-type rice plants. Figure 2 Figure B is Figure 2 The enlarged image in the red frame of Figure A, scale bar: 1 μm; Figure 2 Figure C and Figure 2 Figure D shows the mutant als-1 Transmission electron micrograph of chloroplasts in rice plant leaves. Figure 2 The D graph is Figure 2 The enlarged image in the red frame of Figure C, scale bar: 1 μm; Figure 2 The E graph and Figure 2 The F figure is the mutant als-2 Transmission electron micrograph of chloroplasts in rice plant leaves. Figure 2 The F graph is Figure 2Enlarged view of the red frame in Figure E, scale bar: 1 μm.

[0019] Figure 3 for ALS Analytical statistics of the expression levels of gene patterns in different parts of wild-type rice.

[0020] Figure 4 This is a confocal laser scanning microscopy image of the subcellular localization of ALS protein; Figure 4 Figure A shows the distribution of GFP signal in rice protoplasts. Figure 4 Figure B shows chloroplast autofluorescence. Figure 4 Figure C shows rice protoplasts under bright field. Figure 4 The D diagram is Figure 4 Figure A Figure 4 Figure B and Figure 4 The overlap graph of graph C is Figure 4 Figure E shows the distribution of ALS-GFP signals in rice protoplasts. Figure 4 Figure F shows chloroplast autofluorescence. Figure 4 Figure G shows rice protoplasts under bright field. Figure 4 The H diagram is Figure 4 Figure E Figure 4 The F graph and Figure 4 Overlay of G images; scale bar: 5 μm. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0022] Chloroplasts are the most active organelles in plant aerobic metabolism and the main source of reactive oxygen in the body. Accumulation of reactive oxygen can damage cellular macromolecules and affect crop yields. Plants need an antioxidant defense system to remove reactive oxygen, of which superoxide dismutase (SOD) is the key. Some reactive oxygen scavenging mechanisms have been reported in Arabidopsis, but the fine regulation mechanism of SOD on chloroplast stress in rice is still unclear, so it is still necessary to explore genes to improve the chloroplast development regulatory network.

[0023] The invention provides a rice leaf albino gene ALS Its encoded protein and application. Regulating rice leaf albino gene 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 of the encoded protein is shown in SEQ ID NO.3; the present invention cultivates the ALS The albino mutant plant with missing gene function was found through experiments. ALS The mutation will cause abnormal development of rice chloroplast morphology and structure and a significant reduction in chlorophyll content, and the rice plants will show a lethal phenotype of albinism at the seedling stage.

[0024] Example 1: Obtaining rice albino mutant plants using CRISPR / Cas9 technology 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 of the encoded protein is shown in SEQ ID NO.3 。

[0025] SEQ ID NO.1: SEQ ID NO.2: SEQ ID NO.3: MAFATLVGVGGLSPALFSPSRPLSCSSSTSVSAPFILRAGGGGDARRHGLRRLVTPLRGSACRGESTNSRVLQCANEANVVTEDDIVNDGIDDETASDAEMDEDAEANGDESSGTDEDASVSWIEQQPLPYPSDALEPYISKETVEQHWGVHQNIHVERLNGMIGGSEWEGMSLGQMMLSSFNEGREAPHPPFFHAAQIWN HDFYWRSMQPGGGGKPPERLLKFINRDFGSYDGMIRQFMDAASTQFGSGWVWLCYKTSKLPHVKSRSPIPSDNYGRLVISKSPNAINPLVWGHSPLLAID LWEHAYYLDYEDRRSDYVSTFLEKLVSWETVESRLKKAVQRAVERDEYVSTKHIRKQLLARAKSQIRAMPQQVNGDAREQTSGQEKSLGV*, * represents the stop codon.

[0026] The present invention first ALS ( LOC_Os06g02500 ) The target site is selected at the fourth exon of the gene, and the sequence of the target site is from 1094bp to 1113bp of the sequence shown in SEQ ID NO.1. According to the target site sequence, the knockout forward primer ALS-cas9-F and the knockout reverse primer ALS-cas9-R are designed, and the nucleotide sequences of ALS-cas9-F and ALS-cas9-R are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively;

[0027] SEQ ID NO.4: 5'-ggcaTATTGGCGATCTATGCAACC-3'; SEQ ID NO. 5: 5'-aaacGGTTGCATAGATCGCCAATA-3'.

[0028] Mix 20 µL of 100 µM ALS-cas9-F and ALS-cas9-R at 100°C for 5 minutes, and cool to room temperature. Connect the mixed primers to the restriction endonuclease. Aa The pC1300-Cas9-1gRNA vector was digested to obtain the pCas9-ALS recombinant plasmid vector; the pCas9-ALS recombinant plasmid vector was transformed into the Agrobacterium strain by electroporation EHA105 ; The above Agrobacterium was then genetically transformed into rice variety Zhonghua 11, and the specific operation steps were as follows: (1) Pre-culture: Rice variety Zhonghua 11 was hulled and sterilized, rinsed with sterile water for several times, blown dry, and spread on N6D medium, and cultured at 32°C under continuous light for 15 days.

[0029] (2) Agrobacterium infection: The Agrobacterium strain that has been transformed with the pCas9-ALS recombinant plasmid vector EHA105 (purchased from Beijing Coolaibo Technology Co., Ltd.) and activated culture, and the activated Agrobacterium was cultured overnight with AAM+AS culture medium until OD 600 The callus of Zhonghua 11 was immersed in the Agrobacterium bacterial solution and gently shaken for 1.5 minutes, and the excess bacterial solution was absorbed with sterile filter paper. Finally, the callus was placed in N6D-AS medium with sterile filter paper with AAM and co-cultured at 25°C in the dark for 3 days.

[0030] (3) Callus redifferentiation: The callus obtained in step (2) was rinsed several times with sterile water containing 400 mg / L carboxybenzyl, and then the callus was placed in sterile water containing 400 mg / L carboxybenzyl and shaken on a shaker for 25 minutes. The callus was rinsed again with sterile water containing 400 mg / L carboxybenzyl for 5 times (to remove residual Agrobacterium), and then the callus was placed in sterile water containing 400 mg / L carboxybenzyl and shaken on a shaker for 25 minutes. The callus was rinsed again with sterile water containing 400 mg / L carboxybenzyl for 5 times, and then the callus was placed on sterile filter paper and air-dried. Finally, the callus was inoculated on N6DS medium containing 500 mg / L hygromycin B and 400 mg / L carboxybenzyl and cultured at 32°C with continuous light for two weeks.

[0031] (4) Inducing germination: The vigorously growing callus was transferred to RE-III medium containing 50 mg / L hygromycin B and 250 mg / L carboxybenzyl and incubated at 32°C for 2 weeks under continuous light to induce differentiation.

[0032] (5) Root induction: The differentiated seedlings were transferred to HF medium containing 50 mg / L hygromycin B and 200 mg / L carboxybenzyl to induce rooting.

[0033] (6) The genomic DNA of the transgenic plants was extracted using the CTAB method and used as a template to perform PCR amplification and sequencing of the target sequence and the sequences on both sides using sequencing primers ALS-F and ALS-R. The transgenic plant DNA was amplified using sequencing primers for PCR sequencing identification to obtain a homozygous mutant strain.

[0034] 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; SEQ ID NO.6: 5'-TGGCATGATTGGTGGCAGTG-3'; SEQ ID NO.7: 5'-GAGGCAACTTGCTTGTTTTGTCT-3'.

[0035] The results are as follows Figure 1 A picture~ Figure 1 As shown in Figure C, two mutant strains were obtained by gene knockout. cas-1 and cas-2 Both mutations were nonsense mutations, and both mutant plants showed a lethal phenotype of albinism at the seedling stage.

[0036] Example 2: Determination of Chlorophyll Content Weigh 0.1 g of wild-type plants and mutant plants respectively. als-1 , als-2 Cut the leaves into pieces and place them in 10 ml of 95% anhydrous ethanol. Leave them in the dark at room temperature until the leaves turn white and all chlorophyll precipitates. Repeat three times for each sample. Use 95% anhydrous ethanol as a control and use a UV spectrophotometer to measure the absorption peaks of the samples at 645 nm and 663 nm.

[0037] Chlorophyll was calculated according to the following formula a , Chlorophyll b And total chlorophyll content: 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 extract; W represents the weight of the leaves; D 645 and D 663 They respectively represent the light absorption values ​​read in the UV spectrophotometer; the final calculated chlorophyll content is in mg / g.

[0038] The results are as follows Figure 1 As shown in Figure D, the mutant plants als-1 and als-2 The chlorophyll content in the strain was significantly reduced compared with that in the wild type.

[0039] Example 3: Transmission electron microscopy observation The results of transillumination of wild-type and mutant leaves at the three-leaf stage showed that the mutant als-1 and als-2 The chloroplasts in the chloroplasts are abnormally morphologically developed, lacking thylakoid membranes and stacked grana ( Figure 2 ).

[0040] Example 4: Pattern Expression Analysis Different tissues of the wild type were cooled in liquid nitrogen and ground into powder. Total RNA of different tissues was extracted using a total RNA extraction kit (Axygene, China), and cDNA was synthesized using a ReverTra Ace qPCR-RT kit (TOYOBO, Japan). Real-time fluorescence quantitative PCR (qRT-PCR) experiments were performed using SYBR Green real-time PCR master mix (TOYOBO, Japan) to detect the expression of ribonucleic acid in roots, stems, leaves, leaf sheaths, and panicle tissues of wild-type plants. ALS The results showed that ALS It is expressed in different parts of the wild type, but the highest expression level is in leaves ( Figure 3 ).

[0041] Example 5: Subcellular localization To clarify the subcellular localization of ALS, the ALS coding sequence without the stop codon 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 to construct the ALS-GFP recombinant vector. The recombinant vector was transformed into rice protoplasts extracted from 3-week-old rice seedlings, and the subcellular localization was detected after incubation at 28°C for 16 hours in the dark;

[0042] SEQ ID NO.8: 5'-caggagctcggtaccggatccATGGCGTTCGCCACACTG-3'; SEQ ID NO.9: 5'-gcccttgctcaccatggatccCACCCCTAGGGACTTCTCTTGA-3'.

[0043] Using a confocal laser scanning microscope (LSM 700; Zeiss), we observed that the green fluorescence of ALS-GFP overlapped with the autofluorescence of chloroplasts, indicating that the ALS protein was localized in chloroplasts ( Figure 4 ).

[0044] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0045] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A rice leaf albinism gene ALS The application is characterized in that The rice leaf albinism gene ALS Used for rice breeding, regulating rice chloroplast development or regulating rice leaf color; the rice leaf albino gene ALS The gDNA sequence is shown in SEQ ID NO.1; The rice leaf albinism gene ALS The CDS sequence of the rice leaf albino gene is shown in SEQ ID NO.

2. ALS The amino acid sequence of the encoded protein is shown in SEQ ID NO.

3.

2. A method for cultivating an albino mutant plant, characterized in that: The albino mutant plant is obtained by silencing or knocking out the ALS Obtained after the gene.

3. The cultivation method according to claim 2, characterized in that The albino mutant plant is obtained by knocking out the ALS Obtained after the gene.

4. The cultivation method according to claim 3, characterized in that The following steps are involved: exist ALS Design knockout targets on the gDNA sequence of the gene; A knockout primer set was synthesized according to the knockout target sequence to construct the CRISPR / Cas9 gene editing vector pCas9-ALS; The gene editing vector pCas9-ALS is introduced into Agrobacterium, and the plant is transformed by Agrobacterium-mediated transformation to obtain the albino mutant plant.

5. The cultivation method according to claim 3, characterized in that: The knockout target sequence is bp 1094 to bp 1113 of the sequence shown in SEQ ID NO.

1.

6. The cultivation method according to claim 3, characterized in that: 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; SEQ ID NO.4: 5'-ggcaTATTGGCGATCTATGCAACC-3'; SEQ ID NO. 5: 5'-aaacGGTTGCATAGATCGCCAATA-3'.

7. The cultivation method according to claim 3, characterized in that: The Agrobacterium is Agrobacterium EH105 strains.

8. The cultivation method according to claim 3, characterized in that: The rice variety is Zhonghua 11 or Nipponbare.

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