CN116987714A - Application of OsENODL9 gene in rice grain breeding - Google Patents
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The application relates to the technical field of plant genetic engineering, in particular to application of an OsENODL9 gene in rice grain type breeding, and the rice OsENODL9 gene is knocked out or normal expression is destroyed by a genetic engineering technology, so that a rice variety with ideal grain type is bred, the genome sequence of the rice OsENODL9 gene is shown as SEQ ID NO.1, and the mRNA sequence of the rice OsENODL9 gene is shown as SEQ ID NO. 2. The application utilizes the genetic engineering technology to destroy the normal expression of the OsENODL9, and discovers that the knockout mutation of the OsENODL9 can obviously increase the grain length of rice and reduce the grain width of the rice for the first time, thereby providing a novel molecular breeding approach for developing rice grain breeding by further utilizing the OsENODL9 gene and having wide application prospect.
Description
Technical Field
The application belongs to the technical field of plant genetic engineering, and particularly relates to an application of an OsENODL9 gene in rice grain type breeding, namely a molecular breeding method for creating an ideal grain type rice new variety by knocking out or destroying the normal expression of the OsENODL9 gene.
Background
Rice is a staple food in China and even the world. With the increase of world population and the improvement of living standard of people, how to increase the yield and quality of rice has become the focus of attention of the management departments and researchers related to various countries in the world. Wherein, based on genetic engineering means, the excavation and utilization of key genes for regulating and controlling the yield and quality of rice to develop molecular breeding are important means for improving the yield and quality of rice.
The rice grain mainly comprises grain length, grain width and grain thickness of grains. The formation process of the rice grain shape not only determines the weight/yield of the rice, but also determines the appearance, processing and other qualities of the rice to a certain extent. For example, within a certain range, the rice chalkiness decreases with increasing grain length, but increases with increasing grain width. Therefore, the method has very important significance for developing molecular breeding, cultivating high-yield and high-quality new rice varieties by excavating and utilizing key genes for regulating rice grain types.
At present, many reports on gene cloning and action mechanism research related to rice grain type regulation are reported [ Li et al molecular Networks of Seed Size Control in plants.annual Review of Plant Biology,2019, 70:435-63]. For example, the first previously cloned GS3 gene controlling rice grain length and grain weight, encoding a protein containing OSR domain at the N-terminus and TNFR and VWFC domains at the C-terminus, accomplishes its regulatory function on rice grain length and grain weight by negatively regulating the cell number of rice glumes [ Fan et al gs3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theoretical and Applied Genetics,2006, 112:1164-1171.Sun et al.A G-protein pathway determines grain size in rice Communications,2018,9:851]. Subsequently, the GW2 gene was found to inhibit translation of the protein encoded by the gene after the base mutation of the 5' UTR region thereof, which finally resulted in a lengthening of the grain length of rice grains [ Zhang et al A single nucleotide substitution at-UTR of GSN1 represses its translation and leads to an increase of grain length in line. Journal of Genetics and Genomics,2019, 46:105-108]. The rice grain-type positive control major QTL gene GLW7 is also reported, and the encoded ospl 13 transcription factor can significantly increase grain length of grains, thereby improving rice yield [ Si et al OsSPL13 controls grain size in cultivated rice nature Genetics,2016, 48:447-456]. However, rice grain types belong to complex quantitative traits, and many genes of thousands of synergism may be involved in the formation of rice grain types [ Li and li.signaling pathways of seed size control in plants.current Opinion in Plant Biology,2016, 33:23-32]. Therefore, the rice grain type regulatory genes are excavated and utilized as much as possible, which is helpful for developing molecular breeding so as to improve the yield and quality of rice. The OsENODL9 gene is knocked out by CRISPR technology to change the grain type of rice grains, so that the rice grain type molecular breeding method is hopeful to be used, and no related report is found at present.
Disclosure of Invention
Aiming at the defects and the problems in the prior art, the application aims to provide the application of the OsENODL9 gene in rice grain type breeding, and the application uses the genetic engineering technology to knock out or destroy the normal expression of the OsENODL9 gene, thereby providing a novel molecular breeding method for breeding rice varieties with ideal grain types by utilizing the OsENODL9 gene.
In order to achieve the above purpose, the application adopts the following technical scheme:
the application of the OsENODL9 gene in rice grain type breeding is that the rice OsENODL9 gene is knocked out or the normal expression of the rice OsENODL9 gene is destroyed by a genetic engineering technology, a novel rice variety with ideal grain type is bred, the genome sequence of the rice OsENODL9 gene is shown as SEQ ID NO.1, and the mRNA sequence of the rice OsENODL9 gene is shown as SEQ ID NO. 2.
The application preferably uses CRISPR gene editing technology to knock out or destroy the normal expression of rice OsENODL9 gene, comprising the following steps:
(1) Construction of CRISPR knockout expression vector of OsENODL9 gene:
and (3) utilizing a CRISPR knockout target primer pair, a rice U6a/U6b promoter and a pYLCRISPR/Cas9 plasmid to obtain the CRISPR knockout expression vector of the OsENODL9 gene through enzyme digestion, connection, transformation of escherichia coli competent cells, positive colony PCR detection and sequencing analysis.
(2) Obtaining OsENODL9 gene knockout mutant long-grain/narrow-grain rice plants:
the constructed OsENODL9 gene knockout expression vector is directly transformed into calli/cells of flower 11 in japonica rice by using an agrobacterium-mediated method, transgenic rice plants are obtained through hygromycin screening, resistance callus differentiation and rooting culture, then the transgenic rice with the OsENODL9 gene knockout mutation is identified by using methods of PCR amplification, electrophoresis, sequencing analysis and the like, and is planted together with wild rice in a field seed, and seed comparison analysis is carried out in a mature period to determine that the OsENODL9 gene knockout mutant long-grain/narrow-grain rice plants are obtained.
The CRISPR/Cas9 knockout target of the OsENODL9 gene in the step (1) comprises a primer pair shown as SEQ ID NO.3 and SEQ ID NO. 4; and a primer pair shown as SEQ ID NO.5 and SEQ ID NO. 6.
The specific primer pair sequences of the hygromycin screening gene fragment amplification in the step (2) are shown as SEQ ID NO.7 and SEQ ID NO. 8.
Compared with the prior art, the application has the beneficial effects that:
the application provides the application of the OsENODL9 gene in rice grain type breeding for the first time, and the rice grain is lengthened and narrowed by knocking out or destroying the normal expression of the OsENODL9 gene so as to achieve the purposes of obtaining ideal rice grain type and improving rice quality, thereby providing a new molecular breeding approach for cultivating excellent rice varieties with ideal grain type by utilizing the OsENODL9 gene and having wide application prospect.
Drawings
Fig. 1: is a schematic structural diagram of the pYLCRISPR/Cas9 plasmid. Wherein P is 35s Is a 35S promoter of cauliflower mosaic virus; p (P) ubi Is a maize ubiquitin gene promoter; ccdB is ccdB lethal gene, and after BsaI enzyme digestion, the ccdB is replaced by the sgRNA expression cassette to complete the construction of the knockout expression vector; tnos is a terminator; RB is the right boundary; LB is the left boundary; kan (Kan) R Is a kanamycin resistance gene; HPT is a hygromycin resistance gene; pBR322 ori is the replication initiation site of pBR 322; cas9p is a Cas9 protein gene; SP-L1 and SP-R are colony PCR primer pairs and sequencing primers.
Fig. 2: is the sequencing identification of CRISPR/Cas9 knockout target site and off-target site of the OsENODL9 gene. A: schematic diagram of double-knockout target sequences of OsENODL9 genes; b: sequencing analysis of double-target mutation types (target 1 on the left and target 2 on the right); c: knocking out the possible off-target sequence of the target point 1 and the position of the off-target sequence on the genome; d: knocking out possible off-target sequences of the target 2 and the positions of the off-target sequences on a genome; e: knocking out the sequencing result of the most probable off-target sequence corresponding to the target 1 without mutation; f: and knocking out sequencing results of the most probably off-target sequence corresponding to the target 2 without mutation. WT: is wild rice middle flower 11; CR1: is a mutant in which 1T base insertion occurs in both target 1 and target 2, i.e., a (+T/+T) knockout mutant; CR2: is a mutant in which 1A base insertion occurs in target 1 and 46 base deletion occurs in target 2, i.e., (+A/-46 nt) knockout mutant.
Fig. 3: is the grain length and grain width analysis result of OsENODL9 gene knockout mutant rice. A: the grain length phenotype of the mature OsENODL9 gene knockout mutant rice; b: grain length statistics of mature OsENODL9 gene knockout mutant rice; c: the grain width phenotype of the OsENODL9 gene knockout mutant rice in the mature period; d: grain width statistics results of mature-period OsENODL9 gene knockout mutant rice; WT: middle flower 11 (wild type); CR1 and CR2: knockout mutants of OsENODL9 gene, respectively; * *: p < 0.01.
Detailed Description
The experimental methods referred to in the following examples are conventional experimental methods unless otherwise specified. In the application, primer sequence synthesis and DNA fragment sequencing analysis are completed by Shanghai biological engineering technology Co.
Example 1: construction of knockout expression vector of OsENODL9 gene and rice genetic transformation
The genome DNA sequence SEQ ID NO.1 and the mRNA sequence SEQ ID NO.2 of the rice OsENODL9 gene are obtained in a phytozome database (https:// phytozome-next. Jgi. Doe. Gov /), and then the CRISPR/Cas9 knockout target 1 primer pair SEQ ID NO.3 of the OsENODL9 gene is obtained by utilizing online software CRISPR-P2.0 (http:// CRISPR. Hzau. Edu. Cn/CRISPR2 /). GCCGCAGCAACATTCTTCGACAA (Bsa I cleavage site at the first four positions) and SEQ ID NO.4: AAACTTGTCGAAGAATGTTGCTG (Bsa I cleavage site in the first four positions), the target 2 primer pair SEQ ID NO.5 is knocked out: GTTGAGCGATTCTTTAGACCCCAA (Bsa I cleavage site at the first four positions) and SEQ ID NO.6: AAACTTGGGGTCTAAAGAATCGCT (Bsa I cleavage site in the first four positions); then, carrying out denaturation annealing on the primer pair, respectively connecting the primer pair with rice U6a and U6b promoters, and carrying out 2 rounds of overlapping PCR amplification to obtain 2 sgRNA expression cassettes; then, the gene CRISPR/Cas9 double-target knockout expression vector of the OsENODL9 gene is obtained by enzyme digestion and connection to a pYLCRISPR/Cas9 plasmid (figure 1), and then the method of a precursor [ Hiei et al. Effect transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal,1994,6:271-282]Based on agrobacterium mediating method, transforming flower 11 in japonica rice, obtaining 37 strains of genetic transformation T altogether through callus induction, preculture, co-culture, resistance callus screening, differentiation, rooting culture, seedling hardening, transplanting and the like 0 And (3) extracting genome DNA of the rice plants of the generation by using a CTAB method, and then carrying out PCR identification of hygromycin gene specific primers to determine to obtain transgenic positive rice plants. The specific primer pair sequences for amplifying hygromycin gene fragments are as follows:
HPT-F：5’-CTGAACTCACCGCGACGTCTGTC-3′(SEQ ID NO.7)；
HPT-R：5’-TAGCGCGTCTGCTGCTCCATACA-3’(SEQ ID NO.8)；
the PCR amplification conditions were: 94 ℃ for 4min; [94 ℃ 30sec,62 ℃ 30sec,72 ℃ 50sec ],32 cycles; 7min at 72 ℃.
Example 2: CRISPR/Cas9 knockout mutant molecular identification of OsENODL9 gene
According to CRISPR/Cas9 knockout target sequence information (figure 2A) of OsENODL9 gene, a pair of specific primers (SEQ ID NO.9: GCCCATCTTGGAGCCCATAA and SEQ ID NO.10: CGAGCAGGTACTTGGTGTCG) are designed on rice genome corresponding to the gene, wild type middle flower 11 (WT) and 37 parts of rice genome DNA extracted as above are used as templates, single target genome DNA fragment containing the double knockout target sequence is obtained through amplification, sequencing analysis is carried out, and T with knockout mutation is determined to be obtained 0 The transgenic plant is generated, positive detection of hygromycin gene fragments and sequencing analysis of double target sites are further carried out in the seedling stage of the isolated offspring, so that the OsENODL9 knockout mutant without T-DNA insertion is obtained, and PCR amplification and sequencing analysis are carried out on possible off-target sites of the double targets. Two homozygous knockout mutants (CR 1 and CR 2) without exogenous T-DNA insertion and without off-target phenomena (FIGS. 2B-F) were used in subsequent experiments.
Example 3: the OsENODL9 gene knockout mutation obviously increases grain length and reduces grain width
Subsequently, wild-type middle flower 11 (WT) was planted in the field simultaneously with knockout mutants of OsENODL9 gene (CR 1 and CR 2), found in the mature period: the OsENODL9 knockout mutants CR1 and CR2 were significantly longer in grain length (fig. 3A and B) than wild-type WTs, and significantly narrower in grain width (fig. 3C and D) than wild-type WTs.
Claims (7)
- The application of the OsENODL9 gene in rice grain type breeding is characterized in that the genome DNA sequence of the OsENODL9 gene is shown as SEQ ID NO.1, and the mRNA sequence of the OsENODL9 gene is shown as SEQ ID NO. 2.
- 2. The application of the OsENODL9 gene in rice grain type breeding according to claim 1, wherein the rice OsENODL9 gene is knocked out or normal expression is destroyed, and an ideal grain type rice variety is bred.
- 3. The use of OsENODL9 gene according to claim 2 in rice grain breeding, wherein the genetic engineering technique is CRISPR gene editing technique.
- 4. The use of OsENODL9 gene according to claim 2 in rice grain breeding, wherein the knockout or disruption of OsENODL9 gene normal expression increases rice grain length and decreases rice grain width; the ideal grain type rice variety is a long grain/narrow grain rice plant.
- 5. The use of OsENODL9 gene according to any one of claims 2 to 4 in rice grain breeding, wherein the normal expression of the OsENODL9 gene of rice is knocked out or destroyed by CRISPR gene editing technique to obtain OsENODL9 knocked out mutant long grain/narrow grain rice plants, comprising the steps of:(1) CRISPR/Cas9 knockout expression vector construction of OsENODL 9: the CRISPR knockout target point primer pair, the rice U6a/U6b promoter and the pYLCRISPR/Cas9 plasmid are utilized to obtain a CRISPR/Cas9 knockout expression vector of the OsENODL9 through enzyme digestion, connection, transformation of escherichia coli competent cells, positive colony PCR detection and sequencing analysis;(2) Obtaining OsENODL9 knockout mutant long grain/narrow grain rice plants: the constructed CRISPR/Cas9 knockout expression vector of the OsENODL9 is directly transformed into calli/cells of flower 11 in japonica rice by using an agrobacterium-mediated method, a transgenic rice plant is obtained through hygromycin screening, resistance callus differentiation and rooting culture, and then the transgenic rice with the OsENODL9 knockout mutation is obtained by identification through PCR amplification, electrophoresis and sequencing analysis methods, and is planted together with wild rice in a field, and is subjected to seed comparison analysis in a mature period to determine that the long-grain/narrow-grain rice plant with the OsENODL9 knockout mutation is obtained.
- 6. The use of OsENODL9 gene according to claim 5 in rice grain breeding, wherein the CRISPR/Cas9 knockout target of OsENODL9 gene in step (1) comprises a primer pair as shown in SEQ ID No.3 and SEQ ID No. 4; and a primer pair shown as SEQ ID NO.5 and SEQ ID NO. 6.
- 7. The use of OsENODL9 gene according to claim 5 in rice grain breeding, wherein the specific primer pair sequences amplified by hygromycin screening gene fragments in step (2) are shown as SEQ ID NO.7 and SEQ ID NO. 8.
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| CN202311084051.2A CN116987714A (en) | 2023-08-28 | 2023-08-28 | Application of OsENODL9 gene in rice grain breeding |
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