CN115725597A - Rice grain width and weight regulation gene DWG1 and application thereof - Google Patents
Rice grain width and weight regulation gene DWG1 and application thereof Download PDFInfo
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Abstract
The invention discloses a rice grain width and grain weight regulation gene DWG1 and application thereof, belonging to the field of plant genetic engineering, wherein a nucleotide sequence for coding the gene DWG1 is shown as SEQ ID NO:1, and the amino acid sequence of the protein coded by the protein is shown as SEQ ID NO:2, respectively. The invention also discloses application of the over-expression DWG1 in increasing the grain width and grain weight of rice and improving the rice yield after DWG1 gene recombination is carried out on an expression vector and rice is transformed, and the invention has important significance for the synergistic improvement of the rice yield and quality.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to a rice grain width and grain weight regulating gene DWG1 and application thereof.
Background
The grain weight, the effective spike number, the spike grain number and the setting rate are four major factors forming the rice yield, the grain weight is determined by the grain type and the grain fullness, and the grain weight is less influenced by the environment and is the most stable constituent factor of the rice yield. The rice grain type is a composite trait and mainly comprises grain length, grain width, aspect ratio and grain thickness, and the morphological traits are important components of rice grain weight and directly influence the rice yield.
The rice grain type belongs to quantitative characters and is regulated and controlled by a complex genetic network. At present, the rice grain type is mainly determined by the number of glume cells and the cell size, and is jointly regulated and controlled by cell proliferation and cell expansion pathways. The current research shows that the rice grain type regulatory gene is mainly involved in molecular approaches such as G protein signal, ubiquitin-proteasome, mitogen Activated Protein Kinase (MAPK) signal, plant hormone signal, transcription factor regulation and the like to regulate glume cell proliferation and cell expansion.
In recent years, the cloning and function analysis of the grain type gene provide important gene resources for the molecular design and breeding of rice. With the rapid development of rice molecular breeding technology, part of cloned dominant grain type alleles have been successfully applied to the variety improvement of rice. Although a certain number of grain type genes are cloned in rice at present, the number of genes which are really widely applied in breeding is still small, and the molecular mechanism for grain type regulation is still unclear. Therefore, the method continues to excavate new grain type genes and analyze new regulation and control mechanisms, and has important significance for the synergistic improvement of the yield and the quality of the rice.
Disclosure of Invention
The invention aims to provide a rice grain width and grain weight regulation gene DWG1 and application thereof, and has important significance for the synergistic improvement of rice yield and quality.
In order to achieve the purpose, the invention provides a rice grain width and grain weight regulating gene DWG1, and the nucleotide sequence of the gene DWG1 is shown as SEQ ID NO:1 or a sequence corresponding to SEQ ID NO:1 present at least 90% homology.
Preferably, the nucleotide sequence of the rice grain width and grain weight regulatory gene DWG1 is also included in the nucleotide sequence shown in SEQ ID NO:1 by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence shown in the formula 1.
The invention also provides a protein coded by the rice grain width and grain weight regulating gene DWG1, and the amino acid sequence of the protein is shown as SEQ ID NO:2, respectively.
Preferably, the amino acid sequence of the protein encoded by the rice grain width and grain weight regulatory gene DWG1 is also included in SEQ ID NO:2 by adding, substituting, inserting or deleting one or more amino acids to the amino acid sequence shown in the formula (2).
The invention also provides a recombinant vector containing the nucleotide sequence.
The invention also provides a recombinant engineering bacterium containing the genetic engineering vector.
The invention also provides an application of the recombinant vector or the recombinant engineering bacterium in rice breeding.
Preferably, the above-mentioned use comprises the preparation of transgenic rice lines.
The rice grain width and grain weight regulation gene DWG1 and the application thereof definitely disclose that the DWG1 is a regulation gene of the rice grain width and grain weight by utilizing a CRISPR/Cas9 editing system, solve the problems of rice grain width and grain weight regulation, excellent rice breeding and the like, and have the following advantages:
the invention provides a novel rice grain width and grain weight regulation gene DWG1. The DWG1 is edited by using a CRISPR/Cas9 system to obtain the DWG1-KO mutant, the grain width and the grain weight are obviously reduced, the overexpression DWG1 obviously increases the grain width and the grain weight, the biological function of the gene in the aspect of regulating the grain type and the grain weight of rice is determined, the DWG1 can be applied to the improvement of the grain type and the grain weight of rice, the rice yield is improved, the important breeding and utilization value is achieved, and meanwhile, a new thought is provided for researching a rice grain type and grain weight regulation mechanism.
Drawings
FIG. 1 is a diagram showing the results of gene mapping of rice gene DWG1 of the present invention.
FIG. 2 is a spectrum of the knockout vector of DWG1 gene in the present invention.
FIG. 3 is a diagram of the over-expression vector of DWG1 gene of the present invention.
FIG. 4 is a graph showing the comparison results of rice grain width and grain weight phenotype before and after DWG1 gene knockout in the present invention.
FIG. 5 is a graph showing the comparison results of rice grain width and grain weight phenotype before and after overexpression of DWG1 gene in the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Description of the drawings: r600 is a conventional rice variety;
the methods used in the examples are conventional methods, and the reagents used are conventional reagents unless otherwise specified.
Experimental example 1 obtaining of rice DWG1 mutant
The mutant DWG1 is obtained by mutagenizing rice R600 by Ethyl Methane Sulfonate (EMS) and has stable phenotype through continuous multi-generation selfing. The mutant DWG1 phenotypically has a significantly smaller grain width and weight than the wild type.
Experimental example 2 genetic analysis of Rice Gene DWG1
By the hybridization of DWG1 with R600,obtained F 1 The phenotype is identical to that of R600, thus suggesting that the mutant phenotype is controlled by a pair of recessive genes. F 2 Segregation occurs in mutant phenotype in the population, and mutant phenotype individuals and wild type individuals accord with 1:3, indicating that the mutant character is controlled by a single recessive gene.
Experimental example 3 localization of Rice Gene DWG1
As shown in FIG. 1, based on the gene mapping method of Mutmap combined coseparation verification, the DWG1 gene is located at chromosome 4, the candidate locus number is LOC _ Os04G54440, the coding region of the gene is mutated by G761A, and therefore the gene is classified as the DWG1 candidate gene.
Experimental example 4 Rice knockout of DWG1 Gene and construction of overexpression vector and genetic transformation
1. DWG1 knock-out vector construction
The gRNA target sequence of DWG1 (LOC _ Os04g 54440) knocked out by the CRISPR/Cas9 system is designed by using CRISPR design tool (http:// Rice. Hzau. Edu. Cn/cgi-bin/Rice2/CRISPR _ Rice) of Rice Information portal website (Rice Information GateWay), and the sequence is shown as SEQ ID NO:3, showing: CTTGAACCCGCTGCTGCATTTGG(the underlined TGG is the PAM sequence). Then designing a corresponding sgRNA Oligo, wherein the sequence of the oligonucleotide target is shown as follows,
the sgRNA sequence is as follows (5 '-3'):
UP Oligo(SEQ ID NO：4)：CACCGCTTCTGCTGGGTTTCAGGTTGG；
Low Oligo(SEQ ID NO：5)：AAACCCAAATGCAGCGGGTGTTCAAG。
and (3) adding water to dissolve the synthesized Oligo to 10 mu M, and carrying out vector construction according to a CRISPR/Cas vector construction kit (Baige). Oligo dimers were first prepared as follows:
BufferAneal 18. Mu.L, UP Oligo 1. Mu.L, low Oligo 1. Mu.L, total 20. Mu.L; the temperature was heated at 95 ℃ for 3 minutes and then slowly decreased to 20 ℃ at about 0.2 ℃/sec.
The product is then ligated into CRISPR/Cas9 vector BGK03 (hundred lattices), as follows: h 2 O6. Mu.L, CRISPR/Cas Vector 2. Mu.L, oligo dimer 1. Mu.L, enzyme Mix 1. Mu.L.
Adding 5 μ L of the reaction solution into at least 50 μ L of competent cells, mixing, and standing in ice bath for 30min (without shaking, keeping standing strictly); gently taken out, heat-shocked at 42 ℃ for 60 seconds, and immediately placed on ice for 2 minutes; adding 500. Mu.L of SOB/LB, and culturing at 37 ℃ and 200rpm for 1 hour; an appropriate amount of the bacterial solution was spread on LB plate containing kanamycin and inverted overnight culture was carried out at 37 ℃. Selecting a monoclonal for PCR identification, and for the monoclonal with correct sequencing, amplifying and shaking a bacterial solution, extracting a plasmid by using a kit (Meiji organism) to obtain a knockout vector BGK03-DWG1 of DWG1, wherein the map is shown in figure 2.
2. DWG1 overexpression vector construction
1) Seeds of Nipponbare of rice are planted in a culture box containing complete nutrient solution of the rice and grow for 15 days in a light incubator under the conditions of light and dark for 12 hours respectively, the temperature is 28 ℃ in the day and 23 ℃ in the evening. And (3) taking the overground part of the seedling, and extracting RNA according to the method of the extraction kit for the total RNA of the plants.
2) RNA was reverse transcribed into cDNA according to HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) Kit of Nanjing Novodka.
3) PCR amplification was performed using cDNA as a template using a designed DWG1 primer containing a restriction site and a homology arm sequence, and the primer sequence was as follows (5 '-3'):
pDWG1-F(SEQ ID NO：6)：
GGGGATCCTCTAGAGTCGACATGGCTCCGAAGAAGCGAA；
pDWG1-R(SEQ ID NO：7)：
ACGACGGCCAGTGCCAAGCTTCAAAATCCACCCTGAAAACCA。
the PCR reaction system is as follows:
4 μ L of cDNA, 2 μ L of each of pDGG 1-F and pDGG 1-R primers, 25 μ L of buffer, 1 μ L of DNTPs, 1 μ L of Hi-Fi enzyme and ddH 2 O 15μL。
The PCR reaction procedure was as follows:
pre-denaturation at 95 deg.C for 3min;95 ℃ for 15sec;58 ℃,20sec;72 ℃,1min 35 cycles, final extension 72 ℃,5min.
4) PCR product recovery and product ligation
And (3) recovering the PCR product by using an agarose gel recovery kit (purchased from Meiji organisms), and connecting the recovered fragment with the vector after enzyme digestion.
5) Vector cleavage
Carrying out double enzyme digestion on the vector pCAMBIA1300 by utilizing SalI and Hind III (Saimerlifei), and carrying out a 50 mu L reaction system; mu.L each of the enzymes, buffer 5. Mu.L, plasmid 10. Mu.L, ddH 2 And (3) incubating the mixture at 37 ℃ for 1.5h by using 31 mu L of O, and then recovering the enzyme digestion product by the same method as that of the step 4).
6) Ligation of vector and PCR product
The carrier is constructed by using a homologous recombination method, and the recombinase is purchased from Nanjing Novovzan biology company. System 20 μ L: PCR product 2. Mu.L, linearized vector 1. Mu.L, recombinase 2. Mu.L, buffer 4. Mu.L, ddH 2 O11. Mu.L. Incubate 30min at 37 ℃.
7) Transformation of recombinant vectors
The recombinant vector was transferred to E.coli DH 5. Alpha. Competence, incubated on ice for 30min, heat shock at 42 ℃ for 45sec, added with 500. Mu.L of antibiotic-free liquid LB, incubated at 37 ℃ for 1h. Centrifugation was carried out at 5000rpm, 100. Mu.L of the liquid was left to resuspend the cells, which were uniformly spread on a Carna-resistant LB plate, and the plate was cultured at 37 ℃ for 24 hours.
8) Identification of Positive clones
Selecting a monoclonal for PCR identification, and for the monoclonal with correct sequencing, amplifying and shaking a bacterial solution, extracting a plasmid by using a kit (Meiji organism) to obtain an overexpression vector 35S-DWG1, wherein the map is shown in figure 3.
3. Genetic transformation of rice
The overexpression vector 35S-DWG1 and the knockout vector BGK03-DWG1 are introduced into the agrobacterium EHA105 by a freeze-thaw method. Picking single colony in YEP liquid culture medium (each 50mg/L of kana Km and rifampicin Rif), shaking and culturing for 12-18 h at 28 ℃, then taking 1-5 mL bacterial liquid to 100mL YEP liquid culture medium (containing 100 mu mol/L of acetosyringone), shaking and culturing for 4h, and measuring OD value to be diluted to corresponding concentration (OD = 0.5). The cells were collected from fresh cells at 8000rpm, 4 ℃ and 5min and resuspended in one-third volume of AAM medium. Adding the mixture into a sterilized triangular flask containing the embryogenic callus which is vigorous in growth, soaking for 25min, blowing dry surface bacteria liquid, transferring the callus onto a co-culture medium, and performing dark culture at 28 ℃ for 2-3 d. The co-cultured callus is rinsed by sterile water and sterile water containing 500mg/L cefadroxil Cef, blown on a workbench for about 4 hours and transferred to a pre-culture medium for dark culture at 28 ℃ for 5-7 days. The pre-cultured callus is transferred to a screening culture medium containing hygromycin Hyg and Cef for continuous culture for 3 to 4 weeks, and the resistant callus is transferred to a screening culture medium containing only Hyg for screening for 1 to 2 times. Taking the resistant callus, transferring the resistant callus to a differentiation medium, and irradiating for differentiation at 28 ℃. Transferring the differentiated plantlets to a rooting culture medium, culturing for 3-4 weeks at 28 ℃ under illumination, performing open culture and hardening seedlings for about 7d, and finally transplanting to a field. After the obtained regeneration plant is transplanted to survive, screening the transformation plant by using herbicide or hygromycin; and extracting total DNA of leaves from the positive plants, and further identifying the transformed plants by PCR. And investigating the grain type of the transgenic T1 generation plant, and verifying the function of the DWG1 gene.
Experimental example 5 functional analysis of DWG1
1. Phenotypic analysis of DWG1 knockout transgenic plants
DWG1 is subjected to gene editing by using a CRISPER-Cas9 system in the background of rice ZH11, and successfully-edited mutants KO-1 and KO-2 are obtained through PCR identification, wherein A is inserted after 42 nucleotides of a KO-1 coding region, 40-47 nucleotides of a KO-2 coding region are deleted, and both mutations cause premature translation termination. The transgenic T1 generation plant grain phenotype survey shows that the grain width and grain weight of the DWG1 knockout plant is obviously reduced, and as shown in figure 4, the loss of the DWG1 function is shown to reduce the grain width and grain weight of rice.
2. Phenotypic analysis of DWG1 overexpression transgenic plants
The grain width and thousand grain weight of rice grains of over-expression transgenic lines OE1 and OE2 obtained in the experimental example 4 are significantly higher than those of wild type ZH11, and as shown in FIG. 5, it is shown that DWG1 may positively regulate the grain width and grain weight of rice. The over-expression of DWG1 can increase the grain weight of rice, improve the yield and have important breeding value.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (8)
1. A rice grain width and grain weight regulation gene DWG1 is characterized in that the nucleotide sequence of the gene DWG1 is shown in SEQ ID NO:1 or a sequence corresponding to SEQ ID NO:1 present at least 90% homology.
2. The rice grain width and weight regulatory gene DWG1 of claim 1, wherein the nucleotide sequence of the gene is comprised in the nucleotide sequence set forth in SEQ ID NO:1 by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence shown in the formula 1.
3. The rice grain width and grain weight regulator gene DWG1 protein of claim 1, wherein the protein has the amino acid sequence as shown in SEQ ID NO:2, respectively.
4. The rice grain width and weight regulatory gene DWG1 protein of claim 3, characterized in that the amino acid sequence is further comprised in the amino acid sequence as shown in SEQ ID NO:2 by addition, substitution, insertion or deletion of one or more amino acids.
5. A recombinant vector comprising a nucleotide sequence as claimed in claim 1 or 2.
6. A recombinant engineered bacterium comprising the recombinant vector of claim 5.
7. The recombinant vector of claim 5 or the recombinant engineered bacterium of claim 6 for use in rice breeding.
8. The use of claim 7, wherein the use comprises preparing a transgenic rice line.
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