CN115894646B - OsJDG1 gene and application thereof in regulation of rice grain type and thousand grain weight - Google Patents
OsJDG1 gene and application thereof in regulation of rice grain type and thousand grain weight Download PDFInfo
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Abstract
The invention belongs to the field of plant genetic engineering, and discloses an OsJDG1 gene and application thereof in regulating rice grain type and thousand grain weight. The invention discovers for the first time that the OsJDG1 gene has the function of regulating the grain type and thousand seed weight of rice, the grain length, width and thousand seed weight of transgenic rice are increased by over-expressing the OsJDG1 gene in the rice, and the OsJDG1 gene is subjected to site-directed mutagenesis by utilizing CRISPR/Cas9 technology, so that the grain length of the grain of mutant plants is increased, the grain width is reduced and the thousand seed weight is reduced. The invention provides valuable gene resources for improving the rice yield and improving the appearance quality of rice, and has important significance in rice breeding production.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to an OsJDG1 gene and application thereof in regulating rice grain type and thousand grain weight.
Background
Rice is one of the most important food crops in the world, and is the main food source for more than half of the population worldwide. With the reduction of the cultivated area and the rapid population growth, the improvement of the grain yield has very important significance for guaranteeing the grain safety of China. The grain type of rice comprises the length, width and thickness of grains, which are not only important factors influencing the yield of rice, but also determine the appearance quality of rice. Currently, many QTL/genes associated with the GRAIN form in rice have been successfully cloned, including GRAIN SIZE 3 (GS 3), GRAIN WIDTH AND WEIGHT (GW 2), GRAIN WIDTH (GW 5)/GRAIN SIZE 5ON CHROMOMOMME 5 (GSE 5), etc., and major pathways for regulating the GRAIN form are also disclosed, such as the G protein signaling pathway, the phytohormone pathway, the ubiquitin-proteasome pathway, etc. The novel grain type regulation and control gene is excavated to be beneficial to the breeding of high-yield high-quality rice varieties, and has important production and application values for increasing the rice yield and improving the appearance quality of rice.
Indica and japonica are the two major subspecies of oryza sativa, and the genetic basis of indica subspecies differentiation is mainly represented by differences in their genomes. The genomic sequence of 3010 cultivar was analyzed, and 6,038 genes (japonica-nonglutinous genes) which were present mainly in japonica rice but rarely occur in indica rice were found, and 147 genes (japonica-specific genes) which were unique to japonica rice were found. Based on bioinformatic analysis, it was found that the OsJDG1 gene has a highly homologous sequence only in oryza species and is present in most japonica varieties but not in most indica varieties. However, there is no report on the regulation of OsJDG1 gene on rice grain type and thousand grain weight.
Disclosure of Invention
The invention aims to provide application of a rice gene OsJDG1, in particular to application of the OsJDG1 gene in regulation of rice grain type and thousand grain weight. Over-expressing the OsJDG1 gene in rice, and increasing the grain length, grain width and thousand grain weight of the rice; in rice, the OsJDG1 gene is subjected to site-directed mutagenesis, so that the grain length of the seeds is increased, and the grain width and thousand seed weight of the seeds are reduced. Therefore, the invention provides valuable gene resources for improving the rice yield and improving the appearance quality of rice, and has important significance in rice breeding production.
The technical scheme provided by the invention is as follows:
a rice OsJDG1 protein has an amino acid sequence shown in SEQ ID No. 2.
Amino acid sequence of OsJDG1 protein (SEQ ID No. 2)
MPQPRPPSATAPSPVRRASDPSSTRLGMQIWPSGAVSSIPVLWPPPPIVGRRHLHPTTAGHHLLNPRASSATSDHRLHPRAPATMASTTVASSKLLGIASVLFVCVL. The invention also provides a rice OsJDG1 gene, wherein the rice OsJDG1 gene codes the rice OsJDG1 protein of claim 1, and the nucleotide sequence of the rice OsJDG1 gene is shown as SEQ ID NO. 1.
Nucleotide sequence of OsJDG1 (SEQ ID No. 1)
ATGCCCCAACCTCGTCCGCCAAGCGCCACCGCCCCATCCCCTGTCCGTCGGGCATCGGATCCGTCCTCCACAAGGTTAGGGATGCAGATCTGGCCATCGGGCGCCGTCTCATCCATCCCAGTGCTCTGGCCGCCACCTCCGATCGTCGGGCGACGCCACCTCCATCCTACAACCGCGGGACACCATCTCCTCAATCCCCGAGCATCGTCTGCCACCTCCGACCACCGCCTCCATCCTCGAGCACCCGCCACCATGGCCTCCACCACCGTGGCCTCCTCCAAGCTCCTTGGAATTGCAAGTGTTTTATTTGTTTGTGTTTTATGA。
The invention also provides an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene.
The invention also provides application of the protein or the gene in regulation of rice grain type and thousand grain weight.
Furthermore, according to the CRISPR/Cas9 gene editing technology, the recombinant plasmid pCas9-OsJDG1 is constructed and is introduced into target rice to obtain transgenic rice.
Furthermore, the recombinant plasmid pCas9-OsJDG1 expresses a gene of Cas9 protein and a gene of sgRNA, and a target sequence of the sgRNA is shown in a sequence table as shown in SEQ ID No.3.
sgRNA target sequence (SEQ ID No. 3): ATCCCCTGTCCGTCGGGCAT.
Further, the regulation of the grain size and thousand grain weight of the rice is to increase the grain length, grain width and thousand grain weight of the rice.
The invention also provides a method for cultivating transgenic rice, the rice OsJDG1 gene is overexpressed in the rice, and the grain length, grain width and thousand grain weight of the obtained transgenic rice are increased compared with those of the target rice.
Further, connecting the coding sequence of the OsJDG1 gene to a pCAMBIAI1390 vector driven by a maize ubiquitin promoter, transferring the recombinant plasmid pCAMBIAI1390-OsJDG1 into agrobacterium EHA105 to obtain recombinant agrobacterium, infecting embryogenic callus of flower 11 in rice, and culturing to obtain a transgenic plant; and obtaining a positive OsJDG1 over-expression strain through PCR and qRT-PCR identification.
Furthermore, the upstream primer sequence constructed by the pCAMBIAI1390-OsJDG1 vector is SEQ ID No.15, and the downstream primer sequence is SEQ ID No.16. The OsJDG1 gene is a gene encoding the following protein (a) or (b):
(a) A protein consisting of the amino acid sequence shown in SEQ ID No. 2;
(b) And (3) the protein which is obtained by substituting and/or adding and/or deleting one or more amino acid residues for the sequence shown in SEQ ID No.2, is related to the grain type and/or thousand grain weight of rice and is derived from the SEQ ID No. 2.
The regulation refers to over-expressing the OsJDG1 gene in rice, and increasing the grain length, grain width and thousand grain weight of the rice; in rice, the OsJDG1 gene is subjected to site-directed mutagenesis, so that the grain length of the seeds is increased, and the grain width and thousand seed weight of the seeds are reduced.
The invention also provides a method for cultivating transgenic rice with increased grain length and/or increased grain width and/or increased thousand grain weight of rice seeds, comprising the following steps: constructing a CDS sequence of the OsJDG1 gene on a plant expression vector, and transforming target rice to obtain a positive transgenic plant; the transgenic rice has increased grain length and/or increased grain width and/or increased thousand grain weight as compared to the rice of interest.
The CDS sequence of the OsJDG1 gene is shown as SEQ ID No. 1.
In the invention, the plant expression vector used for over-expressing the OsJDG1 gene is preferably a pCAMBIAI1390 vector driven by a corn ubiquitin promoter.
The invention also provides a method for increasing the grain length and/or reducing the grain width and/or reducing the thousand grain weight of rice, which utilizes CRISPR/Cas9 gene editing technology to mutate OsJDG1 genes in a site-directed manner, so that the grain length of rice seeds is increased and/or the seed width is reduced and/or the thousand grain weight is reduced.
The nucleotide sequence of the CRISPR/Cas9 gene editing technology is shown as a target site of the rice OsJDG1 gene shown in SEQ ID No.3.
The invention is realized by adopting the following technical scheme: and constructing a CRISPR/Cas9 recombinant plasmid, wherein the CRISPR/Cas9 recombinant plasmid expresses a gene of Cas9 protein and a gene of sgRNA, and a target sequence of the sgRNA is shown as SEQ ID No.3. Transferring the constructed recombinant plasmid into target rice, and screening to obtain positive transgenic rice plants. The length of rice grains is increased, the width is reduced, and thousand grain weight is reduced by site-directed mutagenesis OsJDG 1.
Advantageous effects
The rice OsJDG1 protein is reported for the first time in rice, and the analysis of the tissue expression mode of the rice OsJDG1 protein is carried out, and the results of qRT-PCR and GUS staining analysis show that the expression level of the OsJDG1 gene in young rice ears is relatively high. Subcellular localization analysis indicated that OsJDG1 was localized to the cell membrane. The in vitro protein expression experiment shows that OsJDG1 can be expressed in vitro. Overexpression of OsJDG1 gene in rice, increasing rice grain length, grain width and thousand grain weight; in rice, the OsJDG1 gene is subjected to site-directed mutagenesis, so that the grain length of the seeds is increased, and the grain width and thousand seed weight of the seeds are reduced. Therefore, it is expected to be a target gene for increasing the yield of rice and improving the appearance quality of rice.
The invention discloses the biological function of rice OsJDG1 for the first time, the gene positively regulates the size and thousand seed weight of rice seeds, and the overexpression of the gene increases the grain length and grain width of the rice seeds, thereby increasing the thousand seed weight. Knocking out the gene increases grain length but decreases grain width and results in a decrease in thousand kernel weight. The invention provides valuable gene resources for improving the rice yield and improving the appearance quality of rice, and has important significance in rice breeding production.
Drawings
FIG. 1 is an analysis chart of expression of the OsJDG1 protein coding gene in different tissues of flower 11 in japonica rice. FIG. 1A shows the relative expression levels of OsJDG1 gene in stems, roots, leaves, leaf sheaths, nodes and ears of rice; FIG. 1B shows GUS staining results.
FIG. 2 shows subcellular localization analysis of OsJDG1 protein of the present invention.
FIG. 3 shows in vitro expression analysis of OsJDG1 protein of the present invention. FIG. 3A is a photograph of the purified pCZN1-OsJDG1 protein separated by SDS-PAGE after coomassie blue staining. M represents a protein Marker; lane 1: BSA protein control; lane 2 is purified pCZN1-OsJDG1 recombinant protein. FIG. 3B is a diagram showing the purified pCZN1-OsJDG1 protein after Western blot analysis. M represents a protein Marker; lanes 1-2: purified pCZN1-OsJDG1 recombinant protein.
FIG. 4 is a graph showing the relative expression levels in two OsJDG1 overexpressing transgenic lines (OE 1 and OE 2).
FIG. 5 is a graph showing the mutation sites and sequencing peaks in two OsJDG1 homozygous knockout mutant lines (KO 1 and KO 2). FIG. 5A shows the mutation sites in two homozygous knockout mutant lines of OsJDG1, FIG. 5B and FIG. 5C show the sequencing peaks, and FIG. 5D shows the amino acid sequence.
FIG. 6 shows the comparison of the grain type of OsJDG1 mutant line of rice with that of wild-type middle flower 11. Fig. 6A is grain length appearance contrast, fig. 6B is grain width appearance contrast, fig. 6C is grain length contrast, fig. 6D is grain width contrast, and fig. 6E is thousand grain weight contrast.
Detailed Description
The following describes the technical scheme provided by the invention in detail by combining examples, but the invention is not limited to the following. The experimental methods in the following examples are conventional methods unless otherwise specified. The various experimental materials, reagents, carriers, etc., used in the examples described below are commercially available.
Example 1
Expression pattern of OsJDG1 Gene
1. Real-time fluorescent quantitative PCR
The expression level of OsJDG1 gene in stem, root, leaf sheath, node and young spike of booting stage spike differentiation 2, 5, 9, 13, 17, 22 and 25cm in mature stage of Zhonghua 11 was detected by a real-time fluorescence quantitative PCR (qRT-PCR) method. The primer for OsJDG1 gene real-time fluorescence quantitative PCR analysis is designed according to the full-length cDNA, the upstream primer sequence is SEQ ID No.4, and the downstream primer sequence is SEQ ID No.5. OsActin is used as an internal reference gene, the upstream primer sequence of the OsActin is SEQ ID No.6, and the downstream primer sequence of the OsActin is SEQ ID No.7.
SEQ ID No.4：5’-CAAGGTTAGGGATGCAGATCTG-3’；
SEQ ID No.5：5’-GATTGAGGAGATGGTGTCCC-3’；
SEQ ID No.6：5’-GATGACCCAGATCATCGTTTG-3’；
SEQ ID No.7：5’-GGGCGATGTAGGAAAGC-3’；
As a result, as shown in FIG. 1A, the OsJDG1 gene was expressed in the stems, roots, leaves, leaf sheaths, nodes and ears of rice, and the expression level was relatively high in young ears differentiated by 2-9cm from young ears during the booting stage, and gradually decreased as the ears developed. OsActin is used as an internal reference gene, and error bars represent standard deviations of three biological replicates.
2. GUS staining analysis
A promoter fragment (SEQ ID No. 8) of about 2kb upstream of the OsJDG1 gene was ligated to the pCAMBIA1300 vector to drive the expression of the GUS gene. GUS vector is transformed into flower 11 variety of japonica rice for detecting the expression pattern of OsJDG1 gene in different tissues and organs.
Upstream 2kb nucleotide sequence of OsJDG1 gene (SEQ ID No. 8)
AACAATCAAAAGCTACCATCAATCTAATCTTAATGCGTTTTAAATCTTATAAGATTCTGCTGTGCATAGCTGTTCATCCATATATTTAGGTTTGTGTGCTTCAAAAAGACCATCATGTTTGAAGCTATTAACTAATCTATCATTGAGTTAAGGTTTAATTGAAGAGAAATTTGAGGTCATATAATGTCCGAGGATATGAGCTTTTTTTAAGATAATGGATAACAATTAGGTTTTTATTTCCATCAAGTGGTTTTACAGAGCCAAACAATTACAAGAGTTATTGGACTCTAAGCCTCAACACCGAAGAACATAATTAAACTTAGGAGGCTAAGAAAGAAAGCTACTAGAAGAATAAGGTTCACTCGCATTCCAAGTCTTTGCTTCAAACTTGCCACGATAGTTGTTCTCATGGAATTCCCCTGCCACAACAAGAGAGGAGGGGATGGGAGGTGTGGATCTAGTCTGCCGTACCCACTGTCATGCCACCCGATAGCCTCCACTTTCTTTTGCCACCGAGTCTAGTTCTGGACACGGGGATGAAGGAGCTTGCCCACAGCCTGCTGAACACCCACTGTCCCCATCCAGTATTGCGGATCTCTTCCATCTTGTTGTCGGCATGCCTTGCCTATCCTGATCATTGGGCAACATAATTGCCGTCCATAGCGCGGTGCTTGAGGAATACCCCACCAAGCGGCATCCTTGTCTGGATCAGAGCTGTCCACATTGGTTGTATTCCTCTTTTCCATACTTAATATATTGGAAGTCATGGTTTTATTTCTAAGAATTGGATTTATCCTTGTATATATGCGTATACATGTTTTCCTGGAACAATGAAATGAAAGAGGTGATCAAAGTGGAATACCACCTTGGGGTCTCATGGGTTACCCTCATACTCTTGAGGAACCACTCTCGTGGTTGTTTTCCATATTTACGTCATCAGGACAGCATTGAAAACTTATTGTCTTGTTAATTAATCCCTTTATTTTTTATGTTTAGAAATAATTTTGATAATTCATTTTATTAAAAAAATATAATTATTATTTATTTTGTTATGATTTGTTTTATCGTTACAATTATTTTAAGAAGAACTTATATTTTTCATATTTGTACAAAATTTTTAAATAAGATGAATATTAAAATGCATGTCTAAAAGTTAACAACGTTAAACATAGAAAAGATAGAGTGGTTGGTAACTCACTCGACCCAAGCTCTCTGGGATGTCACATGTATTATACCCCTTGTTTATTTTTTTTGTTGAATTGTGAGGTTAGTCAGATGTACAAAACTCCTACCATCATATGTGCCAAAATCGTTCAAAATGTATTTATAATTGTGGAGATTATGGGTACCAAATACCCACACGGCTGACCTAAGGTGGGTTGTAGCTTGGTTAATAATAATTGTACACAACTTGGTCGTAAGGATTTCCTTGCCGGTTAAGGCACACTACGCCAGATCGACCTATTTGTGACGGGTCAAAGTGACTCGTCAATAATGACCTCATCTTTGACCGGTTAAATTAGATCCTGTTCTAGATGAGGTTTAGGAATACAACCTGTCACAGGGATATAATACCTCACCTTTGATGGTTCATATGTAGATCCGACAGAGAACACCTCTGATAGGTATTAAATTTTCATGCCAGTCACAAATGACTTGAGAAAGAGTAAATACTCCCCTCCTCCCGAGACCCTCGTCCTTTCCCTCTCCACCATCTCTTCCTTCTTCCTCTCCAACTCTCTCGTCCTTCTACCTCTCCACCACCCCTACCAACGGCTAAGAGCTGCTACCTCCATCCCCCAATTGCCGAGCGCTGCCTTCGACTGTCGACCGCCGCTGCCTCCATCCCCAATCGCCAGGCGCCGGATCCGTCCTCCACGAGGTCAGGGGATACGGATCTGCTTGCCGAGCACCGTATCCTCCGTCCCTATGCATCACCGCCGCCTTCGACCACCAAGAGTCACTGCCTCCATCCCTCGACTGTCGGGCACTGAATCCAC。
The upstream primer sequence constructed by the GUS vector is SEQ ID No.9, and the downstream primer sequence is SEQ ID No.10.
SEQ ID No.9：5’-CAGGTCGACTCTAGAGGATCCAACAATCAAAAGCTACCATCAATCTAA-3’；
SEQ ID No.10：5’-GGACTGACCACCCGGGGATCCGTGGATTCAGTGCCCGACAG-3’；
As shown in FIG. 1B, GUS staining analysis shows that the OsJDG1 gene is in a constitutive expression mode in stems, leaf sheaths, roots, nodes, glumes and ears of rice, and stronger GUS activity is detected in young ear glumes and young ears, which indicates that the OsJDG1 gene may play an important role in the development of rice grains.
Example 2
OsJDG1 subcellular localization analysis
To determine subcellular localization of OsJDG1 protein, the coding sequence of rice OsJDG1 gene (terminator removed) was ligated to pCAMBIAI1300 vector with GFP tag. The pCAMBIAI1300 vector and OsJDG1-GFP vector were transformed into the japonica rice variety 11, respectively. The GFP fluorescence signal in the root tip of stable T3-generation transgenic plants was observed under a confocal laser microscope.
The upstream primer sequence constructed by OsJDG1-GFP carrier for OsJDG1 subcellular localization is SEQ ID No.11, and the downstream primer sequence is SEQ ID No.12.
SEQ ID No.11：5’-GAGAACACGGGGGACTCTAGAATGCCCCAACCTCGTCCG-3’；
SEQ ID No.12：5’-TTCTCCCTTACCCATGGTACCTAAAACACAAACAAATAAAACACTTG-3’；
The results are shown in FIG. 2, and subcellular localization results show that OsJDG1 protein is localized on the cell membrane. The scale is 10. Mu.m.
Example 3
In vitro expression analysis of OsJDG1 protein
The coding sequence of OsJDG1 gene is fused and connected to pCZN1 vector with His tag. The recombinant plasmid pCZN1-OsJDG1 was transformed into E.coli BL21 strain. Protein expression was induced at 37℃for 4h with the addition of 0.4mM IPTG. SDS-PAGE detection analysis is carried out on the supernatant and the precipitate of the thallus after ultrasonic disruption, and the result shows that the OsJDG1 protein is in inclusion bodies. Further carrying out denaturation, purification and renaturation on the inclusion body protein, and identifying the size of the purified protein by adopting SDS-PAGE and Western Blot analysis.
The upstream primer sequence constructed by the pCZN1-OsJDG1 vector for OsJDG1 in-vitro expression analysis is SEQ ID No.13, and the downstream primer sequence is SEQ ID No.14.
SEQ ID No.13：5’-TGCATCATCATCATCATCATATGATGCCCCAACCTCGTCCG-3’；
SEQ ID No.14：5’-CAGAGATTACCTATCTAGATCATAAAACACAAACAAATAAAACACTTG-3’；
As a result, as shown in FIG. 3, the purified recombinant protein pCZN1-OsJDG1 produced a single band of about 13KD on SDS-PAGE gel. Western blot analysis further confirmed the molecular weight of pCZN1-OsJDG1 fusion protein. Therefore, the OsJDG1 has protein coding capability and can be expressed in vitro.
Example 4
Acquisition and identification of overexpression lines of OsJDG1 Gene
The coding sequence of the OsJDG1 gene is connected to a pCAMBIAI1390 vector driven by a maize ubiquitin promoter, the recombinant plasmid pCAMBIAI1390-OsJDG1 is transferred into agrobacterium EHA105 to obtain recombinant agrobacterium, embryogenic callus of flower 11 in rice is infected, and then the transgenic plant is obtained by cultivation. And obtaining a positive OsJDG1 over-expression strain through PCR and qRT-PCR identification.
The upstream primer sequence constructed by the pCAMBIAI1390-OsJDG1 vector is SEQ ID No.15, and the downstream primer sequence is SEQ ID No.16.
SEQ ID No.15：5’-GCTCGGTACCCGGGGATCTATGCCCCAACCTCGTCCG-3’；
SEQ ID No.16：5’-CAAAACAAAGCTCTGCAGTCATAAAACACAAACAAATAAAACACTTG-3’；
qRT-PCR identification of OsJDG1 expression levels in wild type plants and over-expressed transgenic lines As shown in FIG. 4, two independent OsJDG1 over-expressed strains (OE 1 and OE 2) were obtained with 276-fold and 327-fold expression levels, respectively, of wild type flower 11.
Example 5
Site-directed mutagenesis of OsJDG1 Gene
According to the full-length cDNA sequence of the rice gene OsJDG1, a CRISPR-P online website (http:// cbi.hzau.edu.cn/cgi-bin/CRISPR) is utilized to design a primer fragment, so that a proper sgRNA target sequence is obtained, wherein the SEQ ID No.3 in the sequence table. Single-stranded DNA molecules OsJDG1-pCas9-F (SEQ ID No. 17) and OsJDG1-pCas9-R (SEQ ID No. 18) were synthesized, respectively, and primers SEQ ID No.17 and SEQ ID No.18 were added to a 10. Mu.L system at a concentration of 1pmol, respectively, according to the procedure: annealing was completed at 94℃for 10min and at 15℃for 10 min. The pCas9 vector was digested with restriction enzyme AarI, and the linearized vector backbone was recovered. And (3) carrying out recombination connection on the annealed product and a linearization vector skeleton to obtain a recombinant plasmid pCas9-OsJDG1, transferring the recombinant plasmid pCas9-OsJDG1 into agrobacterium EHA105 to obtain recombinant agrobacterium, infecting embryogenic callus of flower 11 in japonica rice varieties, and culturing the embryogenic callus to obtain plants. The transgenic plants are subjected to PCR and sequencing identification to obtain the homozygous knockout mutant of the OsJDG1 mutation without carrier skeleton.
Specific primers SEQ ID No.19 and SEQ ID No.20 are respectively designed at the upstream and downstream of the OsJDG1 mutation target site, sequencing identification is carried out after PCR amplification, the mutation site and sequencing peak diagram of the OsJDG1 are shown in figure 5, and the obtained two OsJDG1 homozygous mutants are respectively homozygous mutant strains (figures 5A-C) with deletion of 1 base G and insertion of 2 base GT, and the coded amino acid sequences are all terminated in advance (figure 5D);
SEQ ID No.17：5’-AGATGATCCGTGGCAATCCCCTGTCCGTCGGGCATGTTTTAGAGCTATGC-3’；
SEQ ID No.18：5’-GCATAGCTCTAAAACATGCCCGACGGACAGGGGATTGCCACGGATCATCT-3’；
SEQ ID NO.19：5’-CGTATCCTCCGTCCCTATGC-3’；
SEQ ID No.20：5’-AAGCACCTCAATCAGAAACATG-3’；
example 6
Phenotypic analysis of OsJDG1 transgenic plants
When the grain type of OsJDG1 transgenic plants is identified, the result is shown in FIG. 6 (scale 1 cm), compared with the wild type, the grain of the mutant lines becomes more slender, and the grain length is obviously increased, and the grain length of the two mutant lines KO1 and KO2 is respectively increased by 5.53 percent and 4.66 percent; while the grain width is significantly reduced, the grain widths of KO1 and KO2 are respectively reduced by 2.50% and 4.56%. The grain size of the over-expression strain is enlarged, and the grain length and the grain width are obviously increased, the grain length of the two over-expression strains OE1 and OE2 are respectively increased by 2.31 percent and 3.43 percent, and the grain width is respectively increased by 2.30 percent and 2.38 percent. Furthermore, the thousand kernel weight of the kernels of the mutant strain was significantly reduced compared to the wild type; whereas the thousand kernel weight of the over-expressed strain kernels increased significantly, the thousand kernel weights of OE1 and OE2 increased by 8.33% and 11.23%, respectively. Thus, osJDG1 is a novel regulatory factor for controlling pelletization and thousand grain weight.
Claims (9)
1. The rice OsJDG1 protein is characterized in that the amino acid sequence of the rice OsJDG1 protein is shown as SEQ ID NO. 2.
2. A rice OsJDG1 gene is characterized in that the rice OsJDG1 gene codes the rice OsJDG1 protein of claim 1, and the nucleotide sequence of the rice OsJDG1 gene is shown as SEQ ID NO. 1.
3. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the gene of claim 2.
4. Use of the protein of claim 1 or the gene of claim 2 for regulating rice grain type and thousand grain weight.
5. The use according to claim 4, wherein the recombinant plasmid pCas9-OsJDG1 is constructed by using CRISPR/Cas9 gene editing technology, cutting pCas9 vector with restriction enzyme AarI, recovering linearized vector backbone, recombining and connecting annealed product with linearized vector backbone, transferring it into agrobacterium EHA105 to obtain recombinant agrobacterium, and culturing to obtain transgenic plant, wherein the transgenic plant is homozygous knockout mutant of OsJDG1 mutation and no vector backbone, and the rice grain length, grain width and thousand grain weight of the obtained transgenic plant are increased.
6. The use according to claim 5, wherein the recombinant plasmid pCas9-OsJDG1 expresses the gene of Cas9 protein and the gene of sgRNA, the target sequence of which is shown in the sequence table SEQ ID No.3.
7. The use according to claim 4, wherein the regulation of rice grain size and thousand grain weight is an increase in rice grain length, grain width and thousand grain weight.
8. A method for breeding transgenic rice characterized in that the rice OsJDG1 gene of claim 2 is overexpressed in rice, and the resulting transgenic rice has increased grain length, grain width and thousand grain weight as compared to the target rice.
9. The method for cultivating transgenic rice according to claim 8, wherein the coding sequence of OsJDG1 gene is connected to pCAMBIAI1390 vector driven by maize ubiquitin promoter, recombinant plasmid pCAMBIAI1390-OsJDG1 is transferred into agrobacterium EHA105 to obtain recombinant agrobacterium, then the embryogenic callus of flower 11 in rice is infected, and then it is cultivated to obtain transgenic plant; obtaining a positive OsJDG1 over-expression strain through PCR and qRT-PCR identification; the upstream primer sequence constructed by the pCAMBIAI1390-OsJDG1 vector is SEQ ID No.15, and the downstream primer sequence is SEQ ID No.16.
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