CN117051038A - Application of OsUBP7-6 gene in regulation of economic traits of rice and cultivation of high-yield rice varieties - Google Patents
Application of OsUBP7-6 gene in regulation of economic traits of rice and cultivation of high-yield rice varieties Download PDFInfo
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Abstract
The invention discloses application of an OsUBP7-6 gene in regulating economic traits of rice and cultivating high-yield rice varieties. Through constructing an OsUBP7-6 gene overexpression vector and a gene editing vector driven by a constitutive promoter Ubi, infecting rice embryogenic callus by agrobacterium tumefaciens, screening and differentiating to obtain an OsUBP7-6 gene overexpression and knockout plant, and identifying phenotypes related to important economic character grain yield of the two plants, the relation between the OsUBP7-6 gene and the rice grain size is proved. The grain length, grain width and thousand grain weight of the overexpression OsUBP7-6 gene strain are obviously increased, and the grain length, grain width and thousand grain weight of the knock-out OsUBP7-6 gene strain are obviously reduced, so that the OsUBP7-6 gene has potential application value in regulating and controlling the grain size and grain weight of rice and increasing the crop yield, and can generate higher economic benefit.
Description
Technical Field
The invention relates to an application of an OsUBP7-6 gene in regulating and controlling economic traits of rice and cultivating high-yield rice varieties, belonging to the technical field of plant genetic engineering.
Background
The grain weight is one of three factors of rice yield, is determined by grain size (grain length, grain width and grain thickness) and grain filling degree, and the thousand grain weight of rice is quantitative character controlled by multiple genes, and a molecular regulation network is complex, so that new genes for controlling the grain size of rice are discovered through biotechnology means, regulation mechanisms of the thousand grain weight of the rice are analyzed, high-yield germplasm materials are created, important gene resources and theoretical basis are provided for rice high-yield molecular breeding, and important practical significance is achieved.
Ubiquitin-proteasome pathways have been shown to play a key role in regulating organ size, including shape and size of plant seeds (Song et al 2007, wang et al 2017). Ubiquitin can be attached to a suitable substrate and treated by an ATP dependent E1 (ubiquitin inactivating enzyme) -E2 (ubiquitin binding enzyme) -E3 (ubiquitin ligase) enzyme binding cascade (Sadanandom et al 2012). Like many other biochemical reactions, ubiquitination is also a reversible process, and ubiquitin added to a substrate can also be cleaved by Deubiquitinase (DUB) to modify ubiquitination of the substrate protein. Several studies have shown that DUBs play an important regulatory role in plant seed development, WIDE AND THICK GRAIN 1 (WTG 1), encoding an otubain-like protease with deubiquitination activity homologous to OTUB1 in humans, WTG1 controlling rice grain size and thousand kernel weight primarily by affecting glume cell expansion (Huang et al 2017); arabidopsis ubiquitin-specific protease 15 (AtUBP 15) acts as a deubiquitinase, regulating organ and seed size in Arabidopsis by promoting cell proliferation (Du et al 2014). LG1 codes for a constitutively expressed ubiquitin-specific protease OsUBP15 with deubiquitination activity, and increasing the expression level of the OsUBP15 gene can significantly increase the grain width and thousand kernel weight of a transgenic line (Shi et al 2019). However, the current research on related enzymes of ubiquitin-proteasome pathway in rice is insufficient, the functions of most related enzymes of ubiquitin-proteasome pathway in rice are unknown, and whether the related enzymes are involved in rice grain development or not needs further excavation.
Disclosure of Invention
In order to solve the problems, the first object of the invention is to provide an application of an OsUBP7-6 gene in regulating economic traits of rice, and experiments prove that the gene can regulate grain length, grain width and thousand grain weight of rice grains.
The second object of the present invention is to provide an over-expression vector which can realize efficient expression of OsUBP7-6 gene in rice.
The third purpose of the invention is the application of the over-expression vector or the OsUBP7-6 gene in cultivating high-yield rice varieties, and the OsUBP7-6 gene is over-expressed in the rice by a genetic engineering means, so that the grain length, grain width and thousand grain weight of rice grains are obviously increased, and the high-yield rice varieties are obtained.
In order to achieve the above purpose, the application of the OsUBP7-6 gene in regulating and controlling economic traits of rice adopts the following technical scheme:
application of OsUBP7-6 gene in regulation of economic traits of rice: the nucleotide sequence of the OsUBP7-6 gene is as follows: (1) the nucleotide sequence shown in SEQ ID NO. 1; (2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein; the economic character of the rice is rice grain length, grain width and/or thousand grain weight.
The beneficial effects of the technical scheme are that: according to the invention, a specific primer is designed, an OsUBP7-6 gene is obtained through cloning, an overexpression vector and a gene editing vector of the OsUBP7-6 gene are constructed through a genetic engineering means, the OsUBP7-6 gene overexpression plant and a knock-out plant are constructed by transferring the OsUBP7-6 gene into rice through an agrobacterium transformation method, and the OsUBP7-6 gene is involved in the grain development process in rice through comparing rice grains of the OsUBP7-6 gene overexpression plant and the knock-out plant with rice grains of a control group.
As a further improvement, the OsUBP7-6 gene is overexpressed by genetic engineering means, and the grain length, grain width and thousand grain weight of rice are remarkably increased; inhibiting the expression of OsUBP7-6 gene, and remarkably reducing the grain length, grain width and thousand grain weight of rice.
The beneficial effects of the technical scheme are that: through constructing an OsUBP7-6 gene overexpression vector and a gene editing vector driven by a constitutive promoter Ubi, infecting rice embryogenic callus by agrobacterium tumefaciens, screening and differentiating to obtain an OsUBP7-6 gene overexpression and knockout plant, and identifying phenotypes related to important economic character grain yield of the two plants, the relation between the OsUBP7-6 gene and the rice grain size is proved. Wherein the grain length, grain width and thousand grain weight of the over-expressed OsUBP7-6 gene strain are obviously increased, and the grain length, grain width and thousand grain weight of the knockout OsUBP7-6 gene strain are obviously reduced. The size and the weight of the rice grain are important economic characters of rice, the genes for regulating the size and the weight of the rice grain are excavated, the method has important significance for increasing the yield of the rice and improving the economic benefit,
as a further improvement, the overexpression is to construct an OsUBP7-6 gene overexpression vector and to carry out overexpression on the OsUBP7-6 gene in rice.
As a further improvement, the construction method of the over-expression vector is to connect the obtained OsUBP7-6 gene to the pTCK303 vector, and then sequence and identify the gene.
As a further improvement, the inhibition is to construct a gene editing vector to knock out the OsUBP7-6 gene in rice.
In order to achieve the above purpose, the technical scheme adopted by the over-expression vector in the invention is as follows:
an overexpression vector comprising the nucleotide sequence of an OsUBP7-6 gene.
The beneficial effects of the technical scheme are that: the invention constructs the OsUBP7-6 gene over-expression vector driven by the constitutive promoter Ubi, and the vector can realize stable and efficient over-expression of the OsUBP7-6 gene after being transferred into rice, thereby providing a new means for researching the function of the OsUBP7-6 gene.
In order to achieve the above purpose, the technical scheme adopted by the application of the over-expression vector or the OsUBP7-6 gene in the cultivation of high-yield rice varieties is as follows:
the application of the encoding gene OsUBP7-6 of the over-expression vector or rice grain regulatory protein in the cultivation of high-yield rice varieties.
The beneficial effects of the technical scheme are that: the invention constructs a constitutive promoter Ubi-driven OsUBP7-6 gene overexpression vector, overexpresses the OsUBP7-6 gene in rice, and obviously improves the expression level of the OsUBP7-6 gene of an overexpression strain through detection. For T 1 The phenotype related to the grain yield in the harvest period of the generation of the over-expression strain is identified, the grain length, grain width and thousand grain weight of the transgenic strain of the over-expression OsUBP7-6 gene are obviously increased, a new gene resource is provided for cultivating high-yield rice varieties, and a foundation is laid for the accurate cultivation of the high-yield rice varieties.
As a further improvement, the over-expression of the OsUBP7-6 gene remarkably increases the grain length, grain width and thousand grain weight of rice, and a high-yield rice variety is obtained.
The beneficial effects of the technical scheme are that: the grain length, grain width and thousand grain weight of the rice are one of factors influencing the yield of the rice, are also important economic traits of the rice, and have great significance in improving the grain length, grain width and thousand grain weight, improving the yield of the rice and increasing economic benefits of growers.
As a further improvement, the high yielding rice variety is obtained by the following method: and (3) transforming agrobacterium with the over-expression vector to serve as an invasion solution, infecting rice callus, and screening and identifying to obtain rice varieties with obviously increased grain length, grain width and thousand grain weight.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides an application of a coding gene OsUBP7-6 gene for regulating rice grain type protein in regulating rice economic traits. Constructing an OsUBP7-6 gene overexpression vector driven by a constitutive promoter Ubi and a gene editing vector for knocking out the OsUBP7-6 gene, infecting embryogenic callus of rice by utilizing agrobacterium, co-culturing, screening and differentiating to obtain positive plants which are subjected to overexpression and knocking out respectively, identifying phenotypes related to grain yield of the positive plants in the harvest period of the overexpression and knocking out, and verifying the relation between the OsUBP7-6 and the grain size of the rice, wherein the grain length, grain width and thousand grain weight of a transgenic strain of the overexpression OsUBP7-6 gene are obviously increased; the grain length, grain width and thousand grain weight of the strain line of the OsUBP7-6 gene are remarkably reduced. The OsUBP7-6 gene is closely related to rice grain development, participates in regulating and controlling grain length, grain width and thousand grain weight of rice grains, provides new gene resources for a rice grain size regulating molecular network, and lays a theoretical foundation for analyzing a regulating and controlling mechanism of the thousand grain weight of rice.
2. The invention discusses the function of ubiquitin-specific protease in rice variety improvement, thereby providing a theoretical basis for cultivating high-yield rice by OsUBP7-6, in particular to increasing thousand seed weight by adjusting the size of rice seeds. The overexpression vector based on the OsUBP7-6 gene can provide a simple and effective technical means for cultivating high-yield rice varieties, and the OsUBP7-6 gene has potential application value in improving the grain size and thousand seed weight of rice and increasing the crop yield and can be applied to production by utilizing a molecular improvement technology. The cultivated high-yield rice variety has potential industrial application value in increasing crop yield, and can generate higher economic benefit.
Drawings
FIG. 1 is a schematic diagram showing the structure of an OsUBP7-6 gene overexpression vector in example 2 of the present invention;
FIG. 2 shows PCR identification of OsUBP7-6 gene overexpression and knock-out positive plants in example 4 of the present invention (WT represents wild type control plants, osUBP7-6 OE represents OsUBP7-6 gene overexpression positive plants, osUBP7-6 KO represents OsUBP7-6 gene knock-out plants);
FIG. 3 shows the identification of the expression level of OsUBP7-6 5 days after the flowers of the OsUBP7-6 gene overexpression line in example 4 of the present invention (representing P < 0.01);
FIG. 4 shows grain length comparison of wild type control, osUBP7-6 overexpressing transgenic line and OsUBP7-6 knocked-out line after harvest in example 4 of the present invention (WT represents wild type control plant, osUBP7-6 OE represents OsUBP7-6 gene overexpressing positive plant, osUBP7-6 KO represents OsUBP7-6 knocked-out plant);
FIG. 5 shows the grain length difference analysis of wild type control, osUBP7-6 overexpressing transgenic lines and OsUBP7-6 knockout lines of example 4 of the present invention after harvest (WT represents wild type control plants, osUBP7-6 OE represents OsUBP7-6 gene overexpressing positive plants, osUBP7-6 KO represents OsUBP7-6 knockout plants; P < 0.01);
FIG. 6 shows a comparison of grain width after harvesting of wild type control, osUBP7-6 overexpressing transgenic lines and OsUBP7-6 knocked-out lines in example 4 of the present invention (WT represents wild type control plants, osUBP7-6 OE represents OsUBP7-6 gene overexpressing positive plants, osUBP7-6 KO represents OsUBP7-6 knocked-out plants);
FIG. 7 is a graph showing the grain width difference analysis of wild type control, osUBP7-6 overexpressing transgenic lines and OsUBP7-6 knocked-out lines after harvest in example 4 of the present invention (WT represents wild type control plants, osUBP7-6 OE represents OsUBP7-6 gene overexpressing positive plants, osUBP7-6 KO represents OsUBP7-6 knocked-out plants; P < 0.01);
FIG. 8 shows the analysis of thousand kernel weight difference of wild type control, osUBP7-6 overexpressing transgenic lines and OsUBP7-6 knockout lines of example 4 of the present invention after harvest (WT represents wild type control plants, osUBP7-6 OE represents OsUBP7-6 gene overexpressing positive plants, osUBP7-6 KO represents OsUBP7-6 knockout plants; P < 0.01).
Detailed Description
The primers used in the examples of the present invention are shown in Table 1 below.
TABLE 1 primer names and sequences
| Primer name | Primer sequence (5 '-3') |
| OsUBP7-6_F | TCTAGAGGATCCCCGGGTACCATGGCGGAGGCGGCGGGT (shown as SEQ ID NO. 4) |
| OsUBP7-6_R | TTCGAGCTCTCTAGAACTAGTTCAATAAAATGTTTGGGCCGTC (shown as SEQ ID NO. 5) |
| pTCK303_VF | GCCCTGCCTTCATACGCTAT (shown as SEQ ID NO. 6) |
| pTCK303_VR | TAACATAGATGACACCGCGC (shown as SEQ ID NO. 7) |
| Hyg_F | ATGAAAAAGCCTGAACTCACCG (shown as SEQ ID NO. 8) |
| Hyg_R | CTATTTCTTTGCCCTCGGACG (shown as SEQ ID NO. 9) |
| OsUBP7-6RT_F | TTCTGTCAGTGCGGTCAAAC (shown as SEQ ID NO. 10) |
| OsUBP7-6RT_R | TCTACACACCAACGCCTCATC (shown as SEQ ID NO. 11) |
| β_actin_F | GGAAGTACAGTGTCTGGATTGGAG (shown as SEQ ID NO. 12) |
| β_actin_R | TCTTGGCTTAGCATTCTTGGGT (shown as SEQ ID NO. 13) |
| sgRNA-LP | GGCAGGTGGTGGCTGTGGTGATTG (shown as SEQ ID NO. 15) |
| sgRNA-RP | AAACCAATCACCACAGCCACCACC (shown as SEQ ID NO. 16) |
| T3-F | CATACGAACAGATCACTTAA (shown as SEQ ID NO. 17) |
| pC1300-Cas9-F | ACACTTTATGCTTCCGGCTC (shown as SEQ ID NO. 18) |
| CRISPRUBP7-6-JD-F | AGATTTTGGCGGGTTGGCTA (shown as SEQ ID NO. 19) |
| CRISPRUBP7-6-JD-R | TTGACAGCCTTGCACTGCTT (shown as SEQ ID NO. 20) |
The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples, experimental examples and comparative examples were all commercially available, except for the specific descriptions.
Example 1 amplification of OsUBP7-6 Gene sequence
The specific primers were designed in this example, and the OsUBP7-6 gene was successfully amplified using the reverse transcription cDNA of Japanese sunny day as a template, and the specific implementation procedure was as follows:
downloading the genomic DNA sequence of OsUBP7-6 (Os 08g 0478500) on a rice biological website (http:// rapdb. DNA. Affrc. Go. Jp /), removing the 5'UTR end (untranslated region), the 3' UTR end (untranslated region) and the intron sequence of the front part of the sequence, and obtaining the CDS sequence of the OsUBP7-6 gene, wherein the nucleotide sequence is shown as SEQ ID NO.1 (ATG at the forefront end of the sequence is a start codon, TAG at the end is a stop codon, and the stop codon is not translated protein), and the amino acid sequence of the encoded OsUBP7-6 protein is shown as SEQ ID NO. 2.
Primers OsUBP7-6_F (SEQ ID NO. 4) and OsUBP7-6_R (SEQ ID NO. 5) comprising the flanking sequences of a basic vector pTCK303 polyclonal site Kpn I and Sac I are designed, and a target fragment is amplified by taking a reverse transcription cDNA of Japanese sunny as a template. OsUBP7-6CDS fragment amplification System: 1. Mu.L of each of the forward and reverse primer (final concentration: 10. Mu.M) and the DNA template was added with 25. Mu.L of 2 XHi-Fi enzyme and ddH 2 O to 50. Mu.L. PCR reaction conditions: pre-denaturing for 3min at 95 ℃, pre-denaturing for 30s at 95 ℃, annealing for 30s at 55 ℃ and extending for 30s at 72 ℃ for 35 cycles, and finally amplifying to obtain a 2937bp double-chain OsUBP7-6CDS fragment which is used for constructing a subsequent pTCK303-OsUBP7-6 vector.
Example 2 an over-expression vector
In this example, the OsUBP7-6 gene obtained in example 1 was inserted into pTCK303 vector to construct an OsUBP7-6 gene overexpression vector, and the following steps were carried out:
the pTCK303 vector was linearized with Kpn I and Sac I, the T4 ligase ligated vector with the OsUBP7-6 gene fragment, the ligation product was transformed into DH 5. Alpha. Competent cells, and after activation, plates containing 50mg/L kanamycin antibiotic were plated and incubated for 14 hours at 37℃in an inverted state. Several round and full monoclonals are selected, colony PCR verification is carried out by using a universal primer pTCK303_VF (shown as SEQ ID NO. 6) and a universal primer pTCK303_VR (shown as SEQ ID NO. 7) of a pTCK303 vector, the verified bacterial liquid is sent to sequencing, plasmid extraction is carried out correctly after sequencing, and the correct plasmid is named as pTCK303-OsUBP7-6, and the structure of the specific vector is shown in figure 1.
EXAMPLE 3 Gene editing vector
According to the OsUBP7-6 gene sequence, a CRISPR/Cas9 expression vector for knocking out the OsUBP7-6 gene is constructed, and the specific implementation operation is as follows:
1. selection of OsUBP7-6 gene gRNA target sequence and design of upstream and downstream primers of gRNA oligonucleotide chain
Target sites are designed according to the recognition characteristics of a CRISPR/Cas9 system, and a 20bp sgRNA target sequence is designed in an OsUBP7-6 gene sequence, wherein the sgRNA sequence is as follows: GGTGGTGGCTGTGGTGATTG (SEQ ID NO. 14).
The primers upstream and downstream of the gRNA oligonucleotide strand were designed based on the sgRNA sequences shown in Table 1 for the sgRNA-LP (shown as SEQ ID NO. 15) and for the sgRNA-RP (shown as SEQ ID NO. 16).
2. Construction of Gene editing vector
1) Equal amounts of primers upstream and downstream of the gRNA oligonucleotide strand (final concentration 100. Mu.M) were mixed, passed through 5min at 37℃for 5min at 95℃and then reduced to 25℃at a rate of 5℃per min to form complementary double-stranded DNA for subsequent vector construction.
2) The intermediate vector sk-gRNA was digested with the restriction enzyme Aar I and linearized, and a 50. Mu.L digestion system was as follows: sk-gRNA plasmid 2. Mu.g, 10 XCutSmart Buffer 5. Mu.L, aar I10U, add ddH 2 O to 50 mu L, enzyme is inactivated by heat treatment at 65 ℃ for 20min after enzyme digestion for 4h at 37 ℃; after purification the following ligation system was added: 1.5. Mu.L of linear plasmid, 6.5. Mu.L of 200-fold diluted oligonucleotide double-stranded DNA, 1. Mu.L of 10 XBuffer, 1. Mu.L of T4 DNA ligase; the conditions for the connection are: the connection is carried out at 4 ℃ for 12 hours.
3) The ligation product is transformed into E.coli DH5 alpha competent cells, the bacterial liquid is coated on an ampicillin LB culture medium plate containing 50mg/L, and after overnight culture, monoclonal shaking bacteria are selected for propagation. Colony PCR was verified using primers T3-F (shown as SEQ ID NO. 17) and sgRNA-RP (shown as SEQ ID NO. 16).
The PCR system is as follows: 2 XTaq Master mix 5. Mu.L; T3-F (10. Mu.M) 0.4. Mu.L; sgRNA-R (10. Mu.M) 0.4. Mu.L; 1 mu L of monoclonal template; ddH 2 O 3.2μL。The PCR conditions were: pre-denaturation at 94℃for 2min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, final extension at 72℃for 10min, wherein denaturation, annealing and extension are 35 cycles.
4) The correct intermediate vector and final PC-1300-Cas9 were sequenced using restriction endonucleases Kpn I and BamH I linearization. T4 ligase was used to ligate the vector to the fragment, the ligation product was transformed to DH 5. Alpha. Competent, and after activation, plates containing 50mg/L kanamycin antibiotics were plated and incubated for 14 hours at 37℃in an inverted position. Several round and full monoclonals are selected, colony PCR verification is carried out by using pC-1300-Cas9-F (shown as SEQ ID NO. 18) and sgRNA-RP (shown as SEQ ID NO. 16) as universal primers of pC-1300-Cas9 vectors, the verified bacterial liquid is sent to be sequenced, plasmid extraction is carried out correctly after sequencing, and the correct plasmid is named pC-1300-Cas9-OsUBP7-6..
Example 4 application of OsUBP7-6 Gene in regulating economic Properties of Rice
In the embodiment, the constructed gene overexpression vector pTCK303-OsUBP7-6 and the gene editing vector pC-1300-Cas9-OsUBP7-6 are respectively transformed into agrobacterium tumefaciens EHA105 to infect rice callus, and positive plants of overexpression and knockout of the gene OsUBP7-6 are obtained through screening and differentiation. The specific implementation operation is as follows:
1. agrobacterium-mediated rice callus genetic transformation and positive transgenic plant detection
The gene overexpression vector pTCK303-OsUBP7-6 and the gene editing vector pC-1300-Cas9-OsUBP7-6 are respectively introduced into the agrobacterium tumefaciens EHA105 by a heat shock method according to the method reported by Hiei et al (Hiei et al 1997), calli of the rice variety Japanese (Oryza sativa ssp. Japonicac. Nipponbare) are infected with the agrobacterium containing the overexpression vector and the gene editing vector, and rice transgenesis is performed according to the method reported by Nishimura et al (Nishimura et al 2006), and a regenerated seedling is obtained by hygromycin screening. Primers Hyg_F (shown as SEQ ID NO. 8) and Hyg_R (shown as SEQ ID NO. 9) were used for OsUBP7-6 gene overexpression and knockout plant positive selection.
The PCR amplification system and the reaction conditions were as follows:
the PCR system is as follows: 2 XTaq Master mix 5. Mu.L; hyg_F (10. Mu.M) 0.4. Mu.L; hyg_R (10. Mu.M) 0.4. Mu.L; 1 μl of DNA template; ddH2O 3.2. Mu.L. The PCR conditions were: pre-denaturation at 94℃for 2min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s, final extension at 72℃for 10min, wherein denaturation, annealing and extension are 35 cycles. The amplified products are detected by 1% agarose gel electrophoresis, the wild type reference does not have amplified fragments, the amplified fragments of the OsUBP7-6 gene overexpression and knockout plant positive line are 1026bp, and the result is shown in figure 2.
2. Overexpression plant OsUBP7-6 gene expression level and gene knockout plant OsUBP7-6 gene mutation type detection
(1) Overexpression plant OsUBP7-6 gene expression level detection
The expression level of the OsUBP7-6 over-expression plant is detected by adopting a fluorescent quantitative PCR method. Wild type control and transgenic plants (T) 0 Generation) total RNA from seeds 5 days after flowers was extracted with TRIzol (Transgen, ET 121-01), and 1. Mu.g of total RNA was reverse transcribed with reverse transcriptase (Tiangen, KR 103). Beta-actin was used as a reference gene, diluted (1:10) cDNA was used as a template, and 2 was used -ΔΔCt The relative expression levels of the OsUBP7-6 gene were calculated by the method (Livak et al, 2001), and the quantification was performed using primers of OsUBP7-6 RT_F (SEQ ID NO. 10) and OsUBP7-6 RT_R (SEQ ID NO. 11), and the primers used for the fluorescent quantification of beta-actin were beta-actin_F (SEQ ID NO. 12) and beta-actin_R (SEQ ID NO. 13).
The fluorescent quantitative PCR adopts a 20 mu L system, wherein 5 mu L of template cDNA is added after dilution according to the proportion of 1:20, 1.0 mu L of forward primer OsUBP7-6 RT_F and 1.0 mu L of reverse primer OsUBP7-6 RT_R are respectively added, and 3 mu L of ddH is added 2 O, fluorescent dye (Taq Pro Universal SYBR qPCR Master Mix, vazyme, Q712-02) was 10. Mu.L and quantitatively detected using CFX 96Real Time System (BioRad, USA), the results of which are shown in FIG. 3. As shown in FIG. 3, the OsUBP7-6 gene is highly expressed in the OsUBP7-6 OE transgenic line.
(2) Gene knockout plant OsUBP7-6 gene mutation type detection
The gene mutation type detection of the OsUBP7-6 gene knockout plant is carried out by adopting a PCR amplification and product sequencing method. Wild type control and transgenic plants (T) 0 Generation) taking plant leaves to extract genome DNA. For OsUBP7-6 gene knockout positive plants, the target site isA pair of primers CRISPRUBP7-6-JD-F (SEQ ID NO. 19) and CRISPRUBP7-6-JD-R (SEQ ID NO. 20) were designed upstream and downstream for PCR amplification.
The PCR amplification system and the reaction conditions were as follows:
the PCR system is as follows: 2 XTaq Master mix 5. Mu.L; CRISPRUBP7-6-JD-F (10. Mu.M) 0.4. Mu.L; CRISPRUBP7-6-JD-R (10. Mu.M) 0.4. Mu.L; 1 μl of genomic DNA template; ddH2O 3.2. Mu.L. The PCR conditions were: pre-denaturation at 94℃for 2min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, final extension at 72℃for 10min, wherein denaturation, annealing and extension are 35 cycles.
After PCR amplification, the correct bands were sequenced by Shanghai worker using 1% agarose gel electrophoresis, the sequences were read using Contigexpress and aligned with the wild type sequences to obtain the mutant types, and the results are shown in Table 2.
TABLE 2 mutant type statistics for knockout plants
3. Seed phenotype analysis of over-expression plants and gene knockout plants
Respectively selecting OsUBP7-6 gene over-expression plants and gene knockout plants, taking 5 plants from each plant line, randomly selecting 60 mature harvested seeds from each plant (the object detected in phenotype detection is T) 1 Mature grain of the generation), grain length and grain width of the grain were measured using a rice appearance quality detector, and the average was taken 5 times repeatedly, and the results are shown in fig. 4 to 7. Thousand seed weight was determined by weighing 1000 randomly selected seeds, taking the average of 5 replicates, and the results are shown in figure 8.
As can be seen from fig. 4-5, the grain length of the three lines L3, L6 and L9 overexpressing OsUBP7-6 was significantly increased compared to the wild-type control; and the grain length of the two lines L15 and L16 of the gene knockout OsUBP7-6 is obviously reduced compared with that of a wild type control.
As can be seen from fig. 6-7, the grain widths of the three lines L3, L6 and L9 overexpressing OsUBP7-6 were significantly increased over the wild-type control; whereas the grain widths of the two lines L15 and L16 of the gene knockout OsUBP7-6 are extremely reduced compared with the wild type control.
As can be seen from fig. 8, the thousand kernel weight of three lines L3, L6 and L9 overexpressing OsUBP7-6 was significantly increased over the wild-type control; and thousand seed weight of the two lines L15 and L16 of the gene knockout OsUBP7-6 is remarkably reduced compared with that of a wild type control.
The last explanation is: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
- The application of the OsUBP7-6 gene in regulating economic traits of rice is characterized in that: the nucleotide sequence of the OsUBP7-6 gene is as follows: (1) the nucleotide sequence shown in SEQ ID NO. 1; (2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein; the economic character of the rice is rice grain length, grain width and/or thousand grain weight.
- 2. The use of the OsUBP7-6 gene according to claim 1 for regulating economic traits of rice, characterized in that: through the gene engineering means, the OsUBP7-6 gene is over-expressed, and the grain length, grain width and thousand grain weight of rice are obviously increased; inhibiting the expression of OsUBP7-6 gene, and remarkably reducing the grain length, grain width and thousand grain weight of rice.
- 3. The use of the OsUBP7-6 gene according to claim 2 for regulating economic traits of rice, characterized in that: the over-expression is to construct an OsUBP7-6 gene over-expression vector to over-express the OsUBP7-6 gene in rice.
- 4. The use of the OsUBP7-6 gene according to claim 3 for regulating economic traits of rice, wherein: the construction method of the over-expression vector comprises the steps of connecting the obtained OsUBP7-6 gene to the pTCK303 vector, and sequencing and identifying.
- 5. The use of the OsUBP7-6 gene according to claim 2 for regulating economic traits of rice, characterized in that: the inhibition is to construct a gene editing vector and knock out the OsUBP7-6 gene in rice.
- 6. An over-expression vector, characterized in that: the overexpression vector comprises the nucleotide sequence of the OsUBP7-6 gene according to claim 1.
- 7. Use of the overexpression vector or the OsUBP7-6 gene according to claim 6 for cultivating high-yield rice varieties.
- 8. The use of the overexpression vector or the OsUBP7-6 gene according to claim 7 in cultivation of high-yield rice varieties, characterized in that: over-expressing OsUBP7-6 gene to increase rice grain length, grain width and thousand grain weight obviously and obtain high-yield rice variety.
- 9. The use of the overexpression vector or the OsUBP7-6 gene according to claim 7 or 8 in the cultivation of high-yielding rice varieties, characterized in that: the high-yield rice variety is obtained by the following method: and (3) transforming agrobacterium with the over-expression vector to serve as an invasion solution, infecting rice callus, and screening and identifying to obtain rice varieties with obviously increased grain length, grain width and thousand grain weight.
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