CN117070515A - Rice biological clock gene OsPRR37 promoter region guide RNA double-target sequence and application thereof - Google Patents

Rice biological clock gene OsPRR37 promoter region guide RNA double-target sequence and application thereof Download PDF

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CN117070515A
CN117070515A CN202211418088.XA CN202211418088A CN117070515A CN 117070515 A CN117070515 A CN 117070515A CN 202211418088 A CN202211418088 A CN 202211418088A CN 117070515 A CN117070515 A CN 117070515A
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osprr37
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t78pm
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余舜武
王玉岚
杨访问
吴金红
罗利军
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SHANGHAI AGROBIOLOGICAL GENE CENTER
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Abstract

The invention provides a double target sequence of guide RNA of a promoter region of a rice biological clock gene OsPRR37, and the nucleotide sequence of the double target sequence is shown as SEQ ID NO.9 and 10. The invention also provides a rice biological clock gene OsPRR37, the nucleotide sequence of which is shown as SEQ ID NO. 2. The target sequence of the invention can be applied to constructing a CRISPIP/CAS 9 gene editing expression vector with double targets or two single targets, and then simultaneously transforming rice to edit rice genome. The rice genome editing is carried out on the two RNA sequence target positions simultaneously by utilizing the rice biological clock gene OsPRR37 promoter provided by the invention, and the deletion of genome fragments is generated on the upstream promoter of the OsPRR37 gene, so that the expression of the OsPRR37 gene is changed, and the growth period of rice is shortened. The invention also provides a molecular marker for detecting the mutant. The invention provides a new way for creating rice short-term germplasm.

Description

Rice biological clock gene OsPRR37 promoter region guide RNA double-target sequence and application thereof
Technical Field
The invention belongs to the technical field of plant biology, relates to a rice related gene, in particular to a rice biological clock gene OsPRR37 promoter region guide RNA double-target sequence, a method for preparing a rice mutant by using the double-target CRISPR/Cas9-sgRNA system, and application of the rice mutant in rice breeding.
Background
Rice (Oryza sativa L.) is a staple food of our country, and plays the most important role in guaranteeing the food safety crops of our country. However, rice production is challenged. The development of modern industry and the gradual rise of global air temperature increase the probability of frequent extreme climate conditions, shorten the growth period of rice and enhance the capability of plant stress resistance, especially drought resistance and high and low temperature resistance, which is the urgent need at present. The biological clock gene is used as an internal core regulation module of the plant, and plays an important role in regulating and controlling the growth and development process of the plant and the change of the external environment. PRR family genes are one of the core components of biological clocks and play an important role in the growth and development of plants. Among the genes of the PRR family of rice, osPRR37 was the earliest studied in rice. OsPRR37 is capable of controlling flowering by regulating rice sensitivity to photoperiod and also affects plant architecture and grain yield (Wei Hua, wang Yan, liu Baohui. Plant biological clock and research progress for its regulated growth and development. Programming, 2018,53 (4): 456-467). It was found that OsPRR37 has various natural variations in different varieties, including the original genetic variation of wild rice and the mutation obtained after domestication, and these variations are beneficial to the rice to adapt to different living environments better (Koo BH, yoo SC, park JW, kwon CT, lee BD, an G, zhang Z, li J, li Z, paek NC.Nature variation in OsPRR37regulates heading date and contributes to rice cultivation at a wide range of latitudes.mol plant.2013,6 (6): 1877-1888). In addition, PRR family genes have been found to be involved in the drought stress response of plants (Li Jia, liu Yunhua, zhang Yu, chen Chen, yu Xia, yu Shunwu. Effects of drought on daily rhythmic changes in rice biological clock genes and drought stress response genes. Inheritance, 2017,39 (9): 837-846). These suggest that the OsPRR37 gene is a functional gene with abundant genetic effects, and it is possible to shorten the growth period and mutational types of other agronomic traits by editing or modifying the promoter of the gene and constructing an appropriate expression pattern of the gene.
CRISPR/Cas is an acquired immune defense system found in archaebacteria, and the CRISPR/Cas system which is found at present comprises types i, ii and iii, wherein the composition of the type ii system is simpler, and the CRISPR/Cas9 technology modified by the type ii system is a tool which is most effective for precisely editing plant genome with the advantages of easy operation, low cost, high efficiency, high specificity, multiple gene editing and the like, and is widely applied to research on gene functions and genetic improvement of animals, plants, microorganisms and the like. The CRISPR/Cas9 technology is utilized to knock out unfavorable genes or negative regulation genes at fixed points, thereby realizing the precise improvement of target characters and greatly improving the directional genetic improvement efficiency of crops. The technology can also simultaneously carry out gene editing on a plurality of targets, and mutation occurs at a plurality of positions of a genome. If two mutation sites are located adjacent to the same chromosome, this may result in the deletion of a large fragment between the double targets. The deletion of large fragments in the promoter region of the gene can effectively change the expression mode of the target gene, and the current double-target or multi-target CRISPR/Cas9 technology has also been successful on rice (Zeng D, liu T, ma X, wang B, zheng Z, zhang Y, xie X, yang B, zhao Z, zhu Q, liu YG. Quantitative regulation of Waxy expression by CRISPR/Cas9-based promoter and' UTR-intron editing improves grain quality in price. Plant Biotechnol J,2020,18 (12): 2385-2387). However, there are few reports on the improvement of stress resistance, yield and other genes, which are important breeding values for rice fine parents through biological clock genes. The invention can be used for precise breeding of rice for shortening the growth period through gene editing and transformation.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a double-target sequence of guide RNA of a promoter region of a rice biological clock gene OsPRR37 and application thereof, and the double-target sequence of guide RNA of the promoter region of the rice biological clock gene OsPRR37 and a novel method for shortening the rice growth period in the prior art.
The invention provides a rice gene OsPRR37 promoter double-target guide RNA sequence, which comprises the following nucleotide sequences:
37T7:5’-GACGTGGAACAAATGGAAGTGGG-3’(SEQ ID NO.9)
37T8:5’-ATATCTAGTAGCAGTAGCAGCGG-3’(SEQ ID NO.10)。
the invention also provides a rice biological clock gene OsPRR37, the nucleotide sequence of which is shown as SEQ ID NO. 2.
The invention also provides a preparation method of the rice biological clock gene OsPRR37, which utilizes a PCR amplification method to construct an sgRNA expression cassette containing the guide RNA target sequence of claim 1;
loading the two sgRNA expression cassettes onto the same CRISPR/Cas9 vector to obtain a CRISPR/Cas9-sgRNA vector containing a double-target-point sequence;
and transforming the rice callus by using a CRISPR/Cas9-sgRNA vector carrying two targets to obtain a mutant with a fragment deletion of a promoter region of the rice biological clock gene OsPRR37, namely the rice biological clock gene OsPRR37.
The invention also provides a molecular marker for detecting the rice gene OsPRR37 promoter double-target editing generation fragment deletion mutant T78Pm, wherein the nucleotide sequence of a forward primer and a reverse primer of the molecular marker of the rice mutant T78Pm is as follows:
37Pm-1F:5’-ctcggggagcgcaagggagc-3’(SEQ ID NO.31)
37Pm-1R:5’-aggcggaagcgagtaatcgg-3’(SEQ ID NO.32)
37Pm-2F:5’-gcgctccccactcagcggag-3’(SEQ ID NO.33)
37Pm-2R:5’-gcagaatgacgacgaggcac-3’(SEQ ID NO.34)。
the invention also provides application of the molecular marker in identification of rice OsPRR37 promoter region mutant T78Pm and/or rice assisted selection breeding.
Specifically, the amplified fragments of the primers 37Pm-1F and 37Pm-1R carrying the T78Pm mutant material are 189bp, while the rice material not carrying the T78Pm mutant is 236bp; the amplified fragments of the primers 37Pm-2F and 37Pm-2R are 220bp, while the rice material without the T78Pm mutation cannot amplify the fragments, and the material meeting the two detection results is a T78Pm plant with the deletion of the specific DNA fragment.
Further, the molecular markers 37Pm-1 and 37Pm-2 of the mutant T78Pm are generated by adopting the double-target editing of the rice gene OsPRR37 promoter, a pair primer is synthesized in 300bp at the upstream and downstream of the corresponding genome position, and the amplified sequence fragment contains the molecular marker of the rice OsPRR37 promoter mutant T78 Pm.
The invention also provides application of the rice biological clock gene OsPRR37, which is characterized in that: deletion of the promoter fragment of the gene OsPRR37 can change the expression mode of the gene OsPRR 37; the change of the OsPRR37 gene expression mode can shorten the rice growth period.
The invention is based on the fact that a part of gene derived from the OsPRR37 of the rice is an important biological clock gene, and has important roles in the growth and the stress resistance regulation of the rice. The invention aims to provide an application of OsPRR37 gene expression mode change in shortening the growth period of rice.
The invention provides a method for changing the expression mode of important genes by manually editing rice genome through double targets to cause large fragment deletion. The invention also comprises the steps of constructing single-target CRISPR/Cas9 vectors by adopting other methods, simultaneously introducing two single-target CRISPR/Cas9 vectors into the same agrobacterium for transforming rice during genetic transformation of the rice, and the final effect is similar to that of a double-target CRISPR/Cas9 vector. The invention obviously changes the growth period of the rice by modifying the OsPRR37 promoter to form mutant alleles, and the results show that the OsPRR37 gene has important application value in rice molecular breeding. The genome editing is used for accurately improving the OsPRR37 promoter, so that the gene which can be applied to the rice with stable yield and high yield and proper growth period in the current plant biotechnology can be expanded, and an accurate breeding means is provided for the accurate breeding of improved rice.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a double-target-spot guide RNA sequence of a rice biological clock gene OsPRR37 promoter region, which can realize convenient and efficient rice genome editing and generate new deletion mutant materials.
2. The large-fragment deletion mutant of the promoter region of the biological clock gene OsPRR37 of the rice can shorten the growth period of the rice, and is beneficial to the accurate improvement of rice varieties.
Drawings
FIG. 1 shows the distribution of cis-acting elements associated with stress response in the promoter region of the OsPRR37 gene.
Fig. 2 shows a schematic diagram of double-target cleavage of promoter region of ospr 37 gene. In order to screen mutants capable of shortening the growth period and other agronomic traits, 8 gene editing sites (37T 1-37T 8) are selected in the 1000bp interval upstream of the rice OsPRR37 gene promoter, and each two sites are combined into a double-target CRISP/CAS9 editing system for gene editing so as to screen mutants with large fragment deletion. The OsPRR37 genome comprises 11 exons (exon), fig. 1 shows only the OsPRR37 gene promoter and the first 4 exon structures, blue arrows indicate the upstream 1000bp promoter region, boxes indicate exons, dark boxes indicate the gene coding region, and straight lines indicate introns (introns); T1-T8 respectively represent genome positions corresponding to 37T1-37T8sgRNA, two adjacent sites form a pair of double-target sgRNA sites, and 8 sites are combined into an OsPRR37 promoter region 4 pair of double-target sgRNA editing sites.
FIG. 3 shows a schematic diagram of 37T7 and 37T8 double editing vector construction.
FIG. 4 shows that the T78Pm mutant in the field had spike and flower earlier than the wild type plant. Under normal growth conditions, T78Pm and control heading stage phenotypes. T78Pm is a double-target homozygous large fragment deletion mutant of the promoter region T7 and T8 of the OsPRR37, and WT is a receptor parent.
FIG. 5 shows the identification of T78Pm and other mutant materials using PCR methods. 37Pm-1 shows an electrophoresis pattern of PCR products generated using 37Pm-1F and 37Pm-1R primers, and 37Pm-2 shows an electrophoresis pattern of PCR products generated using 37Pm-2F and 37Pm-2R primers. Line1 is a T78Pm mutant, line 2 and Line 3 are other mutants Cr1 and Cr2, line 4 is a receptor parent, line M is DNA der, and the sizes are 500bp,250bp and 100bp respectively. In 37Pm-1, the T78Pm mutant amplifies 189bp single band, and Cr1, cr2 and Shanghai dry 2B amplify 236bp; in 37Pm-2, the fragment amplified by the T78Pm mutant is 220bp, and the fragments of Cr1, cr2 and Shanghai dry 2B cannot be amplified.
Detailed Description
The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand.
Both the pYLsgRNA-OsU a/LacZ, pYLsgRNA-OsU6b/LacZ plasmid and the pYLCRISPR/Cas9Pubi-H vector were provided by the university of agricultural university of North China, university of agricultural science, college of science (Ma et al, 2015,Molecular Plant,DOI:10.1016/j.molp.2015.04.007); the rice strain is Shanghai 2B (cultivated by agricultural biological gene center of Shanghai city of this unit, registered in national Rice data center, https:// www.ricedata.cn/variety /), which is a maintainer line of three-line hybrid rice Ganyou No.2, and can be used for further cultivation of sterile line.
Example 1: promoter cis-acting element analysis and gene editing target site sgRNA design
1.1 promoter cis-acting element analysis
OsPRR37 (LOC_Os 07g 49460) and its upstream 1kbp promoter sequence (shown in SEQ ID NO. 1) were found from national Rice data center (CHINA RICE DATA CENTRE), and the gene was located on chromosome 7 of rice at 29616705bp and 29629223bp, with a total length of 12.52kbp. A number of elements related to plant flowering and stress response were found by cis-acting element analysis of the 1kbp promoter before the transcription initiation point of OsPRR37 gene using the plant pan 3.0 webpage, wherein 11 cis-acting elements related to stress were widely present in the initiation codon region of OsPRR37 gene, including 30 NF-YB (nucleic factor-YB), 1 LEA-5 (late embryogenesis abundant), 3 bHLH (basic helix-helix), 5 bZIP (basic region-nucleic acid sequence), 2C 2H2, 3 GATA, 2 HD-ZIP (Homeodomain-nucleic acid sequence), 2 Myb/SANT, 2 TBP (TATA binding protain), 1 WRKY and 1 WRKDS box. By analysis of the positions of these cis-acting elements, they were found to be more intensively distributed at the first 600bp of the OsPRR37 gene (fig. 1). Then, the gene editing target is designed mainly in the range of the first 600 bp.
1.2 guide RNA target sequence selection and primer design
The present example relates to the creation of rice biological clock gene OsPRR37 promoter region deletion mutants based on CRISPR-Cas9 technology. In order to screen mutants capable of shortening the growth period and other agronomic traits, 8 gene editing sites (37T 1-37T 8) are selected from the upstream-600 of the gene promoter of the rice biological clock gene OsPRR37 (ID: LOC_Os07g 49460) to the first exon region, the sgRNA of 8 promoter regions of the OsPRR37 is designed in total, and the designed sgRNA target sequences are subjected to rice genome database comparison to exclude nonspecific target cutting sites. Each two adjacent sites are combined into a double-target design sgRNA guide sequence, which is used for constructing a CRISP/CAS9 editing system so as to screen large fragment deletion mutants. The corresponding sgRNA sequences and adaptor-added primer sequences are shown in table 1 below, and adaptor-added primers were used to construct CRISP/CAS9 editing vectors.
TABLE 1 sgRNA leader sequences and primer sequences for constructing vectors thereof
Example 2 construction of double target sequence sgRNA expression cassettes
Double targets the construction of double target sequence sgRNA expression cassettes was described by way of example for 37T1 and 37T2, with other 37T3 and 37T4, 37T5 and 37T6, 37T7 and 37T8 double target vectors constructed similarly, replacing the corresponding primers, corresponding primers being shown in table 1, with the last behaving reverse primer and the next behaving forward primer for each sgRNA designation. Referring to the method of Ma et al (Ma et al 2015,Molecular Plant,DOI:10.1016/j. Molp.2015.04.007), a first round of PCR reactions was performed to amplify fragments corresponding to the two targets. For 37T1 target, 2-5ng of pYLsgRNA-OsU a/LacZ plasmid (1 ul) is taken as a template, and PCR amplification is carried out in two reaction systems respectively: U-F and adaptor-added sgRNA reverse primers were used to amplify OsU a-target fragments, and gR-R and adaptor-added sgRNA forward primers were used to amplify sgRNA-target fragments.
The PCR product is detected by electrophoresis, the OsU a-target fragment is about 700bp, and the gRNA-target fragment is about 131 bp. For T2, 2-5ng of pYLsgRNA-OsU b plasmid is added as a template, and PCR amplification is carried out in two reaction systems respectively: U-F and adaptor-added sgRNA reverse primers were used to amplify OsU b-target fragments, and gR-R and adaptor-added sgRNA forward primers were used to amplify sgRNA-target fragments. The PCR product is detected by electrophoresis, the OsU6 b-target fragment is about 600bp, and the gRNA-target fragment is about 131 bp.
The PCR reaction generally uses high fidelity polymerase with a reaction system of 1uL plasmid template, 2.5uL 10 XBuffer, 0.5uL polymerase, 1uL 25mM MgSO 4 2.5uL of 2mM dNTPs,10uM of each of the front and rear primers 0.5uL, and dd H was added 2 O to 25uL; the PCR amplification procedure was: 95℃2min,98℃10s,58℃15s,68℃20s 25 cycles. A second round of PCR reaction was then performed, ligating the 37T1 and 37T2 expression cassettes into the same PCR fragment. The first round of 4 PCR products were taken with 1. Mu.l each as template, and the primers U-GAL, pgs-GA2, U-GA2 and Pgs-GAR were added for the second round of PCR,28-30 cycles, in which 4 fragment-pooled sgRNA expression cassette fragments with 37T1 and 37T2 were generated by overlapping PCR. The size of the product is about 1.5Kb, gel electrophoresis detection and gel cutting recovery are carried out, and the product is the biological clock gene OsPRR37 promoter region double-target-spot sequence 37T1 and 37T2-sgRNA expression cassette.
The target fragment containing the 37T1 and 37T2 double-target sequence sgRNA expression cassette is recombined with the linearized pYLCRISPR/Cas9 vector to obtain the NanjinopranExamples of ClonExpress rapid cloning recombinases from Biotechnology Inc.: 4 μl of 5 XCE II Buffer,200ng of linearized pYLCRISPR/Cas9Pubi-H plasmid, 200ng of PCR product of the double target sequence-sgRNA recovered above, 2 μl of Exnase TM And II, finally adding water to 20 μl, carrying out a warm bath for 30min at 37 ℃, converting 5 μl of the reaction mixture into escherichia coli, coating an LB plate containing kanamycin for screening positive clones, picking positive monoclonal on the next day for sequencing verification, and extracting plasmids for preservation. The vector shown in the plasmid is shown in opinion FIG. 3, agrobacterium EHA105 was transformed, and finally rice callus transformation experiments were performed.
Example 3: genetic transformation of rice
3.1 seed Disinfection
Removing the shell of mature Shanghai dry 2B rice seeds, putting into a sterile triangular flask, soaking in 75% alcohol for 1-2min, and washing with sterile water for 2 times; sterilizing with 30% NaClO for 30min, shaking, washing with sterile water for 3-4 times, sucking excessive water with sterile filter paper, inoculating the seeds onto callus induction medium (MS+2, 4-D2.0 mg/L), culturing about 30 grains per dish, and dark culturing at 28deg.C.
3.2 subculture
After induction for nearly 1 month, the rice grows yellow and enlarged callus, scutellum is removed, and the callus is transferred to fresh callus induction medium (MS+2, 4-D2.0 mg/L) for subculture. And (3) carrying out subculture once every 2 weeks, and carrying out subculture for 2-4 times to obtain the tender yellow granular embryogenic callus suitable for transgenosis. After 2 weeks of subculture, embryogenic particles were selected for genetic transformation.
3.3 cultivation of Agrobacterium
Single colonies were picked on transformation plates and cultured in 1ml Agrobacterium medium. 1ml of the above culture was added to 50ml of Agrobacterium medium (containing the corresponding antibiotics), and incubated at 200rpm and 28℃for 5-6hr until OD600 was 0.6-1.0, and acetosyringone (AS, final concentration 100. Mu.M) was added 2hr before the end of the incubation. Taking the bacterial liquid at room temperature at 4000rpm for 10min, discarding the supernatant, adding MS liquid culture medium (containing AS 100 uM) to resuspend the bacterial body, and culturing for 2hr under the same condition AS above, so that the OD600 = 0.5-1 of the bacterial liquid can be used for transforming the callus. As=acetostinone.
3.4 Co-cultivation
The embryogenic callus of rice is immersed in the agrobacterium liquid for 20-30min (using the product of example 2), then the moisture is absorbed by sterile absorbent paper, the infected callus is placed on a co-culture medium (MS+2, 4-D2.0 mg/L+AS 100 uM), and is dark-cultured at 28 ℃ for three days.
3.5 bacterial washing
The co-cultured callus is washed 3 times by sterile water, soaked in MS liquid culture medium containing Cef/CN 400mg/L for 20-30min, and transferred to sterile filter paper for drying.
3.6 selection culture
The callus with the water absorbed was inoculated on a selection medium (MS+2, 4-D2.0 mg/L+Hyg30mg/L+Cef400 mg/L). After 3 weeks, the newly grown calli were selected and inoculated onto selection medium (MS+2, 4-D2.0 mg/L+Hyg50mg/L+Cef250mg/L) and selected for 2 weeks.
3.7 differentiation culture
The resistant callus obtained after 2 times of selection is transferred to a pre-differentiation culture medium (N6+KT2.0 mg/L+NAA 0.2mg/L+6-BA 2.0mg/L+Hyg30 mg/L+Cef200mg/L+agar 9g/L+sucrose 45 g/L) for dark culture for about 10 days, and then transferred to a differentiation culture medium (N6+KT2.0 mg/L+NAA 0.2mg/L+6-BA 2.0mg/L+Hyg30 mg/L+agar 4.5 g/L+sucrose 30 g/L) for light culture.
3.8 rooting culture
About 1-2 months, seedlings about 2cm high were transferred to rooting medium (1/2MS+Hyg 15 mg/L+agar 4.5 g/L+sucrose 20 g/L) to induce adventitious roots.
3.9 transplanting of transgenic seedlings
When the seedlings grow to 10cm high, the seedlings are taken out, the attached solid culture medium is washed by sterile water and is transferred into soil, the seedlings are covered by a glass cover for several days just before beginning, and the glass cover is taken down after the plants are strong, and the seedlings are cultivated in a greenhouse.
Example 4: identification of transgenic plants
In example 3, a total of 140 hygromycin positive T0 generation plants were obtained from 4 transformation experiments with 4 vectors, yielding an average of 35 plants per transformation experiment. To further examine the gene editing effect of these plants, the genomic DNA of the transgenic rice plants obtained in example 3 was extracted by CTAB method of Jr Stewart et al (1993). According to the genome structure, see FIG. 1, primers were designed at both ends of the gene editing site for amplifying the editing region fragments, respectively. PCR amplification was performed on the OsPRR37 promoter region mutant site using sequencing primers 37P1CR and 37P1CR for T1-T4 site plants, and using sequencing primers 37P2CR and 37P2CR for T5-T8 site plants. Wherein the primer sequences are as follows:
37P1CF 5’-gcttctggccaagagtgttcc-3’(SEQ ID NO.27)
37P1CR 5’-tcatttctcctcaaaacactcc-3’(SEQ ID NO.28)
37P2CF 5’-gcttgccttttctgactcgc-3’(SEQ ID NO.29)
37P2CR 5’-cgaacggggttgaggcggag-3’(SEQ ID NO.30)。
the reaction system is as follows: 20ng of rice genome DNA template, 10uL Taq PCR Mastermix (Tiangen Biochemical technology (Beijing) Co., ltd.), front and rear primers of 10uM 1uL each, and dd H was supplemented 2 O to 20uL. The PCR amplification procedure was: pre-denatured at 95℃for 5min, then 30 cycles at 95℃for 30s,60℃for 30s, and 72℃for 45s, and finally extended at 72℃for 5min.
Sequencing result analysis of PCR amplified products shows that in each transformation event, double peaks are detected in an OsPRR37 promoter region, the probability of gene editing is over 95 percent, but due to double-target editing, homozygous mutant plants are not detected in 4 transformation events, so that the T0 generation plants need to be further propagated for one generation, and homozygous single plants are searched in the T1 generation plants.
After the seeds of the T1 generation are collected, DNA is planted and sampled, the sequencing result of the PCR amplification product identifies the plants with homozygous mutation sites in the T1 generation, and the statistics of the detected homozygous mutation types are shown in Table 2. The result shows that the mutation type generated by gene editing is rich, besides the indel mutation of 1-5nt at the single point, the deletion of a long fragment exists, wherein the maximum of the generated fragment is 80bp, further shows that the gene editing is efficient, the probability of the generated fragment deletion is higher than 30%, and the genotype of the long fragment base deletion is very likely to change the expression mode and the function of the OsPRR37.
TABLE 2 mutation of OsPRR37 promoter region Gene editing plants
Note that: -representing a deletion, + representing an insertion, "," representing a number of deleted bases at different positions, and letters representing bases.
Example 5: phenotype investigation of OsPRR37 promoter region mutant plants
In order to clarify what kind of influence is caused to agronomic characters in the mature stage of rice by different gene editing types, observation of field phenotype is carried out on T2 generation OsPRR37 gene promoter editing plants, and the obvious difference between partial OsPRR37 gene promoter variation plants and controls in the aspects of rice plant height, tillering, flowering time and the like is found. In order to shorten the growth period of Shanghai-arid 2B, we observed the mutation type with shortened growth period, found that a single plant (FIG. 4) with 20 days earlier growth period heading exists in the 37T7/8 double-target mutation type, which is named as T78Pm, and the strain lacks 47bp in the promoter region of the OsPRR37 gene, but the growth period change does not reach a significant difference in other types of mutants.
The phenotype experiment shows that the specific mutant T78Pm generated by the deletion of the fragments generated by the double targets 37T7 and 37T8 can obviously shorten the rice growth period, and is an important material for precise breeding of rice molecules.
Example 6: detection of T78Pm molecular marked rice genetic material
The genomic DNA of the genetic material of the rice to be tested is extracted by a conventional CTAB method. The test rice variety T78Pm mutant and parent Shanghai 2B. The primer pair of T78Pm mark 2 is utilized to carry out PCR amplification on the rice variety to be tested,
37Pm-1F:5’-ctcggggagcgcaagggagc-3’(SEQ ID NO.31)；
37Pm-1R:5’-aggcggaagcgagtaatcgg-3’(SEQ ID NO.32)；
37Pm-2F:5’-gcgctccccactcagcggag-3’(SEQ ID NO.33)；
37Pm-2R:5’-gcagaatgacgacgaggcac-3’(SEQ ID NO.34)；
the PCR amplification system is as follows: 20ng of rice genome DNA template, 10uL Taq PCR Mastermix (Tiangen Biochemical technology (Beijing) Co., ltd.), front and rear primers of 10uM 1uL each, and dd H was supplemented 2 O to 20uL. The PCR reaction procedure was: pre-denatured at 95℃for 5min, then subjected to 35 cycles of 95℃30s,60℃30s,72℃30s, and finally extended at 72℃for 5min.
8uL of the PCR product was applied to 2% agarose gel electrophoresis. As shown in FIG. 5, in the marker 37Pm-1, the T78Pm mutant amplifies 189bp single band, and the mutants Cr1, cr2 and the control Shanghai dry 2B amplify 236bp, so that the difference is obvious; in the marker 37Pm-2, the T78Pm mutant can amplify 220bp band of fragment, but the mutants Cr1, cr2 and the control Shanghai dry 2B cannot amplify fragment. It was shown that markers 37Pm-1 and 37Pm-2 are effective in identifying the T78Pm mutant.
The invention is not limited to the above examples, and similar results can be obtained by performing a PCR test similar to that of example 6 on the pair primers synthesized within 300bp upstream and downstream of the corresponding genomic positions of 37Pm-1F/R and 37 Pm-2F/R.
The technical solutions of the above embodiments of the present invention can be cross-combined with each other to form a new technical solution, and in addition, all technical solutions formed by adopting equivalent substitution fall within the scope of protection claimed by the present invention.

Claims (8)

1. The double target sequence of the guide RNA of the promoter region of the biological clock gene OsPRR37 of the rice is characterized in that the nucleotide sequence is as follows:
37T7:5’-GACGTGGAACAAATGGAAGTGGG-3’
37T8:5’-ATATCTAGTAGCAGTAGCAGCGG-3’。
2. a rice biological clock gene OsPRR37 has a nucleotide sequence shown in SEQ ID NO. 2.
3. The method for preparing the rice biological clock gene OsPRR37 as defined in claim 2, which is characterized in that: constructing an sgRNA expression cassette containing the guide RNA target sequence of claim 1 by using a PCR amplification method;
loading the two sgRNA expression cassettes onto the same CRISPR/Cas9 vector to obtain a CRISPR/Cas9-sgRNA vector containing a double-target-point sequence;
transforming rice callus with CRISPR/Cas9-sgRNA vector carrying two targets to obtain a mutant with fragment deletion of the promoter region of the rice biological clock gene OsPRR37, namely the rice biological clock gene OsPRR37 in claim 2.
4. The molecular marker for detecting the rice gene OsPRR37 promoter double-target editing generation fragment deletion mutant T78Pm is characterized in that the nucleotide sequence of a forward primer and a reverse primer of the molecular marker of the rice mutant T78Pm is as follows:
37Pm-1F:5’-ctcggggagcgcaagggagc-3’
37Pm-1R:5’-aggcggaagcgagtaatcgg-3’
37Pm-2F:5’-gcgctccccactcagcggag-3’
37Pm-2R:5’-gcagaatgacgacgaggcac-3’。
5. the molecular marker of claim 4 applied to identification of rice OsPRR37 promoter region mutant T78Pm and/or rice assisted selection breeding.
6. The use according to claim 5, characterized in that: the amplified fragments of the primers 37Pm-1F and 37Pm-1R are 189bp carrying the T78Pm mutant material, and the rice material which does not carry the T78Pm mutation is 236bp; the amplified fragments of the primers 37Pm-2F and 37Pm-2R are 220bp, while the rice material without the T78Pm mutation cannot amplify the fragments, and the material meeting the two detection results is a T78Pm plant with the deletion of the specific DNA fragment.
7. The use according to claim 5, characterized in that: the molecular markers 37Pm-1 and 37Pm-2 of the mutant T78Pm generated by double-target editing of the rice gene OsPRR37 promoter according to claim 4 are adopted to synthesize a pair primer in 300bp at the upstream and downstream of the corresponding genome position, and the amplified sequence fragment contains the molecular marker of the rice OsPRR37 promoter mutant T78 Pm.
8. The use of a rice biological clock gene ospr 37 according to claim 2, characterized in that: deletion of the promoter fragment of the gene OsPRR37 can change the expression mode of the gene OsPRR 37; the change of the OsPRR37 gene expression mode can shorten the rice growth period.
CN202211418088.XA 2022-11-14 2022-11-14 Rice biological clock gene OsPRR37 promoter region guide RNA double-target sequence and application thereof Pending CN117070515A (en)

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