Rice ospil11 gene mutant, preparation method and application thereof

Abstract

The application discloses a rice OsPIL11 gene mutant and a preparation method and application thereof, relates to the technical field of genetic engineering, and the rice OsPIL11 gene mutant comprises a gene with a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The application adopts CRISPR / Cas9 gene editing technology to target knockout of a rice OsPIL11 gene, and two rice OsPIL11 mutants are obtained through differentiation screening, so that the rice heading stage can be advanced, and the breeding cycle of an early heading type variety is greatly shortened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a rice OsPIL11 gene mutant and a preparation method and application thereof. BACKGROUND

[0002] Rice is one of the important food crops, and provides food sources for more than half of the population in China. The rice planting area in China is vast, and there are great differences in climate among different regions. Due to the different climate conditions such as light and temperature, the rice varieties in different planting areas are significantly different. The heading stage is the process of the conversion of rice from vegetative growth to reproductive growth, which is jointly regulated by variable external environmental factors and complex internal gene networks. Rice is a facultative short-day plant, and short-day promotes heading, while long-day delays the heading of rice. The appropriate heading stage is of great significance for maintaining high yield and stable yield of rice. Breeding and domesticating rice varieties with different heading stages is beneficial to expanding the regional adaptability of rice and improving yield.

[0003] Phytochrome-interacting factors (PIFs) play an important role in multiple processes such as germination and flowering of plants. Six PIF transcription factors (OsPIL11-OsPIL16) have been identified in rice, among which, overexpression of OsPIL13 under drought conditions can make the internode of rice elongate; overexpression of OsPIL14 can promote the elongation of the rice mesocotyl in the dark and improve salt tolerance; OsPIL15 can positively regulate drought tolerance of rice, and negatively regulate grain size, tiller angle and sheath blight resistance; OsPIL16 plays a certain role in regulating grain size of rice, and OsPIL11 and OsPIL12 need to be further studied.

[0004] With the rapid development of genomics, molecular genetics and other disciplines, a large number of rice heading stage related genes have been cloned, such as GHD7, DTH8, EHD1, Hd3a, RFL, Hd1, DTH7, etc. Studies have shown that these heading stage genes mainly regulate rice heading through two pathways, respectively, the OsGI-Hd1-Hd3a pathway which is conserved in the GI-CO-FT pathway in Arabidopsis, and the Ehd1 pathway which is specific to rice. There are problems such as long breeding cycle and long heading stage of rice in the current rice variety breeding. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide a rice OsPIL11 gene mutant and a preparation method and application thereof. The CRISPR / Cas9 gene editing technology is used to target knockout the rice OsPIL11 gene, and two rice OsPIL11 mutants are obtained through differentiation screening, which can make the heading stage of rice advance, and greatly shorten the breeding cycle of early heading type varieties.

[0006] The application provides the following technical solutions:

[0007] In a first aspect, a rice OsPIL11 gene mutant is provided, which comprises a gene having a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0008] In a second aspect, a preparation method of the rice OsPIL11 gene mutant is provided, which comprises the following steps: performing targeted editing on a selected site of a nucleotide sequence of an OsPIL11 gene of a target plant to obtain a mutant having a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0009] Further, the method comprises the following steps:

[0010] selecting an editing target site of the rice OsPIL11 gene and designing an upstream primer and a downstream primer;

[0011] constructing an intermediate vector targeting the OsPIL11 based on the upstream primer and the downstream primer;

[0012] constructing a final vector targeting the OsPIL11 based on the intermediate vector targeting the OsPIL11;

[0013] transforming the final vector targeting the OsPIL11 into a competent cell of Agrobacterium tumefaciens EHA105, and infecting a callus of the rice to regenerate a transgenic rice plant, and screening to obtain a rice plant with a shortened heading stage.

[0014] Further, the nucleotide sequence of the target site is shown in SEQ ID NO. 3.

[0015] Further, the nucleotide sequence of the upstream primer OsPIL11-gRNA-F is shown in SEQ ID NO. 4, and the nucleotide sequence of the downstream primer OsPIL11-gRNA-R is shown in SEQ ID NO. 5.

[0016] Further, the method for constructing the intermediate vector targeting the OsPIL11 comprises the following steps:

[0017] denaturing and annealing the mixed upstream primer and downstream primer to form double-stranded DNA with sticky ends;

[0018] performing enzyme cutting on the SK-gRNA vector with a restriction endonuclease Aar·I to obtain a vector with sticky ends;

[0019] connecting the vector and the double-stranded DNA with T4 ligase to obtain a ligation product, and performing transformation and detection on the ligation product to obtain the intermediate vector targeting the OsPIL11.

[0020] Further, the method for constructing the final vector targeting OsPIL11 comprises the following steps:

[0021] The pC1300-Cas9 vector is subjected to enzyme digestion with restriction endonuclease Kpn·I and BamH·I;

[0022] The intermediate vector targeting OsPIL11 is subjected to enzyme digestion with restriction endonuclease Kpn·I and Bgl·II, and the fragment is recovered;

[0023] The recovered fragment is connected to the pC1300-Cas9 vector subjected to enzyme digestion to obtain a connection product, and the connection product is subjected to transformation and detection to obtain the final vector targeting OsPIL11.

[0024] Further, the method for screening comprises the following steps: amplifying the genomic fragment of the transgenic rice plant by using OsPIL11 sequencing F-specific primers and OsPIL11 sequencing R-specific primers, sequencing, screening the rice mutant plant, and then obtaining the rice plant with shortened heading stage through agronomic trait investigation.

[0025] Further, the nucleotide sequence of the OsPIL11 sequencing F-specific primer is shown in SEQ ID NO. 6, and the nucleotide sequence of the OsPIL11 sequencing R-specific primer is shown in SEQ ID NO. 7.

[0026] In a third aspect, the application provides a use of the rice OsPIL11 gene mutant in the first aspect or prepared by the method in the second aspect in shortening the heading stage of rice.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] The application adopts the CRISPR / Cas9 gene editing technology to target knockout of the rice OsPIL11 gene, selects an editing target site of the rice PIF transcription factor OsPIL11 and designs upstream primers and downstream primers, and constructs an intermediate vector targeting OsPIL11 and a final vector targeting OsPIL11 in sequence; the final vector targeting OsPIL11 is transfected into rice callus, and two rice OsPIL11 mutants are obtained through differentiation and screening, that is, the targeted knockout of the rice PIF transcription factor OsPIL11 is completed; the method can make the heading stage of rice advance, and eliminates the work of hybridization and breeding, and can be applied to rapid breeding of early-heading rice varieties, and greatly shortens the breeding cycle of early-heading varieties. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The sequence analysis of the OsPIL11 gene and the design of the gene editing target point in the embodiments of the application are performed;

[0030] Figure 2 Target site sequence of OsPIL11 mutant in the embodiments of the present application;

[0031] Figure 3 Sequencing peak chart of wild type Nipponbare and OsPIL11 mutant target site in the embodiments of the present application;

[0032] Figure 4 Plant photos of wild type Nipponbare and OsPIL11 mutant at heading stage in the embodiments of the present application, the scale is 10 cm;

[0033] Figure 5 Columnar contrast chart of heading time of wild type Nipponbare and OsPIL11 mutant in the embodiments of the present application. DETAILED DESCRIPTION

[0034] The present application is further described below in conjunction with the drawings. The following examples are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0035] The gene editing method used in the present embodiment is the CRISPR / Cas9 system provided by Wang Kjian research group of China National Rice Research Institute. The specific target sequence of OsPIL11 genome is selected, and according to the gRNA primer design principle, GGCA is added before the forward sequence and AAAC is added before the reverse complementary sequence, which is connected into the intermediate vector SK-gRNA; then the intermediate vector is enzyme digested, the gRNA fragment containing the target site is recovered and connected into the final vector pC1300-Cas9, and after sequencing, the recombinant vector is transformed into rice by Agrobacterium-mediated genetic transformation method (Liu et al. 1998).

[0036] OsPIL11 gene is located on the 12th chromosome of rice, the coding region CDS is 1338 bp long, and it encodes 445 amino acids, and the coding region sequence is shown as SEQ ID NO. 8.

[0037] SEQ ID NO. 8:

[0038]

[0039] The following examples use japonica rice variety Nipponbare as an example, using Agrobacterium to transform the designed vector into the Nipponbare genome, and screening to obtain genetically modified rice plants. The specific method is as follows:

[0040] Step 1, RNA target sequence design.

[0041] According to the genomic sequence of rice transcription factor gene OsPIL11 and the principle of designing target sites of CRISP / Cas9 technology, a guide RNA target sequence is designed, the nucleotide sequence of which is shown as SEQ ID NO. 3, which is located at the 238-257 base of the coding region of OsPIL11 gene, as shown in Figure 1 .

[0042] SEQ ID NO. 3: 5'-GACCTGTTCACCGAGCTGTT-3'.

[0043] Step 2, design of upstream and downstream primers of SK-gRNA sequence.

[0044] According to the SK-gRNA sequence in step 1, the nucleotide sequence of the upstream primer OsPIL11-gRNA-F is shown as SEQ ID NO. 4, and the nucleotide sequence of the downstream primer OsPIL11-gRNA-R is shown as SEQ ID NO. 5.

[0045] SEQ ID NO. 4: 5'-GGCAGACCTGTTCACCGAGCTGTT-3';

[0046] SEQ ID NO. 5: 5'-AAACAACAGCTCGGTGAACAGGTC-3'.

[0047] Step 3, construction of intermediate vector targeting OsPIL11.

[0048] An equal amount of OsPIL11-gRNA upstream primer and downstream primer (final concentration 100 μM) was mixed, and after 95℃ annealing for 5 min, it was gradually cooled to room temperature to form complementary double-stranded DNA, which was used for subsequent vector construction.

[0049] After the SK-gRNA vector was digested with restriction endonuclease Aar·I at 37℃ for 12h, a vector with sticky ends was formed, and the specific enzyme digestion system is shown in Table 1.

[0050] Table 1 Enzyme digestion system

[0051] Ingredient Amount 10x buffer Aar.I 2 μL 50x oligonudeotide 0.4 μL Aar.I 0.6 μL Vector SK-gRNA 8 μL ddH2O 9 μL

[0052] The SK-gRNA vector digested with restriction enzymes and the DNA forming complementary double strands were connected with T4 ligase to construct an intermediate vector. The connection system is shown in Table 2.

[0053] Table 2 Connection system

[0054] Ingredient Amount SK-gRNA (Aar.I) 2 μL 10x T4 DNA ligase buffer 1 μL Primer annealing product 6.5 μL T4 ligase 0.5 μL

[0055] After connection at 22°C for 1 h, transformation was performed. E. coli transformation: 10 μl of the connection product was taken into a 1.5 ml centrifuge tube, 50 μl of E. coli DH5α competent cells were added, and the pipette was mixed; it was placed on ice for 30 min, and then heat shock at 42°C in a constant temperature water bath for 45 s, and then quickly placed on ice for 2 min; 500 μl of LB liquid medium without antibiotics was added to each tube; it was cultured at 37°C and 220 rpm for 1 h; after high-speed centrifugation for 15 s, the supernatant was removed, and only a small amount of solution was left, which was resuspended with the pipette and then completely coated on LB solid medium containing A + , and cultured overnight at 37°C. The next day, single colonies were selected for sequencing.

[0056] Step 4, construction of the final vector targeting OsPIL11.

[0057] The pC1300-Cas9 vector was digested with Kpn·I and BamH·I at 37°C for 1 h. The specific digestion system is shown in Table 3.

[0058] Table 3 Digestion system

[0059]

[0060]

[0061] The SK-gRNA vector (intermediate vector targeting OsPIL11) with the target fragment obtained in step 3 was digested with restriction enzymes Kpn·I and Bgl·II at 37°C for 1 h, and the fragment was recovered. The specific digestion system is shown in Table 4.

[0062] Table 4 Digestion system

[0063] Ingredient Amount Intermediate vector with the fragment of interest 15 μL Kpn.I 1 μL Bgl.II 1 μL 10x buffer 5 μL ddH2O 28 μL

[0064] The recovered fragment was mixed with the pC1300-Cas9 vector digested with restriction enzymes, and connected at 22°C for 30 min to obtain the final vector containing the target site sequence. The connection system is shown in Table 5.

[0065] Table 5 Connection system

[0066] Ingredient Amount pC1300-Cas9 (Kpn.I and BamH.I) 1.5 μL 10x T4 DNA ligase buffer 1 μL T4 ligase 0.5 μL Fragment of interest cut from SK-gRNA 7 μL

[0067] Step 5, transform the final vector targeting OsPIL11 into Agrobacterium tumefaciens EHA105 competent cells: take 2 μL of recombinant plasmid with a cooled sterile gun head and add it to 100 μL of Agrobacterium competent cells after mixing; place on ice for 5 min, then put it into liquid nitrogen for quick freezing for 5 min; then transfer it to a 37°C water bath for 5 min; add 500 μL of LB medium without any antibiotic, cultivate at 28°C for 2-3 h to recover the bacteria; high-speed centrifuge for 15 s. Remove the supernatant and only leave a small amount of solution, resuspend and coat on LB medium containing K + antibiotics, and cultivate at 28°C for 24-36 h.

[0068] The successfully constructed OsPIL11 gene-specific knockout vector was sent to Jiangsu Baige Gene Technology Co., Ltd. for transgenic rice receptor Nipponbare, and transgenic plants were obtained, which were then transplanted to a transgenic rice test field.

[0069] After the obtained regenerated plants were transplanted and survived, T0 generation transgenic plants were obtained. After extracting genomic DNA from T0 generation rice leaves by CTAB method, the DNA sequence of the regenerated plants was amplified by PCR using OsPIL11 sequencing-F (nucleotide sequence as shown in SEQ ID NO. 6) and OsPIL11 sequencing-R primers (nucleotide sequence as shown in SEQ ID NO. 7), and sequencing identification was performed, and the sequencing results are shown in Figure 3 .

[0070] SEQ ID NO. 6: 5'-TAGGGTTCGTGCGATTCTTGG-3';

[0071] SEQ ID NO. 7: 5'-CGGCATCATCCTGCTCTGG-3'.

[0072] Plants with mutation phenomena detected in T0 generation were collected for seed, and T1 generation was planted. DNA was extracted from T1 generation plant leaves, and the above detection primers were used for detection to determine that the OsPIL11 gene mutation type was homozygous mutation; hygromycin detection primer-F (nucleotide sequence as shown in SEQ ID NO. 9) and hygromycin detection primer-R (nucleotide sequence as shown in SEQ ID NO. 10) were designed.

[0073] SEQ ID NO. 9: GGACTTCGGGGCAGTCCT;

[0074] SEQ ID NO. 10: CGATGTAGGAGGGCGTGG.

[0075] The presence of the hygromycin fragment was detected to determine whether the foreign vector T-DNA fragment was present. Finally, two types of OsPIL11 mutant (i.e., ospil11-1 and ospil11-2, as shown in Figure 2 The seeds of the plants, which were homozygous mutants and negative for hygromycin detection, were collected.

[0076] Mutant ospil11-1: One base "T" was deleted at the 254th position of the coding region of the OsPIL11 gene, and the nucleotide sequence of the ospil11-1 is shown in SEQ ID NO. 1.

[0077] SEQ ID NO. 1:

[0078]

[0079] Mutant ospil11-2: deletion of four bases "GAGC" at positions 250-253 of the coding region of the OsPIL11 gene, the nucleotide sequence of which is shown as SEQ ID NO. 2.

[0080] SEQ ID NO. 2:

[0081]

[0082] The T1 generation plants screened are collected for seeds, and the T2 generation is planted. At the mature stage of the T2 generation, the agronomic traits of wild type plants and two mutant types of plants are investigated and photographed, and it is found that the two OsPIL11 gene mutants have an early heading date, as shown in Figure 4 and Figure 5 Compared with the wild type, the heading date of the ospil11-1 mutant is advanced by 4.21 days, and the heading date of the ospil11-2 strain is advanced by 3.26 days.

[0083] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A preparation method of a rice OsPIL11 gene mutant for shortening the heading stage of rice, characterized in that, Targeted editing is performed on a site selected from the nucleotide sequence of the OsPIL11 gene of a target plant to obtain a mutant having a gene with a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the method comprises the following steps: An editing target site of a rice OsPIL11 gene is selected, and an upstream primer and a downstream primer are designed; An intermediate vector targeting the OsPIL11 is constructed based on the upstream primer and the downstream primer; A final vector targeting the OsPIL11 is constructed based on the intermediate vector targeting the OsPIL11; The final vector targeting the OsPIL11 is transformed into competent cells of Agrobacterium tumefaciens EHA105, and the transformed cells are used to infect calli of rice, and transgenic rice plants are regenerated, and screened to obtain rice plants with shortened heading stage; The nucleotide sequence of the target site is shown in SEQ ID NO. 3, the nucleotide sequence of the upstream primer OsPIL11-gRNA-F is shown in SEQ ID NO. 4, and the nucleotide sequence of the downstream primer OsPIL11-gRNA-R is shown in SEQ ID NO.

5.

2. The production method according to claim 1, characterized by, The method for constructing the intermediate vector targeting the OsPIL11 comprises: The upstream primer and the downstream primer are mixed and denatured to anneal to form double-stranded DNA with sticky ends; The SK-gRNA vector is digested with restriction endonuclease Aar·I to obtain a vector with sticky ends; The vector and the double-stranded DNA are connected by T4 ligase to obtain a ligation product, and the ligation product is transformed and detected to obtain the intermediate vector targeting the OsPIL11.

3. The preparation method according to claim 1, characterized in that, The method for constructing the final vector targeting the OsPIL11 comprises: The pC1300-Cas9 vector is digested with restriction endonucleases Kpn·I and BamH·I; The intermediate vector targeting the OsPIL11 is digested with restriction endonucleases Kpn·I and Bgl·II, and the fragment is recovered; The recovered fragment is connected to the digested pC1300-Cas9 vector to obtain a ligation product, and the ligation product is transformed and detected to obtain the final vector targeting the OsPIL11.

4. The method of claim 1, wherein, The method for screening comprises: amplifying the genomic fragment of the transgenic rice plant by using OsPIL11 sequencing F-specific primers and OsPIL11 sequencing R-specific primers, sequencing, screening rice mutant plants, and then investigating agronomic traits to obtain rice plants with shortened heading stage.

5. The production method according to claim 4, characterized by, The nucleotide sequence of the OsPIL11 sequencing F-specific primer is shown in SEQ ID NO. 6, and the nucleotide sequence of the OsPIL11 sequencing R-specific primer is shown in SEQ ID NO.

7.

6. The use of the rice OsPIL11 gene mutant prepared by the method of any one of claims 1-5, characterized in that, The rice OsPIL11 gene mutant is applied to shorten the heading stage of rice, and the variety of the rice OsPIL11 gene mutant is japonica rice variety Nipponbare.

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