Application of rice OsSPL10 gene in controlling rice panicle sprouting

Abstract

The application discloses application of a rice OsSPL10 gene in regulation of rice panicle sprouting, and belongs to the technical field of molecular biology and plant genetic engineering. The OsSPL10 gene is directionally edited by using CRISPR / Cas9 technology, a recombinant expression vector for specific knockout of a target site of the OsSPL10 gene is constructed, Nipponbare (NIP) is taken as a background, and three homozygous mutant strains ospl10-1, ospl10-2 and ospl10-3 of the OsSPL10 gene function deficiency type mutation are obtained by transgenesis. A panicle sprouting simulation experiment is carried out in a high-temperature and high-humidity environment set by a culture box, and it is found that the panicle sprouting resistance of the OsSPL10 mutant material is significantly higher than that of a wild type. The application provides a method for improving the panicle sprouting resistance of rice, and can accelerate the cultivation process of a new rice variety with panicle sprouting resistance.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and plant genetic engineering, specifically relating to the application of the rice OsSPL10 gene in regulating rice panicle germination. Background Technology

[0002] Rice is one of my country's most important food crops, with about 60% of the population relying on it as a staple food. Ensuring safe rice production is crucial for safeguarding my country's food security and agricultural development. Good seed germination is directly related to rice seedling emergence rate, seedling uniformity, and seedling vigor. Panicle sprouting is a special germination phenomenon where seeds germinate on the parent plant before harvest. This phenomenon occurs in major rice-producing regions worldwide, including China, India, and Japan. It is particularly severe in southern my country's rice-growing areas, where high temperatures and heavy rainfall are frequent during the rice ripening and harvesting periods. Panicle sprouting not only affects the seed value of rice but also significantly impacts its yield, storage, and processing quality. Besides climatic factors, the genetic characteristics of rice varieties are also a key factor influencing panicle sprouting. Therefore, targeted breeding and creation of germplasm resistant to panicle sprouting are among the most economical and effective ways to solve this problem, and are increasingly attracting the attention of breeders.

[0003] Currently, research progress on rice panicle sprouting is relatively slow, mainly due to the scarcity of sprouting-resistant genes with breeding value and the still unclear genetic regulatory network. Therefore, discovering new sprouting resistance-related genes and rapidly applying them to improve rice panicle sprouting resistance can effectively solve the serious problem of sprouting in rice production, which is of great significance for ensuring my country's food security. The CRISPR / Cas9 system is a site-directed genome editing tool that has matured in recent years, enabling precise improvement of crop traits through rapid, site-specific alterations to plant genes. Summary of the Invention

[0004] This invention provides a method for cultivating rice resistant to panicle germination using gene editing technology. The method uses NIP as the recipient material, and employs CRISPR / Cas9 technology to directionally edit the rice OsSPL10 gene, constructing an OsSPL10 gene knockout recombinant vector. This vector is then transformed into rice callus tissue containing the NIP recipient material using Agrobacterium-mediated transformation. After screening and molecular identification, homozygous mutant lines are obtained. Experiments using these lines' panicles are conducted to determine germination rate and germination time, verifying that the screened OsSPL10 knockout homozygous mutant rice plants exhibit enhanced resistance to panicle germination.

[0005] The present invention adopts the following technical solution,

[0006] A rice OsSPL10 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] A rice OsSPL10 gene, wherein the rice OsSPL10 gene encodes the rice OsSPL10 protein as described in claim 1, and the nucleotide sequence of the rice OsSPL10 gene is shown in SEQ ID NO.2.

[0008] The present invention also provides expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines for the above-mentioned genes.

[0009] The present invention also provides the application of the above-mentioned protein or gene in regulating rice panicle germination.

[0010] Furthermore, the regulation of rice panicle germination is to reduce the rice panicle germination rate.

[0011] Furthermore, a target-specific knockout CRISPR / Cas9 editing vector was constructed, and the constructed CRISPR-Cas9-OsSPL10 vector was transformed into Agrobacterium. Genetically engineered bacteria containing this CRISPR-Cas9-OsSPL10 vector were obtained through screening. These genetically engineered bacteria were then used to transform rice callus and obtain regenerated rice seedlings. The genetically engineered bacteria containing the CRISPR-Cas9-OsSPL10 vector were transferred into the recipient rice callus tissue to be edited. Through hygromycin screening, transgenic rice plants were obtained. The germination rate of the resulting transgenic rice panicles decreased under a set high-temperature and high-humidity environment.

[0012] Furthermore, using CRISPR / Cas9 technology, the nucleotide sequence was edited at position 111bp-130bp of the first exon of the rice OsSPL10 gene as shown in SEQ ID NO.2, with the gRNA target site sequence being 5'-GATGAGCGGTAGGATGAACG-3'.

[0013] Furthermore, the amplification primer sequences are as follows:

[0014] F sequence: 5'-ggcaGATGAGCGGTAGGATGAACG-3'

[0015] R sequence: 5'-aaacCGTTCATCCTACCGCTCATC-3'.

[0016] Furthermore, the primer sequences for identification are as follows:

[0017] F sequence: GACAACCAACACGCCAATA;

[0018] R sequence: GCCGCGAAGTCCAAGC.

[0019] The present invention also provides a method for breeding rice resistant to panicle sprouting, characterized in that the above-mentioned rice OsSPL10 gene is knocked out in rice, and the resulting transgenic rice has reduced panicle sprouting compared with the target rice.

[0020] This invention also provides a method for cultivating rice resistant to panicle sprouting using gene editing technology. The method involves targeted editing of the rice OsSPL10 gene using CRISPR / Cas9 gene engineering technology, transforming a CRISPR-Cas9-OsSPL10 vector with a target site specifically knocked out into a recipient rice variety, and then screening to obtain transgenic improved rice plants to regulate panicle sprouting. The specific process is as follows:

[0021] (1) Construction of CRISPR-Cas9-OsSPL10 vector: A target-specific knockout CRISPR / Cas9 editing vector was constructed targeting the OsSPL10 gene. The gene editing target was designed as follows: Figure 1 As shown;

[0022] (2) Construction of engineered bacteria: The constructed CRISPR-Cas9-OsSPL10 vector was transformed into Agrobacterium, and genetically engineered bacteria containing this CRISPR-Cas9-OsSPL10 vector were obtained by screening.

[0023] (3) Transformation of rice callus by genetically engineered bacteria and obtaining regenerated rice seedlings: Genetically engineered bacteria containing the CRISPR-Cas9-OsSPL10 vector were screened and transferred into the recipient rice callus tissue to be edited. Transgenic rice plants were obtained by screening with hygromycin.

[0024] (4) Using OsSPL10 sequencing-F specific primers and OsSPL10 sequencing-R specific primers, the genome fragments of the transgenic rice plants obtained in process 3 were amplified, sequenced, and rice mutant plants were screened.

[0025] Further defining the rice OsSPL10, it is characterized by being located on the rice Chr6 chromosome, with coordinates 27114985-27117417, a CDS length of 1281 bp, and a sequence as shown in SEQ ID NO.2.

[0026] Further defining the process (1), characterized in that the nucleotide sequence of the rice OsSPL10 gene, as shown in SEQ ID NO.2, is edited at 518bp-539bp using CRISPR / Cas9 technology, with the gRNA target site sequence being 5'-GATGAGCGGTAGGATGAACG-3', as shown in SEQ ID NO.3. The corresponding primer F sequence is designed as 5'-ggcaGATGAGCGGTAGGATGAACG-3', as shown in SEQ ID NO.4, and the R sequence is designed as 5'-aaacCGTTCATCCTACCGCTCATC-3', as shown in SEQ ID NO.5. The pC1300-Cas9 vector is digested with Kpn I and BamHI, and the gRNA is digested with Kpn I and Bgl II. The gRNA fragment is then recovered and linked to the pC1300-Cas9 vector to construct the knockout recombinant vector CRISPR-Cas9-OsSPL10.

[0027] Further defining the process (2), characterized in that the constructed CRISPR-Cas9-OsSPL10 vector is transformed into Agrobacterium strain EHA105 by freeze-thaw method, and genetically engineered bacteria containing this CRISPR-Cas9-OsSPL10 vector are obtained by kanamycin screening.

[0028] Further defining the process (3), characterized in that: rice callus tissue with Nipponbare (NIP) as background is infected with Agrobacterium EHA105 containing the CRISPR-Cas9-OsSPL10 vector and cultured in a 28℃ culture room for 3 days; after washing away Agrobacterium with liquid culture medium, the rice callus is placed on a selection medium containing suitable antibiotics for culture; after 3-4 weeks of culture, resistant callus is obtained, the resistant callus is differentiated into seedlings, planted in rice fields, and T0 generation positive plants with OsSPL10 gene functional defect mutation are screened to obtain positive plants.

[0029] Further defining the process (4), the nucleotide sequence of the OsSPL10 sequencing-F specific primer is shown in SEQ ID NO.6, namely GACAACCAACACGCCAATA. The nucleotide sequence of the OsSPL10 sequencing-R specific primer is shown in SEQ ID NO.7, namely GCCGCGAAGTCCAAGC. The obtained T1 generation OsSPL10 mutants have three mutation types, named knockout mutant lines osspl10-1, osspl10-2, and osspl10-3, as follows: Figure 3 As shown, all of these lead to premature termination of protein translation.

[0030] Beneficial effects

[0031] This invention constructs a knockout vector for the OsSPL10 gene, transforms rice callus tissue using Agrobacterium-mediated transformation to obtain OsSPL10 knockout rice plants, and screens homozygous knockout rice lines through sequencing. A panicle sprouting verification experiment is then conducted to determine the germination rate. This invention is the first to propose the effect of the OsSPL10 gene on panicle sprouting in rice. Knockout mutant lines of the rice SPL transcription factor family gene OsSPL10 exhibit delayed panicle sprouting compared to wild-type NIP, indicating that OsSPL10 is one of the important factors controlling panicle sprouting in rice. The transformation vector can be applied to improve the trait of resistance to panicle sprouting in rice, which has important application value in the field of crop breeding and is of positive significance for preventing yield and quality reduction in rice. Attached Figure Description

[0032] Figure 1 Sequence analysis of the OsSPL10 gene and design of gene editing targets;

[0033] Figure 2 Sequencing diagram of OsSPL10 transgenic plants and wild-type target sites;

[0034] Figure 3 A schematic diagram of the amino acid changes in the osspl10-1 to osspl10-3 mutants;

[0035] Figure 4 The budding status of the spikelets in OsSPL10 transgenic plants and wild-type plants;

[0036] Figure 5 This is a statistical chart showing the germination rate of spikelets from OsSPL10 transgenic plants and wild-type plants. Detailed Implementation

[0037] The present invention and its embodiments are described below. This description is not restrictive, and actual embodiments are not limited thereto. All technologies implemented based on the above description of the present invention fall within the protection scope of the present invention.

[0038] Example 1

[0039] 1. Construction of rice OsSPL10 gene knockout vector

[0040] 1) Determining the target sequence. Based on the CRISPR / Cas9 target design principles, the PAM site was chosen as the target sequence for NGG knockout. The gRNA target sequence was determined to be 5'-GATGAGCGGT AGGATGAACG-3' (SEQ ID NO. 3) at 111bp-130bp of the first exon of the OsSPL10 gene. The gene editing target design is as follows: Figure 1 As shown;

[0041] 2) Design the corresponding primer F and R sequences. The F sequence is...

[0042] 5'-ggcaGATGAGCGGTAGGATGAACG-3' (SEQ ID NO.4), R sequence is

[0043] 5'-aaacCGTTCATCCTACCGCTCATC-3' (SEQ ID NO. 5);

[0044] 3) Construction of intermediate carrier

[0045] The A.SK-gRNA vector was digested with Aar I (Ferment) to form a vector with sticky ends. The specific digestion system is shown in Table 1 below.

[0046] Element Added amount 10×buffer AarI 2ul 50×oligonudeotide 0.4ul Aar I 0.6ul SK-gRNA 8ul <![CDATA[ddH20]]> 9ul

[0047] After mixing BF and R primers, denature at 95°C for 5 min and anneal for 30 s to form complementary double-stranded DNA, which will be used for subsequent vector construction.

[0048] C. The enzyme-digested SK-gRNA vector and the DNA fragment from step B above were ligated at 22°C for 1 hour, and then transformed into E. coli competent cells DH5α to obtain an intermediate vector. The specific ligation system is shown in Table 2 below.

[0049]

[0050] D. Use common primers T7 or T3 for sequencing to verify correctness.

[0051] 4) The intermediate vector obtained in process C was digested with restriction endonucleases Kpn I and Bgl II at 37℃ for 30 min, and the fragment was recovered. The specific digestion system is shown in Table 3 below:

[0052] Element Added amount The intermediate carrier obtained in step 3 10ul Kpn I 0.5ul Bgl II 0.5ul 2×buffer 2ul <![CDATA[ddH20]]> 7ul

[0053] 5) The pC1300-Cas9 vector was digested with restriction endonucleases Kpn I and BamHI at 37℃ for 30 min. The digestion system is shown in Table 4 below:

[0054] Element Added amount pC1300-Cas9 10ul Kpn I 0.5ul BamH I 0.5ul 2×buffer 2ul

[0055] 6) Mix the fragment obtained in step 4 and the pC1300-Cas9 vector digested in step 5 for 30 min and then ligate to obtain the final vector containing the target site sequence. The specific ligation system is shown in Table 5 below:

[0056] Element Added amount pC1300-Cas9 (product of step 5) 1.5ul SK-gRNA (product of step 4) 7ul 10x T4 DNA Iigase buffer 1ul T4 ligase 0.5ul

[0057] The ligation product was transformed into competent E. coli DH5α cells, and positive clones were detected by colony PCR. Identification and sequencing confirmed that the knockout recombinant vector was successfully constructed.

[0058] 2. Genetic transformation and screening of OsSPL10 gene knockout rice

[0059] 1) Callus transfer. The constructed knockout recombinant vector CRISPR-Cas9-OsSPL10 was transferred into Agrobacterium tumefaciens EHA105 competent cells using the freeze-thaw method. Genetically engineered bacteria containing this CRISPR-Cas9-OsSPL10 vector were obtained by screening with kanamycin and rifampin and then infected with Nipponbare NIP rice callus tissue.

[0060] 2) Screening for resistant callus. The above callus was cultured in a 22℃ incubator for 3 days. After washing away Agrobacterium with liquid culture medium, the rice callus was placed on a screening medium containing suitable antibiotics and cultured for 3-4 weeks to obtain resistant callus.

[0061] 3) The resistant callus was differentiated into seedlings, planted in paddy fields, and T0 generation positive plants with OsSPL10 gene functional defect mutation were screened.

[0062] 4) Sequencing identification. The knockout rice was identified by sequencing. The nucleotide sequence of the OsSPL10 sequencing-F specific primer is: GACAACCAACACGCCAATA, as shown in SEQ ID NO.6. The nucleotide sequence of the OsSPL10 sequencing-R specific primer is: GCCGCGAAGTCCAAGC, as shown in SEQ ID NO.7.

[0063] 5) Sequence alignment. Sequencing results were analyzed using Snap Gene software, and the expected gene sequences were aligned. Three homozygous mutants were identified through sequencing analysis and named osspl10-1, osspl10-2, and osspl10-3. Specifically, osspl10-1 had a deletion of an 'A' at position 17 bp of the edited target sequence; osspl10-2 had a deletion of 'GA' at positions 16-17 bp of the edited target sequence; and osspl10-3 had 'ATGAA' changed to 'C' at positions 14-18 bp of the edited target sequence. The sequences of these three mutants and the control NIP rice are as follows: Figure 2 As shown. Figure 3 As shown, all three mutants resulted in premature termination of OsSPL10 protein translation.

[0064] 3. Rice panicle germination experiment

[0065] 1) Select 15 ears of each of transgenic plants osspl10-1, osspl10-2, osspl10-3 and wild control NIP with the same maturity, growth and other conditions as much as possible, and wash them three times with running water.

[0066] 2) All rice panicles were uniformly soaked in water to simulate the high temperature and rainy environment of the field. They were then treated in an incubator with suitable light and temperature. The germination standard was defined as the bud protruding 1mm. The germination status of the panicles was counted every 12 hours, and the germination percentage (GP) was calculated. The statistics were collected for a total of 8 days.

[0067] 3) Results as follows Figure 4 , 5 As shown, the panicle germination rates of the knockout mutants osspl10-1, osspl10-2, and osspl10-3 were reduced by 25.55%, 49.26%, and 18.15% respectively compared to the wild type's 59.70%, all significantly lower than the control NIP, indicating that panicle germination occurred later than in the wild type. This suggests that the OsSPL10 gene is involved in regulating panicle germination in rice, and that targeted knockout of the OsSPL10 gene can enhance rice's resistance to panicle germination, providing a new direction for research on delaying panicle germination in rice.

[0068] SEQ ID NO.1MMSGRMNAAGDESPFPFGAMQAPGPGAYVGFDHGAAAVAAAAAAAQRAGMLQHHHHHMYDGLDFAAAMQFGGGQDAPPHPQLLALPPSMAAPPPPPMPMPLQMPMTMPMPGDVYPALGIVKREGGGGGQDAAAGRIGLNLGRRTYFSPGDMLAVDRLLMRSRLGGVFGLGFGGAHHQPPRCQAEGCKADLSGAKHYHRRHKVCEYHAKASV VAASGKQQRFCQQCSRFHVLTEFDEAKRSCRKRLAEHNRRRRKPAAAATTAVAAAKDAAAAPVAAGKKPSGGAATSYTGDNKNVVSMSAAKSPISSNTSVISCLPEQGKHAAAAARPTALTLGGAPPHESSAPQIGAMLHHHHHHHQQDHMQVSSLVHINGGGGGGSNNILSCSSVCSSALPSTATNGEVSDQNNDNSHNNGGNNNNMHLFEVDFM

[0069] SEQ ID NO.2

[0070]

[0071] SEQ ID NO.3

[0072] gatgagcggt aggatgaacg

[0073] SEQ ID NO.4

[0074] ggcagatgag cggtaggatg aacg

[0075] SEQ ID NO.5

[0076] aaaccgttca tcctaccgct catc

[0077] SEQ ID NO.6

[0078] gacaccac acgccata

[0079] SEQ ID NO.7

[0080] gccgcgaagt ccaagc

Claims

1. Oryza sativa OsSPL10 application of a combination of gene mutants in reducing the rate of panicle sprouting in rice, characterized in that, Rice OsSPL10 The gene mutant combination consists of osspl10-1, osspl10-2, osspl10-3 mutants, wherein osspl10-1 deletes one base 'A' at the 17th bp of the edited target site sequence, osspl10-2 deletes 'GA' at the 16th-17th bp of the edited target site sequence, and osspl10-3 changes 'ATGAA' at the 14th-18th bp of the edited target site sequence to 'C'; the gRNA target site sequence at 111th-130th bp of the 1st exon of the OsSPL10 gene is 5'-GATGAGCGGT AGGATGAACG-3' (SEQ ID NO. 3); the nucleotide sequence of the rice OsSPL10 gene is shown in SEQ ID NO. 2; the rice OsSPL10 gene encodes the rice OsSPL10 protein, and the amino acid sequence of the rice OsSPL10 protein is shown in SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Constructing a CRISPR / Cas9 editing vector for target site-specific knockout, transforming the constructed CRISPR-Cas9-OsSPL10 vector into Agrobacterium, and obtaining a genetically engineered bacterium containing the CRISPR-Cas9-OsSPL10 vector through screening; transforming the genetically engineered bacterium into rice callus and obtaining regenerated rice seedlings: transforming the genetically engineered bacterium containing the CRISPR-Cas9-OsSPL10 vector obtained through screening into a recipient rice callus to be genetically edited, and regenerating transgenic rice plants through hygromycin screening; the obtained transgenic plants have a reduced rice panicle germination rate.

3. Use according to claim 2, characterized in that, The CRISPR / Cas9 technology is used to site-specifically edit a nucleotide sequence of a rice OsSPL10 gene first exon 111bp-130bp shown in SEQ ID NO. 2, and the gRNA target site sequence is 5'-GATGAGCGGTAGGATGAACG-3'.

4. Use according to claim 2, characterized in that, The corresponding primer sequence of the designed gRNA target site sequence is: F sequence: 5'-ggcaGATGAGCGGTAGGATGAACG-3', R sequence: 5'-aaacCGTTCATCCTACCGCTCATC-3'.

5. Use according to claim 2, characterized in that, Identifying OsSPL10 The primer sequences for the gene mutants are: F sequence: GACAACCAACACGCCAATA; R sequence: GCCGCGAAGTCCAAGC.

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