Application of rice OsIAA19 gene in improving grain shape and increasing yield of rice

Abstract

The application discloses application of a rice OsIAA19 gene in improving grain shape and increasing yield, and relates to the technical field of plant genetic engineering. The application is achieved by editing, interfering or knocking out the rice OsIAA19 gene, reducing or destroying the biological function of the rice OsIAA19 gene, and obtaining a rice strain with improved grain shape and increased yield. It is proved that knocking out the OsIAA19 gene in rice can significantly increase the grain length, and further increase the thousand-grain weight of rice, does not affect other main agronomic traits, and does not affect the cooking and eating quality of rice, so the application has good breeding application potential and is of great significance to high-yield breeding of rice.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, and in particular to the application of the rice OsIAA19 gene in improving rice grain shape and increasing yield. Background Technology

[0002] Plant hormones, also known as plant growth substances or plant regulators, are trace organic substances produced by plants that play a crucial regulatory role in plant growth, development, and adaptation to environmental changes. The plant hormone family mainly includes: auxins, gibberellins, cytokinins, ethylene, abscisic acid, jasmonic acid, brassinolide, and salicylic acid. Auxin, as the first plant hormone discovered, is essential for plant growth and development, such as vascular tissue formation, apical dominance, root formation, tropism, cell division, differentiation, and flower and fruit development. Therefore, auxins are extremely important and deserve the attention of scientists.

[0003] Elucidating the biosynthesis and signal transduction mechanisms of auxin is crucial for a better understanding of plant growth and development, responses to environmental changes, and applications in crop breeding. Auxin synthesis involves two complex pathways: tryptophan-dependent and tryptophan-independent pathways. Compared to the synthetic pathway, significant progress has been made in the study of auxin signal transduction and transport mechanisms. TIR1 / AFB-mediated auxin transcriptional regulation, also known as classical auxin signal transduction, is the most widely studied and best-characterized auxin signaling pathway. This pathway mainly includes three core transduction components: auxin receptors TIR1 / AFBs, transcriptional repressors Aux / IAA, and transcription factors ARFs. In short, under low auxin concentrations, the repressor protein Aux / IAA binds to the active transcription factor ARF to form a heterodimer, inhibiting ARF's transcriptional activation activity. Conversely, under high auxin concentrations, auxin binds to the TIR1 / AFB receptor, degrading the Aux / IAA protein through ubiquitination, thereby releasing ARF transcription factors that activate the transcription of auxin-responsive genes.

[0004] The Aux / IAA protein family consists of plant-specific transcriptional repressors that primarily regulate root and leaf formation, phototropism, gravity, and apical dominance in plants. Rice is a monocotyledonous model plant and one of the world's most important food crops, with more than half of the world's population relying on rice as their staple food. High quality and high yield are the primary goals of rice breeding. Given the crucial role of auxins in regulating plant growth and development, functional studies of the rice Aux / IAA family genes are essential.

[0005] Grain shape, as a key agronomic trait affecting rice yield and quality, has received widespread attention from rice geneticists and breeders. To date, numerous grain shape genes have been cloned in rice, involving various regulatory mechanisms, including the G protein pathway, the ubiquitin-proteasome pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone pathways. Among the plant hormone family, auxin (BR) is considered an important positive regulator of rice grain shape, with many BR biosynthetic or signaling components involved in controlling grain shape. However, research on the role of auxin in rice grain shape regulation remains limited. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0007] Therefore, the present invention provides an application of the rice OsIAA19 gene in improving rice grain shape and increasing yield. The coding region sequence of the OsIAA19 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by OsIAA19 is shown in SEQ ID NO.2.

[0008] Furthermore, the rice OsIAA19 gene is a constitutively expressed gene, and its expression level in seeds increases with seed development; the protein encoded by the rice OsIAA19 gene is located in the cell nucleus.

[0009] Furthermore, the application involves editing, interfering with, or knocking out the rice OsIAA19 gene to reduce or disrupt its biological function, thereby obtaining rice lines with improved grain shape and increased yield.

[0010] Furthermore, the application involves using the CRISPR / Cas9 system to knock out the rice OsIAA19 gene, thereby obtaining rice lines with improved grain shape and increased yield.

[0011] Furthermore, the knockout of the rice OsIAA19 gene does not affect the rice plant architecture.

[0012] Furthermore, the knockout of the OsIAA19 gene in rice does not affect the cooking and eating quality of the rice.

[0013] Furthermore, the method for knocking out the rice OsIAA19 gene using the CRISPR / Cas9 system includes:

[0014] OsIAA19 gene primer design and denaturation annealing; wherein, the sequence of OsIAA19 gene primer OsIAA19-cas9-F is shown in SEQ ID NO.3, and the sequence of OsIAA19 gene primer OsIAA19-cas9-R is shown in SEQ ID NO.4.

[0015] The digested SK-gRNA vector and primer annealing products were ligated using homologous recombinase to construct the intermediate vector SK-gRNA-OsIAA19 containing the target OsIAA19 gene fragment.

[0016] The digested intermediate vector SK-gRNA-OsIAA19 was ligated with the digested pC1300-Cas9 vector using homologous recombinase to construct the final vector pC1300-OsIAA19-Cas9, which was then screened and sequenced for verification.

[0017] The final vector pC1300-OsIAA19-Cas9 was transformed into Agrobacterium, and then Agrobacterium-mediated rice genetic transformation was used to transfer the Agrobacterium containing the pC1300-OsIAA19-Cas9 vector into rice callus tissue. After resistance screening and tissue culture, rice plants were obtained.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: the application of the rice OsIAA19 gene of the present invention in improving rice grain shape and increasing yield demonstrates good breeding application value to a certain extent and is of great significance to rice production. Attached Figure Description

[0019] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application.

[0020] Figure 1 The CRISPR / Cas9-mediated OsIAA19 gene editing sites and mutation types provided in this embodiment of the invention.

[0021] Figure 2 The sequencing results of the osiaa19 mutant sequence provided in this embodiment of the invention.

[0022] Figure 3 The Cas9 gene detection results provided in this embodiment of the invention.

[0023] Figure 4 The tissue expression of the OsIAA19 gene provided in this embodiment of the invention.

[0024] Figure 5 Subcellular localization of the OsIAA19 protein provided in this embodiment of the invention.

[0025] Figure 6 Phenotypic diagram of rice plants after knocking out the OsIAA19 gene, provided in this embodiment of the invention.

[0026] Figure 7 The statistical bar chart of rice plant phenotypic data after knocking out the OsIAA19 gene provided in this embodiment of the invention includes plant height, tillering, flag leaf length and flag leaf width as the main indicators.

[0027] Figure 8 The rice grain shape diagram after knocking out the OsIAA19 gene provided in this embodiment of the invention.

[0028] Figure 9 The statistical bar chart of rice grain shape data after knocking out the OsIAA19 gene provided in this embodiment of the invention mainly includes grain length and grain width.

[0029] Figure 10 A bar chart showing the statistical data of 1,000 grain weight of rice after knocking out the OsIAA19 gene, provided in this embodiment of the invention.

[0030] Figure 11 A bar chart illustrating the determination of rice quality traits after the OsIAA19 gene was knocked out, provided in this embodiment of the invention. Detailed Implementation

[0031] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0032] Unless otherwise specified, the experimental methods in the following examples were performed according to standard procedures, and the materials used were all commercially available products.

[0033] This invention provides a detailed description of the application of the rice OsIAA19 gene in improving rice grain shape and increasing yield, as detailed below:

[0034] (I) Rice OsIAA19 gene

[0035] The OsIAA19 gene is located on chromosome 5 of rice, with gene numbers Os05g0559400 (NCBI number) and LOC_Os05g48590 (MSU number). The full-length CDS of the OsIAA19 gene coding region is 846 bp, and the coding region sequence of the OsIAA19 gene is shown in SEQ ID NO.1. The OsIAA19 gene encodes 281 amino acids, and the amino acid sequence encoded by OsIAA19 is shown in SEQ ID NO.2.

[0036] (II) Knockout of the OsIAA19 gene using the CRISPR / Cas9 system

[0037] To knock out the rice OsIAA19 gene, we constructed the intermediate vector SK-gRNA-OsIAA19 and the final vector pC1300-OsIAA19-Cas9. After denaturation and annealing with pre-designed OsIAA19 primers, the digested vector and the annealed primer products were ligated using a homologous recombinase to construct the intermediate vector. The successfully constructed intermediate vector containing the target OsIAA19 fragment was then used to construct the final vector pC1300-OsIAA19-Cas9.

[0038] Specifically, the preparation methods of the CRISPR / Cas9 gene knockout intermediate vector SK-gRNA-OsIAA19 and the final vector pC1300-OsIAA19-Cas9 are as follows:

[0039] (1) The SK-gRNA vector was digested with restriction endonuclease Aar I. OsIAA19 primer annealing primers were designed. The OsIAA19 target gene primer annealing recovery product was ligated with the digested SK-gRNA vector using the T4 ligation method to obtain the intermediate vector SK-gRNA-OsIAA19.

[0040] (2) The vector pC1300-Cas9 was digested with Kpn I and BamHI. The intermediate vector SK-gRNA-OsIAA19 containing the target OsIAA19 gene fragment was digested with Kpn I and Bgl II restriction endonucleases. The digested vector pC1300-Cas9 and the digested intermediate vector SK-gRNA-OsIAA19 were ligated using homologous recombinase to obtain the final vector pC1300-OsIAA19-Cas9. This was then transformed into DH5α Escherichia coli competent cells, screened on LB agar plates with kanamycin, and verified by sequencing.

[0041] The primers for OsIAA19 primer annealing design are as follows:

[0042] sequence name sequence Serial Number OsIAA19-cas9-F 5-GGCATAGTCTCGCGCTTCGAGCGG-3 SEQ ID NO.3 OsIAA19-cas9-R 5-AAACCCGCTCGAAGCGCGAGACTA-3 SEQ ID NO.4

[0043] (3) The final vector pC1300-OsIAA19-Cas9 was transformed into Agrobacterium, and then Agrobacterium-mediated rice genetic transformation was used to transfer the Agrobacterium containing the pC1300-OsIAA19-Cas9 vector into rice callus tissue. After resistance selection and tissue culture, rice plants were obtained. The test results are shown in […]. Figure 1 and Figure 2As can be seen, the OsIAA19 gene coding region in the created gene-edited rice osiaa19-1 and osiaa19-2 was deleted by 2 and 8 bases, respectively, resulting in a frameshift mutation in the target gene. Furthermore, molecular identification of the Cas9 gene showed that the Cas9 gene had been successfully isolated from the osiaa19-1 and osiaa19-2 mutants. Figure 3 Therefore, it does not contain any traces of genetic modification.

[0044] Specifically, it includes:

[0045] Construction of engineered bacteria: The pC1300-OsIAA19-Cas9 vector was transformed into Agrobacterium strain EHA105 by heat shock. Agrobacterium containing the pC1300-OsIAA19-Cas9 vector was obtained by screening with kanamycin, ampicillin and rifampin.

[0046] Transformation of rice callus with pC1300-OsIAA19-Cas9 vector to obtain rice regenerated seedlings: Rice callus tissue was infected with EHA105 containing pC1300-OsIAA19-Cas9 vector and cultured in a 28℃ incubator for 3 days. After washing away Agrobacterium with liquid culture medium, the rice callus was placed on a selection medium containing suitable antibiotics for culture. After two rounds of culture, resistant callus was obtained. The resistant callus was transferred to differentiation medium for differentiation culture to obtain seedlings. The differentiated seedlings were transferred to rooting medium for culture and transplanted after hardening.

[0047] Molecular detection of rice OsIAA19 gene knockout plants: Forward and reverse primers were designed before and after the target site of the OsIAA19 gene DNA sequence. Amplification was performed using these primers, and sequencing was conducted to identify whether the gene knockout material was successfully created. Simultaneously, the Cas9 gene sequence was analyzed to screen for stable mutant plants lacking the Cas9 gene.

[0048] The detection primer sequences are as follows:

[0049] sequence name sequence Serial Number OsIAA19-F 5-GCACGCATGCTTACTTGGC-3 SEQ ID NO.5 OsIAA19-R 5-CTCTCTCCTCCCCTTCTCCAA-3 SEQ ID NO.6 Cas9-1F 5-CACCATCTACCACCTGAGAA-3 SEQ ID NO.7 Cas9-1R 5-CGAAGTTGCTCTTGAAGTTG-3 SEQ ID NO.8

[0050] The PCR product of OsIAA19-F / R is 497bp, and the PCR product of Cas9-1F / R is 600bp.

[0051] Example 1: OsIAA19 Spatiotemporal Representation Mode

[0052] Roots, stems, leaves, sheaths, young panicles, and seeds at the post-flowering stage of wild-type rice plants were sampled. The samples were lysed in liquid nitrogen, and RNA was extracted. After reverse transcription, the OsIAA19 gene was analyzed for tissue expression patterns using designed qRT-PCR primers. The results showed that OsIAA19 is a constitutively expressed gene, and its expression level gradually increased with seed development. Figure 4 The relevant qRT-PCR primer sequences are as follows:

[0053] sequence name sequence Serial Number OsIAA19-QRT-F 5-AGGTTAGCATGGATGGAGCC-3 SEQ ID NO.9 OsIAA19-QRT-R 5-TCCCAAGGAAGATCACCAACA-3 SEQ ID NO.10

[0054] Example 2: Subcellular localization results of OsIAA19 protein

[0055] To understand the specific site of OsIAA19 protein expression in cells, we employed a strategy of expressing the OsIAA19-GFP fusion protein. First, we constructed the pC2300-35s-eGFP vector, then transformed it into tobacco leaves. Finally, we observed the green fluorescence signal under a laser confocal microscope after infecting the tobacco leaves. The results showed that OsIAA19 was specifically expressed in the cell nucleus. Figure 5 The primer sequences for constructing the relevant vector are as follows:

[0056] sequence name sequence Serial Number OsIAA19-eGFP-F 5-ATTTGGAGAGGACAGGGTACCATGCCGCCGCCGCTCGAA-3 SEQ ID NO.11 OsIAA19-eGFP-R 5-CACCATGGTACTAGTGTCGACTTTGTTCTGACCTGTCTGTTCCAG-3 SEQ ID NO.12

[0057] Example 3: Effects of OsIAA19 knockout on rice plant architecture

[0058] To investigate whether OsIAA19 affects rice plant architecture, we studied the agronomic traits of the osiaa19 mutant and the wild-type control ZH11. The investigated agronomic traits included plant height, number of tillers, flag leaf length, and width. The results showed that the osiaa19 mutant exhibited no significant changes in plant height, tiller number, leaf length, and leaf width. Figure 6 , Figure 7 This indicates that regulating the OsIAA19 gene does not affect the rice plant architecture.

[0059] Example 4: Effect of OsIAA19 knockout on rice grain shape

[0060] To understand whether OsIAA19 affects rice grain shape, we measured the grain length and width of the osiaa19 mutant and the wild-type control ZH11. We initially screened harvested mature grains, removing those with abnormal development. Then, we used a rice appearance quality analyzer (SC-E, Wanshen, China) to scan and analyze the grain length and width, and photographed and archived typical grains. Figure 8 The results showed that the osiaa19 mutant had increased grain length, while the grain width did not change significantly. Figure 9).

[0061] Example 5: Effect of OsIAA19 knockout on the thousand-grain weight of rice

[0062] To understand the changes in thousand-grain weight of the osiaa19 mutant, we selected 1000 mature grains each from the osiaa19 mutant and the wild-type control ZH11, weighed them, and repeated the process at least three times. The results showed that the thousand-grain weight of the osiaa19 mutant was significantly increased. Figure 10 Therefore, the OsIAA19 gene is of great value for increasing rice yield.

[0063] Example 6: Effects of OsIAA19 knockout on the physicochemical properties related to the cooking and eating quality of rice

[0064] The physicochemical properties of the OsIAA19 gene knockout material were measured, and the results showed that knocking out OsIAA19 had no significant effect on the amylose content (AC) and gel consistency (GC) of rice. Figure 11 The results indicate that knocking out OsIAA19 does not affect the cooking and eating quality of rice.

[0065] In summary, the rice OsIAA19 gene in this invention is a constitutively expressed gene. The expression level of the OsIAA19 gene is basically consistent in young panicle tissues of different lengths. However, during seed development, the expression level of the OsIAA19 gene gradually increases. Knocking out the OsIAA19 gene can increase rice grain length and thousand-grain weight, while basic agronomic traits such as plant height, tillering, flag leaf length, and flag leaf width, as well as cooking and eating quality, remain unchanged. Therefore, the OsIAA19 gene can specifically regulate rice grain length and thousand-grain weight, and knocking out the rice OsIAA19 gene demonstrates good breeding application value to a certain extent, which is of great significance for rice production.

[0066] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. Application of the rice OsIAA19 gene in improving rice grain shape and increasing yield, wherein the coding region sequence of the OsIAA19 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the OsIAA19 gene is shown in SEQ ID NO.2; The application involves knocking out the rice OsIAA19 gene, thereby disrupting its biological function and obtaining rice lines that increase grain length and yield.

2. The application of the rice OsIAA19 gene according to claim 1 in improving rice grain shape and increasing yield, characterized in that, The application involves using the CRISPR / Cas9 system to knock out the OsIAA19 gene in rice, resulting in rice lines with improved grain shape and increased yield.

3. The application of the rice OsIAA19 gene according to claim 2 in improving rice grain shape and increasing yield, characterized in that, The knockout of the rice OsIAA19 gene does not affect the rice plant architecture.

4. The application of the rice OsIAA19 gene according to claim 2 in improving rice grain shape and increasing yield, characterized in that, The knockout of the OsIAA19 gene in rice does not affect the cooking and eating quality of the rice.

5. The application of the rice OsIAA19 gene according to claim 2 in improving rice grain shape and increasing yield, characterized in that, The method for knocking out the rice OsIAA19 gene using the CRISPR / Cas9 system includes: OsIAA19 gene primer design and denaturation annealing; The digested SK-gRNA vector and primer annealing products were ligated using homologous recombinase to construct the intermediate vector SK-gRNA-OsIAA19 containing the target OsIAA19 gene fragment. The digested intermediate vector SK-gRNA-OsIAA19 was ligated with the digested pC1300-Cas9 vector using homologous recombinase to construct the final vector pC1300-OsIAA19-Cas9, which was then screened and sequenced for verification. The final vector pC1300-OsIAA19-Cas9 was transformed into Agrobacterium, and then Agrobacterium-mediated rice genetic transformation was used to transfer the Agrobacterium containing the pC1300-OsIAA19-Cas9 vector into rice callus tissue. After resistance screening and tissue culture, rice plants were obtained.

6. The application of the rice OsIAA19 gene according to claim 5 in improving rice grain shape and increasing yield, characterized in that, The sequence of the OsIAA19 gene primer OsIAA19-cas9-F is shown in SEQ ID NO.3, and the sequence of the OsIAA19 gene primer OsIAA19-cas9-R is shown in SEQ ID NO.4.

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