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

Abstract

The invention discloses application of a rice OsIAA19 gene in improving rice grain shape and increasing yield, and relates to the technical field of plant genetic engineering. According to the application, the rice OsIAA19 gene is edited, interfered or knocked out, so that the biological function of the rice OsIAA19 gene is reduced or damaged, and a rice strain capable of improving the rice grain shape and increasing the yield is obtained. The OsIAA19 gene of the rice is used as a marker, and it is proved that knockout of the OsIAA19 gene in the rice can significantly increase the grain length of the rice, so that the thousand grain weight of the rice is increased, other main agronomic traits are not affected, and the cooking taste quality of the rice is also not affected, so that the OsIAA19 gene has good breeding application potential and has important significance on high-yield breeding of the rice.

Description

Technical Field

[0001] The present application relates to the technical field of plant genetic engineering, and in particular to the application of rice OsIAA19 gene in improving rice grain shape and increasing yield. Background Art

[0002] Plant hormones, also known as plant growth substances or plant regulators, are trace organic substances produced by plants, which play a key regulatory role in plant growth and development and adaptation to environmental changes. The plant hormone family mainly includes: auxin, gibberellin, cytokinin, ethylene, abscisic acid, jasmonic acid, brassinolide, salicylic acid. As the first plant hormone discovered, auxin is essential for plant growth and development, such as vascular tissue formation, apical dominance, root formation, tropism, cell division, differentiation, and the development of flowers and fruits. Therefore, auxin is extremely important and deserves the attention of scientists.

[0003] Elucidating the biosynthesis and signal transduction mechanisms of auxin is of great significance for better understanding plant growth and development, response to environmental changes, and application in crop breeding. The synthesis of auxin involves two complex pathways, namely, tryptophan-dependent and tryptophan-independent pathways. Compared with the synthesis pathway, great progress has also 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 well characterized auxin signal transduction pathway. This pathway mainly includes three core transduction components, namely, auxin receptors TIR1 / AFBs, transcriptional repressors Aux / IAA, and transcription factors ARFs. In short, in a low-concentration auxin environment, the repressor protein Aux / IAA binds to the active transcription factor ARF to form a heterodimer, inhibiting the transcriptional activation activity of ARF. Conversely, in a high-concentration auxin environment, auxin binds to the TIR1 / AFB receptor and degrades the Aux / IAA protein through ubiquitination, thereby releasing the ARF transcription factor and activating the transcription of auxin-responsive genes.

[0004] The Aux / IAA protein family is a plant-specific transcriptional repressor that is mainly involved in regulating the formation of plant roots and leaves, phototropism, gravitropism, and apical dominance of shoots. Rice is a monocotyledonous model plant and one of the most important food crops in the world. More than half of the world's population relies on rice as their staple food. High quality and high yield are the main goals of rice breeding. Given the key role of auxin in regulating plant growth and development, it is crucial to conduct functional research on rice Aux / IAA family genes.

[0005] Grain shape, as a key agronomic trait affecting rice yield and quality, has received extensive attention from rice geneticists and breeders. To date, many grain shape genes have been cloned in rice, involving multiple regulatory mechanisms, including the G protein pathway, ubiquitin-proteasome pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone pathway. In the plant hormone family, BR is considered to be an important positive regulator of rice grain shape, and many BR biosynthesis or signal transduction components are involved in controlling grain shape. However, there are still few studies on the involvement of auxin in the regulation of rice grain shape. 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] To this end, the present invention provides an application of a 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 localized in the cell nucleus.

[0009] Furthermore, the application is to obtain a rice strain with improved rice grain shape and increased yield by editing, interfering with or knocking out the rice OsIAA19 gene, thereby reducing or destroying the biological function of the rice OsIAA19 gene.

[0010] Furthermore, the application is to use the CRISPR / Cas9 system to knock out the rice OsIAA19 gene to obtain a rice strain with improved rice grain shape and increased yield.

[0011] Furthermore, knocking out the rice OsIAA19 gene does not affect the rice plant type.

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

[0013] Furthermore, the method of knocking out the rice OsIAA19 gene by using the CRISPR / Cas9 system comprises:

[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 the primer annealing product were connected using homologous recombinase to construct an intermediate vector SK-gRNA-OsIAA19 containing the target OsIAA19 gene fragment;

[0016] The intermediate vector SK-gRNA-OsIAA19 after restriction digestion was connected with the pC1300-Cas9 vector after restriction digestion using homologous recombinase to construct the final vector pC1300-OsIAA19-Cas9, which was screened and sequenced for verification;

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

[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 has, to a certain extent, demonstrated good breeding application value and is of great significance to rice production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present application.

[0020] Figure 1 The embodiments of the present invention provide CRISPR / Cas9-mediated OsIAA19 gene editing sites and mutation types.

[0021] Figure 2 The sequencing results of the osiaa19 mutation sequence provided in the embodiments of the present invention.

[0022] Figure 3 The Cas9 gene detection results provided by the embodiments of the present invention.

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

[0024] Figure 5 The subcellular localization of the OsIAA19 protein provided in the embodiments of the present invention.

[0025] Figure 6 The phenotype of rice plants after knocking out the OsIAA19 gene provided in an embodiment of the present invention.

[0026] Figure 7 The embodiment of the present invention provides a statistical bar chart of phenotypic data of rice plants after knocking out the OsIAA19 gene, and the main indicators include plant height, tillering, flag leaf length and flag leaf width.

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

[0028] Fig. 9 The statistical histogram of rice grain shape data after knocking out the OsIAA19 gene provided in the embodiment of the present invention has the main indicators of grain length and grain width.

[0029] Fig.10 The embodiment of the present invention provides a statistical bar graph of the thousand-grain weight data of rice grains after knocking out the OsIAA19 gene.

[0030] Fig.11 A bar graph showing the determination of rice quality traits after knocking out the OsIAA19 gene provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] In order to better understand the above-mentioned technical scheme, the technical scheme of the embodiments of the present application is described in detail below through the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical scheme of the embodiments of the present application, rather than limitations on the technical scheme of the present application. In the absence of conflict, the embodiments of the present application and the technical features in the embodiments may be combined with each other.

[0032] The experimental methods in the following examples without specifying specific conditions were all carried out according to conventional procedures, and the materials and materials used were all commercially available products.

[0033] The present invention provides a detailed introduction to the application of rice OsIAA19 gene in improving rice grain shape and increasing yield, as follows:

[0034] (I) Rice OsIAA19 gene

[0035] The OsIAA19 gene is located on rice chromosome 5, with the gene number Os05g0559400 (NCBI number), LOC_Os05g48590 (MSU number). The CDS of the coding region of the OsIAA19 gene is 846 bp in length, 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 OsIAA19 gene using CRISPR / Cas9 system

[0037] To knock out the rice OsIAA19 gene, we constructed an intermediate vector SK-gRNA-OsIAA19 and a final vector pC1300-OsIAA19-Cas9. After the designed OsIAA19 primers were denatured and annealed, the vector cut with enzymes and the primer annealing products were connected with homologous recombinase to construct the intermediate vector, and then the successfully constructed intermediate vector containing the target OsIAA19 fragment was constructed into 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 of the OsIAA19 gene are as follows:

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

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

[0041] Among them, the primers designed for OsIAA19 primer annealing are as follows:

[0042] sequence name sequence Sequence 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 the Agrobacterium-mediated rice genetic transformation method was used to transform the Agrobacterium containing the pC1300-OsIAA19-Cas9 vector into rice callus tissue, and rice plants were obtained after resistance screening and tissue culture. The test results are shown in Figure 1 and Figure 2, it can be seen that the coding region of the OsIAA19 gene in the gene-edited rice osiaa19-1 and osiaa19-2 was missing 2 and 8 bases respectively, causing frameshift mutations in the target gene. In addition, the molecular identification results of the Cas9 gene showed that the Cas9 gene in the osiaa19-1 and osiaa19-2 mutants had been successfully separated ( Figure 3 ), and therefore does not contain traces of genetically modified organisms.

[0044] Specifically include:

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

[0046] pC1300-OsIAA19-Cas9 vector was used to transform rice callus and obtain rice regenerated seedlings: rice callus tissue was infected with EHA105 containing pC1300-OsIAA19-Cas9 vector and co-cultured in a culture room at 28°C for 3 days. After washing away Agrobacterium with liquid culture medium, the rice callus was placed on a screening culture medium containing appropriate antibiotics for culture. After two rounds of culture, resistant callus was obtained, and the resistant callus was transferred to a differentiation culture medium for differentiation culture to obtain seedlings. The differentiated seedlings were transferred to a rooting culture medium for culture and transplanted after hardening.

[0047] Molecular detection of rice OsIAA19 gene knockout plants: forward and reverse primers were designed based on the target site of the OsIAA19 gene DNA sequence, and these primers were used for amplification and sequencing to identify whether the gene knockout material was successfully created. At the same time, the Cas9 gene sequence was tested to screen for stable mutant plants without the Cas9 gene.

[0048] The detection primer sequences are as follows:

[0049] sequence name sequence Sequence 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 was 497 bp, and the PCR product of Cas9-1F / R was 600 bp.

[0051] Example 1 Spatiotemporal expression pattern of OsIAA19

[0052] The roots, stems, leaves, sheaths, panicles and seeds of wild-type rice plants were sampled and crushed in liquid nitrogen for RNA extraction. After RNA reverse transcription, the tissue expression pattern of the gene was analyzed using the designed OsIAA19 gene qRT-PCR primers. The results showed that OsIAA19 is a constitutively expressed gene, and its expression level gradually increases with the progress of seed development ( Figure 4 ). The relevant qRT-PCR primer sequences are as follows:

[0053] sequence name sequence Sequence 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] In order to understand the specific location of OsIAA19 protein expression in cells, we adopted the strategy of expressing OsIAA19-GFP fusion protein. First, we constructed the pC2300-35s-eGFP vector, and then transferred the constructed expression vector into tobacco leaves. Finally, we placed the infected tobacco leaves under a laser confocal microscope to observe the green fluorescence signal. The results showed that OsIAA19 was specifically expressed in the cell nucleus ( Figure 5 ). The relevant vector construction primer sequences are as follows:

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

[0057] Example 3 Effect of knocking out OsIAA19 on rice plant morphology

[0058] To understand whether OsIAA19 affects rice plant type, we studied the agronomic traits of the osiaa19 mutant and the wild-type control ZH11. The agronomic traits investigated included plant height, tiller number, and sword leaf length and width. The results showed that there were no significant changes in plant height, tiller number, leaf length, and leaf width of the osiaa19 mutant ( Figure 6 , Figure 7 ), indicating that regulating the OsIAA19 gene did not affect rice plant type.

[0059] Example 4 Effect of knocking out OsIAA19 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 screened the harvested mature grains, removed the grains with abnormal development, and then used a rice appearance quality detector (SC-E, Wanshen, China) to scan and analyze the length and width of rice grains, and selected typical grains to take photos and keep ( Figure 8 The results showed that the grain length of the osiaa19 mutant increased, while the grain width did not change significantly ( Fig. 9).

[0061] Example 5 Effect of knocking out OsIAA19 on rice thousand-grain weight

[0062] To understand the changes in thousand-grain weight of the osiaa19 mutant, we selected 1,000 mature grains of the osiaa19 mutant and the wild-type control ZH11, weighed them, and repeated at least three times. The results showed that the thousand-grain weight of the osiaa19 mutant increased significantly ( Fig.10 ). Therefore, the OsIAA19 gene is of great value for increasing rice yield.

[0063] Example 6 Effect of knocking out OsIAA19 on the physicochemical properties related to rice cooking and eating quality

[0064] The results of the determination of physical and chemical indices related to cooking and eating quality of OsIAA19 knockout materials showed that knockout of OsIAA19 had no significant effect on the amylose content (AC) and gel consistency (GC) of rice ( Fig.11 ), indicating that knocking out OsIAA19 did not affect the cooking and eating quality of rice.

[0065] In summary, the rice OsIAA19 gene in the present invention is a constitutively expressed gene. In panicle tissues of different lengths, the expression level of the OsIAA19 gene is basically the same. During the seed development stage, the expression level of the OsIAA19 gene gradually increases. Knocking out the OsIAA19 gene can increase the length of rice grains and improve the thousand-grain weight, while the basic agronomic traits such as plant height, tillering, sword leaf length, sword leaf width and the cooking and eating quality remain unchanged. Therefore, the OsIAA19 gene can specifically regulate the length and thousand-grain weight of rice grains. Knocking out the rice OsIAA19 gene reflects good breeding application value to a certain extent, which is of great significance to rice production and application.

[0066] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed. The above are only preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application. The above are only preferred implementation methods of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present application, and these improvements and variations should also be regarded as the protection scope of the present application.

Claims

1. Application of rice OsIAA19 gene in improving rice grain shape and increasing rice 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.

2. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 1, characterized in that: The rice OsIAA19 gene is a constitutively expressed gene, and the expression level in seeds increases as the seeds develop.

3. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 1, characterized in that: The protein encoded by the rice OsIAA19 gene is located in the cell nucleus.

4. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 1, characterized in that: The application is to obtain a rice strain with improved rice grain shape and increased yield by editing, interfering with or knocking out the rice OsIAA19 gene to reduce or destroy the biological function of the rice OsIAA19 gene.

5. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 4, characterized in that: The application is to use the CRISPR / Cas9 system to knock out the rice OsIAA19 gene, thereby obtaining a rice strain with improved rice grain shape and increased yield.

6. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 5, characterized in that: The knockout of the rice OsIAA19 gene does not affect the rice plant type.

7. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 5, characterized in that: After the rice OsIAA19 gene is knocked out, the cooking and eating quality of the rice is not affected.

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

9. The use of the rice OsIAA19 gene in improving rice grain shape and increasing yield according to claim 8, 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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