A rice OsEns150 gene and its application in improving rice plant type, quality and enhancing resistance to pre-harvest sprouting

Abstract

The present invention belongs to the technical field of plant genetic engineering, and discloses a rice OsEns150 gene and its application in improving rice plant type, quality and enhancing pre-harvest sprouting resistance; the rice OsEns150 gene is specifically expressed in seeds, and knocking out this gene can reduce the plant height of rice to a certain extent, but has no effect on grain traits such as rice grain shape, 1000-grain weight and seed setting rate; knocking out the OsEns150 gene can significantly improve the cooking and eating quality and nutritional quality of rice, including reducing the amylose content and gelatinization temperature of rice, increasing the gel consistency and protein content of rice, and improving its viscosity, etc.; in addition, compared with the wild-type rice control, knocking out the OsEns150 gene slightly delays the germination speed of rice, but has no effect on the germination rate, but can significantly enhance the pre-harvest sprouting resistance of rice. The OsEns150 gene has good application prospects in the practice of cultivating new rice varieties with high quality, high yield and multiple resistances.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice OsEns150 gene and its application in improving rice plant type, quality and enhancing pre-harvest sprouting resistance. Background Art

[0002] Rice has a long cultivation history. It is not only the staple food of the Chinese people but also one of the three major staple foods in the world, so it is often called the "world food". With the growth of the population, the reduction of arable land area and the frequent occurrence of extreme weather, high and stable yield of rice is crucial for ensuring national food security. In addition, with the continuous improvement of people's living standards, the demand for high-quality rice is also increasing. Therefore, breeding new rice varieties with high quality, high yield and multiple resistances has become the most important goal of rice breeding.

[0003] Rice seeds are not only the direct food for humans but also directly affect food production. Good dormancy and germination characteristics of rice seeds are the key to ensuring high quality and high yield of rice. Whether the seeds can germinate smoothly and sprout quickly after sowing, whether the root system is sound after sprouting, and whether they can quickly meet the standards of early, strong and full seedlings will all affect the growth status of rice and the final harvest. In addition, good dormancy characteristics of rice seeds are also very important. If the dormancy is too strong, the seeds are not easy to germinate, while if the dormancy is too shallow or is released inappropriately, pre-harvest sprouting is likely to occur under high temperature and high humidity weather before harvest. Pre-harvest sprouting will cause significant losses in rice yield and serious decline in quality. In addition, pre-harvest sprouting seeds basically lose their seed value, and at the same time, because multiple metabolic pathways inside them have been activated, the seed vigor, nutrition, processing quality and storage characteristics also decrease significantly. Therefore, improving the germination and dormancy characteristics of rice seeds can lay an important foundation for high-quality and stable yield of rice.

[0004] Recently, rice quality has gradually become a hot topic of concern to consumers. The indicators for evaluating the quality of rice mainly include amylose content (AC), gel consistency (GC), gelatinization temperature (GT) and protein content (PC), etc. Generally speaking, the variety with a lower amylose content in rice has a higher eating quality value. Gel consistency is an important factor affecting the cooking quality of rice, the hardness and softness of cooked rice and the taste. And the protein content in rice is the key factor affecting the nutritional quality of rice. The higher the protein content, the higher its nutritional value. However, at the same time, there is a significant negative correlation between the protein content in rice and the eating quality. Varieties with too high protein content often have hard cooked rice, poor taste and bad eating quality. Therefore, balancing and coordinating the eating quality and nutritional quality of rice is the key to improving the comprehensive quality of rice.

[0005] Therefore, cloning important genes that can be used to improve traits such as rice germination, dormancy and rice quality is crucial for cultivating excellent new rice varieties with high quality, high yield and multiple resistances by using modern breeding methods such as molecular design breeding. SUMMARY OF THE INVENTION

[0006] The present invention addresses the problems existing in the above-mentioned prior art and provides a rice OsEns150 gene and its application in improving rice plant type, quality, and enhancing resistance to pre-harvest sprouting.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a rice OsEns150 gene. The OsEns150 gene is located on chromosome 12 of rice, and the gene number is Os12g0464400 (NCBI number), LOC_Os12g27830 (MSU number). The full length of the coding region CDS of the OsEns150 gene is 1050 bp, and the gene structure includes 6 exons and 5 introns. The coding region sequence of the OsEns150 gene is shown in SEQ ID NO.1:

[0009] ATGGTGGATTTGGTGAACGGCGTGCTCAACTGGGTGGCGACGCCGGCCATGGTGG

[0010] CCAGCCTGCTGCTCTTCTACCCGCCCTACTACCTCTTCAAGACCGTCCACTCCTTCC

[0011] TCTCCTACCTCTTCCCCGACGACCTCGCCCGCAAGGTCGTCCTCATCACCGGCGCC

[0012] TCCTCCGGCATCGGCGAGCAATTAGCATACAACTATGCTCTGAACCGGGCATCATT

[0013] GGTCCTTGTTGCAAGAAGAGAATGGAGCCTGCGTAAAGTTGCCGATCAAGCGTTC

[0014] GAGCTTGGAGCACCTGATGTGATCATTCTTCCGGGCGACGTTGCGAATCCTGAAGA

[0015] CTGCAAAAGATTTGTTCAGACCGCAATCGATCACTACGGGCGATTGGACCATCTTG

[0016] TGTGCAACGCTGGCATCGCAAGTGTTGGCGCGTTTCAGGAGATTCCAGATGTTACT

[0017] AACTACAGCTCTCAATTTGATGTGAACTTCTGGGGTTCAGTTCAGTCAACTTTTGA

[0018] AGCTCTCCCTCATCTGAAAAGGAGCCGAGGAAGAATCGTTGTTACTGCGTCGGCA

[0019] ACCGGATGGAATCCTGTTCCAAGAATGACCTTCTACAATGCTGCCAATGCTGCACT

[0020] GATAAACTTCTACGAGACGCTGCGGACAGAGCTTGGTAGCCAAGTTGGAATCACA

[0021] ATTGTAACACCTGGGTGGATCGAGTCTGAGATGTCAAAAGGGAAATTTCTCAAGG

[0022] ATCATGGTGAAATGGAGGTCGATCAAGAAATGCGAGATGCTCAAATTGGTTTATTT

[0023] CCCGTGGAGTACGCGAAGAATTGCGCAAAAGCCATGGTACAAGCGGTTCGGCAAG

[0024] GGAAGCGTTGTCTCACCGTGCCACCATGGTTCAGCACAATGTACCTGTGGAGGGTA

[0025] TTCGCACCGGAGGTCGTCGAGTTCTGCTACCGCCTCCTGTACATGCACCGCCATGG

[0026] TGGTAGCCAAGCTGATGCGCCGAGCAAGAAGATGGCTGAGGCTGGTGGAAAGAAGCTCTTGTATCCAACGTCGCTGCGCTCTGATGACATCAAGGATGAGTGA;

[0027] The OsEns150 gene encodes 349 amino acids, and the amino acid sequence encoded by the OsEns150 is shown in SEQ ID NO.2:

[0028] MVDLVNGVLNWVATPAMVASLLLFYPPYYLFKTVHSFLSYLFPDDLARKVVLITGAS

[0029] SGIGEQLAYNYALNRASLVLVARREWSLRKVADQAFELGAPDVIILPGDVANPEDCKR

[0030] FVQTAIDHYGRLDHLVCNAGIASVGAFQEIPDVTNYSSQFDVNFWGSVQSTFEALPHL

[0031] KRSRGRIVVTASATGWNPVPRMTFYNAANAALINFYETLRTELGSQVGITIVTPGWIE

[0032] SEMSKGKFLKDHGEMEVDQEMRDAQIGLFPVEYAKNCAKAMVQAVRQGKRCLTVP

[0033] PWFSTMYLWRVFAPEVVEFCYRLLYMHRHGGSQADAPSKKMAEAGGKKLLYPTSLRSDDIKDE。

[0034] In a second aspect, the present invention provides the recombinant vector pC1300-OsEns150-Cas9 of the rice OsEns150 gene. The pC1300-OsEns150-Cas9 contains the OsEns150 gene, the vector system is CRISPR / Cas9, and the system contains the intermediate vector SK-gRNA and the final vector pC1300-Cas9. The knockout of the OsEns150 gene is achieved by using the CRISPR / Cas9 gene editing system.

[0035] In a third aspect, the present invention provides a method for preparing the recombinant vector pC1300-OsEns150-Cas9. The method is as follows: After digesting the intermediate vector SK-gRNA with the restriction endonuclease Aar I, it is ligated with the complementary primers of the target gene after denaturation and annealing using T4 ligase to obtain the intermediate vector SK-gRNA-OsEns150; The correctly identified SK-gRNA-OsEns150 intermediate vector by sequencing is double-digested with Kpn I and Bgl II and ligated with the final vector pC1300-Cas9 double-digested with Kpn I and BamH I.

[0036] Furthermore, the specific target site sequence for editing the OsEns150 gene is 5-GAACGGCGTGCTCAACTGGG-3, and the complementary primer sequences of the target gene are as follows:

[0037] Sequence Name Sequence Sequence Number OsEns150-cas9-F 5-GGCAGAACGGCGTGCTCAACTGGG-3 SEQ ID NO.3 OsEns150-cas9-R 5-AAACCCCAGTTGAGCACGCCGTTC-3 SEQ ID NO.4 。

[0038] Fourthly, the present invention provides the application of the rice OsEns150 gene in improving rice plant type, cooking and eating quality and nutritional quality of rice, and enhancing the resistance of rice to pre-harvest sprouting.

[0039] The method of the above application is as follows: knockout the rice OsEns150 gene to improve rice plant type, cooking and eating quality and nutritional quality of rice, and enhance the resistance of rice to pre-harvest sprouting, so that the gene expression level of OsEns150 in the target rice is changed, and then rice plants with different phenotypes are obtained.

[0040] The present invention has the following beneficial effects: (1) The rice OsEns150 gene in the present invention is specifically expressed in rice seeds, and its protein is localized in the cytoplasm and nucleus; by specifically editing the rice OsEns150 gene to create ens150 mutant rice, after knocking out the OsEns150 gene, the plant height of rice can be reduced to a certain extent, which is beneficial to the lodging resistance of plants, but has no effect on grain traits such as rice grain shape, 1000-grain weight and seed setting rate; in addition, the amylose content and gelatinization temperature of rice are significantly reduced, and the gel consistency is significantly increased, significantly improving the cooking and eating quality of rice.

[0041] (2) Knocking out the OsEns150 gene in the present invention can significantly improve the cooking and eating quality and nutritional quality of rice, including reducing the amylose content and gelatinization temperature of rice, increasing the gel consistency and protein content of rice, and improving its viscosity, etc.; after knocking out the OsEns150 gene, the protein content of rice is significantly increased, enhancing the nutritional quality of rice to a certain extent.

[0042] (3) Compared with the wild-type rice control in the present invention, knocking out the OsEns150 gene slightly delays the germination of rice seeds without affecting the final germination rate, but can significantly enhance the resistance of rice to pre-harvest sprouting, thereby ensuring the high-quality and stable yield of rice, and having good breeding application value; the OsEns150 gene has good application prospects in the practice of cultivating new rice varieties with high quality, high yield and multiple resistances. Description of the Drawings

[0043] Figure 1 is the spatio-temporal expression pattern of the OsEns150 gene.

[0044] Figure 2 is the subcellular localization result of the OsEns150 protein.

[0045] Figure 3 It is a comparison of the plant heights of wild-type Zhonghua 11 and the ens150 mutant rice.

[0046] Figure 4 It is a comparison of the grain traits of wild-type Zhonghua 11 and the ens150 mutant rice, including grain length, grain width, grain thickness, 1000-grain weight, and seed setting rate.

[0047] Figure 5 It is a comparison of the physicochemical properties of the cooking and eating quality of wild-type Zhonghua 11 and the ens150 mutant rice, including AAC (apparent amylose content), GC (gel consistency), DSC (thermodynamic properties of rice flour), and RVA (Rapid Visco-Analyzer, rice viscosity).

[0048] Figure 6 It is a comparison of the protein content of wild-type Zhonghua 11 and the ens150 mutant rice.

[0049] Figure 7 It is a comparison of the seed germination characteristics of wild-type Zhonghua 11 and the ens150 mutant rice.

[0050] Figure 8 It is a comparison of the pre-harvest sprouting resistance of wild-type Zhonghua 11 and the ens150 mutant rice. Specific implementation mode

[0051] To understand the present invention, the following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0052] In the following examples, the experimental methods without specific conditions are all carried out according to conventional steps, and the materials and reagents used are all commercially available products.

[0053] Example 1

[0054] Temporal and spatial expression pattern of OsEns150

[0055] Samples were taken from the roots, stems, leaves, leaf sheaths of wild-type rice plants and developing seeds at different stages. After the samples were placed in liquid nitrogen and crushed into powder, total RNA was extracted. After reverse transcription, the temporal and spatial specific expression analysis of the OsEns150 gene was carried out using the designed OsEns150 gene-specific qRT-PCR primers. The results showed that OsEns150 is a gene specifically expressed in rice seeds, and the expression level increases continuously with the progress of seed development ( Figure 1 ). The qRT-PCR primer sequences of the target gene OsEns150 and the internal reference gene Actin1 are as follows:

[0056] Sequence Name Sequence Sequence Number OsEns150-qRT-F 5-AAGAAGAGAATGGAGCCTGCGTAAA-3 SEQ ID NO.7 OsEns150-qRT-R 5-GTAACATCTGGAATCTCCTGAAACG-3 SEQ ID NO.8 Actin1-qRT-F 5-CCAAGGCCAATCGTGAGAAGA-3 SEQ ID NO.9 Actin1-qRT-R 5-AATCAGTGAGATCACGCCCAG-3 SEQ ID NO.10 .

[0057] Example 2

[0058] Subcellular localization results of OsEns150

[0059] To understand the specific location of the expression product of the OsEns150 gene in cells, we fused the OsEns150 gene with the GFP-tagged gene, constructed the OsEns150-GFP expression vector and transferred it into Agrobacterium tumefaciens, and then used the Agrobacterium-mediated transformation method to transfer it into tobacco leaves. After 48 hours, the transformed leaf tissues were selected and placed under a laser confocal microscope to observe the fluorescence signal. The results showed that the OsEns150 protein was expressed in both the nucleus and cytoplasm( Figure 2 ). The primer sequences for the relevant vector construction are as follows:

[0060] Sequence Name Sequence Sequence Number OsEns150-eGFP-F 5-ATTGGAGAGGACAGGGTACCATGGTGGATTTGGTGAACGG-3 SEQ ID NO.11 OsEns150-eGFP-R 5-CACCATGGTACTAGTGTCGACCTCATCCTTGATGTCATCAGAGCG-3 SEQ ID NO.12

[0061] Example 3

[0062] Construction of the OsEns150 gene recombinant vector pC1300-OsEns150-Cas9

[0063] First, a specific sequence containing NGG was screened out in the exon region of OsEns150 as the editing target, and a pair of complementary primers was designed. GGCA was added before the forward sequence, and AAAC was added before the reverse complementary sequence. After the intermediate vector SK-gRNA was digested with the restriction endonuclease Aar I, it was ligated with the complementary primers of the target gene after denaturation and annealing using T4 ligase, and then transformed into Escherichia coli. After colony PCR identification and sequencing verification, it was digested with the restriction endonucleases Kpn I and Bgl II and ligated with the final vector pC1300-Cas9 digested with Kpn I and BamH I. The complementary primer sequences of the OsEns150 target site are as follows:

[0064] Sequence Name Sequence Sequence Number OsEns150-cas9-F 5-GGCAGAACGGCGTGCTCAACTGGG-3 SEQ ID NO.3 OsEns150-cas9-R 5-AAACCCCAGTTGAGCACGCCGTTC-3 SEQ ID NO.4 .

[0065] Example 4

[0066] Application of the rice OsEns150 gene in improving rice plant type, rice cooking and eating quality, and nutritional quality, and enhancing rice pre-harvest sprouting resistance

[0067] (1) Construction of engineering bacteria: The pC1300-OsEns150-Cas9 vector was transformed into the Agrobacterium tumefaciens strain EHA105 by heat shock method, and the Agrobacterium tumefaciens containing the pC1300-OsEns150-Cas9 vector was obtained through kanamycin screening;

[0068] (2) Transformation of rice calli with the pC1300 - OsEns150 - Cas9 vector and obtaining regenerated rice seedlings: Infect rice calli with Agrobacterium tumefaciens EHA105 containing the pC1300 - OsEns150 - Cas9 vector, and co - culture them in an incubator at 28 °C for 3 days. After washing off the Agrobacterium with liquid medium, place the rice calli on a selection medium containing appropriate antibiotics for culture. After two rounds of culture and screening, resistant calli can be obtained. Transfer the resistant calli to a differentiation medium for differentiation culture to obtain small seedlings, and transfer the differentiated small seedlings to a rooting medium for rooting culture. Finally, after hardening off, transplant them to the field.

[0069] Molecular identification of rice plants with edited OsEns150 gene: Design a pair of specific identification primers before and after the editing target site of the OsEns150 gene. Use this pair of primers to amplify the target gene fragment containing the editing target site, and then perform sequencing identification to confirm whether there is a change in the number of bases in the target gene, and screen transgenic rice with frameshift mutations (i.e., changes in the number of bases that are not multiples of 3) for subsequent phenotype and function analysis.

[0070] The sequences of the detection primers are as follows:

[0071] Sequence Name Sequence Sequence Number Ens150-casJ-F 5-GTGGAGCAAGCCAACCAAAG-3 SEQ ID NO.5 Ens150-casJ-R 5-GATACACAGAGCAGAGCCCG-3 SEQ ID NO.6

[0072] The PCR product is 202 bp.

[0073] Example 5

[0074] Investigation of the plant type of rice plants after knocking out OsEns150

[0075] To understand whether knocking out the OsEns150 gene has an impact on the rice plant type, we measured the plant height in the field of the homozygous mutant materials with frameshift mutations in the OsEns150 gene editing and their wild - type control Zhonghua 11. The results showed that knocking out OsEns150 can reduce the rice plant height to a certain extent ( Figure 3 ), indicating that knocking out the OsEns150 gene has the potential to enhance the lodging resistance of rice.

[0076] ZH11 ens150-1 ens150-4 Plant height (cm) 103.94±2.30 93.40±1.34 97.90±2.91

[0077] Example 6

[0078] Investigation of rice grain traits after knocking out the OsEns150 gene

[0079] We systematically investigated the grain traits of the ens150 mutant rice and its wild - type control Zhonghua 11, mainly measuring and analyzing the grain length, grain width, grain thickness, 1000 - grain weight, and seed - setting rate of the grains. The results showed that knocking out the OsEns150 gene does not affect the grain shape, 1000 - grain weight, and seed - setting rate of rice ( Figure 4) It shows that genetic regulation of the OsEns150 gene has no effect on rice grain traits.

[0080] ZH11 ens150-1 ens150-4 Grain length (mm) 7.48±0.16 7.55±0.14 7.57±0.12 Grain width (mm) 3.18±0.10 3.25±0.1 3.17±0.08 Grain thickness (mm) 2.27±0.04 2.23±0.0 2.22±0.06 1000-grain weight (g) 24.76±0.09 24.48±0.18 23.94±0.26 Seed setting rate (%) 89.18±1.43 84.60±4.15 90.47±3.28

[0081] Example 7

[0082] Investigation on improving the physicochemical quality traits of rice by knocking out the OsEns150 gene

[0083] The physicochemical properties of rice from the ens150 mutant rice and its wild-type control Zhonghua 11 were measured. The results showed that knocking out the OsEns150 gene could significantly reduce the apparent amylose content and gelatinization temperature of rice, while the gel consistency increased significantly and the viscosity was also significantly improved ( Figure 5 ). The above results indicate that the cooking and eating quality of ens150 mutant rice has been significantly improved. In addition, compared with the wild-type control Zhonghua 11, the protein content of ens150 mutant rice has also increased to a certain extent ( Figure 6 ). The above results show that knocking out the OsEns150 gene can improve the cooking and eating quality and nutritional quality of rice simultaneously.

[0084] ZH11 ens150-1 ens150-4 Apparent amylose content (%) 18.13±0.21 17.2±0.34 16.86±0.54 Gel consistency (mm) 87.66±6.65 101±3.6 103.3±6.43 Protein (%) 7.91±0.08 8.61±0.09 9.08±0.07

[0085] Example 8

[0086] Knocking out the OsEns150 gene only slightly delays rice seed germination

[0087] Under normal germination conditions, the germination rate of ens150 mutant seeds is slightly slower than that of the wild-type control (less than 12 h), and the final germination rate is the same as that of the wild-type control, and all can germinate completely ( Figure 7 ). Therefore, knocking out the OsEns150 gene has little effect on the normal germination of rice seeds.

[0088] Example 9

[0089] Knocking out the OsEns150 gene can significantly enhance the pre-harvest sprouting resistance of rice

[0090] The main panicles of rice from the ens150 mutant and its wild-type control were directly harvested in the field about 30 days after flowering, and the pre-harvest sprouting of rice was promoted by simulating high temperature and high humidity conditions in the laboratory. The results showed that the pre-harvest sprouting rate of the OsEns150 gene knockout materials was significantly lower than that of the wild-type control Zhonghua 11 at each detection time point, especially after long-term treatment, and the difference between the two was more significant ( Figure 8 ). The results of this experiment show that knocking out the OsEns150 gene can significantly enhance the pre-harvest sprouting resistance of rice. Therefore, the OsEns150 gene has great application value in improving the pre-harvest sprouting resistance of rice varieties.

[0091] The above are only the preferred embodiments of the present invention. However, they are not intended to limit the present invention. Those skilled in the art can make possible changes and modifications to the present invention by using the technical content disclosed above, or modify it into equivalent embodiments with equivalent changes. Without departing from the technical content of the present invention, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention all fall within the protection scope of the technical solution of the present invention.

Claims

1. Application of knocking out rice OsEns150 gene in reducing rice plant height, improving rice cooking and eating quality and nutritional quality, and enhancing rice pre-harvest sprouting resistance, characterized in that, the amino acid sequence encoded by the OsEns150 gene is as shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, the coding region sequence of the OsEns150 gene is as shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, the knocking out is carried out using the vector pC1300-Cas9.

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