Use of loc_os03g43100.2 gene in improving rice traits

Abstract

The application belongs to the field of genetic engineering, and provides application of LOC_Os03g43100.2 gene and the protein coded by the gene in improving rice traits, and a method for cultivating transgenic rice with improved grain size and / or yield. The application obtains transgenic rice with grain length, grain width of matured rice, grain length, grain width of brown rice, and thousand-grain weight of the transgenic rice being obviously greater than those of wild-type rice, and the improvement effect on the traits of rice grains is very obvious, and the application is helpful to clarify the relationship between grain type and rice yield, and lays a theoretical foundation for cultivating high-yield rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to the application of the LOC_Os03g43100.2 gene in improving rice traits. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most widely cultivated food crops in the world, with more than half of the global population relying on it as a staple food. However, current factors such as rapid population growth, unstable climate, frequent natural disasters, and a continuous shrinking of arable land have led to an increasingly severe global food crisis. Improving rice yield has always been a key focus for breeders. Since the "Second Green Revolution," exploring new germplasm resources that can significantly increase rice production capacity has been considered crucial to solving the current problem. Molecular breeding based on the analysis of yield- and quality-related genes and their regulatory networks has become an effective way to overcome the bottleneck in rice yield breeding. Among these factors, rice grain weight is a crucial factor in yield, and there is a positive correlation between rice grain weight and grain shape. Rice grain shape refers to the three-dimensional structure composed of the length, width, and thickness of the grain. Grain shape is not only a key factor determining rice yield but also an important agronomical trait affecting the appearance and quality of rice. Therefore, the discovery and utilization of rice grain shape regulatory genes are of great significance for the breeding of high-yield rice varieties.

[0003] Current research indicates that several genes involved in the ubiquitin-proteasome pathway play a crucial role in the regulation of rice grain shape. For example, GW2 encodes a novel E3 ubiquitin ligase with ubiquitination activity, thereby affecting grain shape. In rice G proteins, the Gα and Gβ subunits are encoded by the RGA1 / D1 and RGB1 genes, respectively, while the Gγ subunit is encoded by RGG1, RGG2, GGC2, DEP1, and GS3. These subunits exhibit antagonistic or synergistic interactions among genes in the regulation of grain size and shape. A natural variation of C>A in exon 2 of GS3 produces a premature terminator, predicting that it encodes a non-functional protein containing only 55 amino acid residues of a partial OSR structure, leading to accelerated longitudinal division of glume cells and thus increased grain length. Under the stimulation of extracellular signaling factors, the MAPK pathway activates downstream response factors through a three-stage phosphorylation reaction: first, MAPKKK phosphorylates MAPKK; activated MAPKK further phosphorylates MAPK; and finally, activated MAPK phosphorylates downstream substrates. The MAPK pathway is highly conserved in eukaryotes. In recent years, it has been found that the rice MAPK cascade reaction composed of OsMKKK10-OsMKK4-OsMAPK6 is closely related to the regulation of rice grain type and panicle type.

[0004] Brassinosteroids (BRs) are a class of sterol compounds initially isolated from rapeseed pollen, influencing multiple physiological processes in plant growth and development. Studies have shown that BRs play a crucial role in regulating grain size. The BR signaling pathway positively regulates grain size in rice. Deletion mutants related to BR synthesis and signal transduction inhibit plant growth and development, resulting in phenotypes such as dwarfing, darker leaf color, smaller leaf angles leading to an upright posture, and smaller panicles and grains.

[0005] Currently, there are some reports on using certain genes in the rice genome to improve rice grain shape, but there is no application of using the rice LOC_Os03g43100 gene for rice grain shape improvement. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention provides the application of the LOC_Os03g43100.2 gene in improving rice traits.

[0007] The first aspect of this invention provides any of the following applications of the LOC_Os03g43100.2 gene or related biological materials:

[0008] 1) Regulating rice yield;

[0009] 2) Regulating the traits of rice grains;

[0010] 3) Rice breeding;

[0011] The sequence composition of the LOC_Os03g43100.2 gene is shown in Sequence 1 of the sequence listing.

[0012] The second aspect of this invention provides any of the following applications of the protein encoded by the LOC_Os03g43100.2 gene:

[0013] 1) Regulating rice yield;

[0014] 2) Regulating the traits of rice grains;

[0015] 3) Rice breeding;

[0016] The sequence composition of the protein encoded by the LOC_Os03g43100.2 gene is shown in Sequence 2 of the sequence listing.

[0017] The third aspect of the present invention provides a method for cultivating transgenic rice with larger grains and / or increased yield, characterized in that: the method includes the operation of transferring the LOC_Os03g43100.2 gene or related biological materials into the target rice.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. This invention is the first to fuse the Ubiquitin promoter, the CDS sequence of the LOC_Os03g43100.2 gene, and the GFP coding sequence, and transform this fused gene into rice, thereby improving rice grain shape. This helps to clarify the relationship between grain shape and rice yield, and lays a theoretical foundation for breeding high-yield rice varieties.

[0020] 2. The mature grain length and width of the transgenic rice obtained by the method of the present invention, the grain length and width of the brown rice, and the thousand-grain weight are all significantly greater than those of wild-type rice, and the improvement effect on the traits of rice grains is very obvious. Attached Figure Description

[0021] Figure 1 The image shows the LOC_Os03g43100.2-GFP-pCambia1390 vector from Example 1.

[0022] Figure 2 This is an electrophoresis image of the PCR products detected in transgenic plants in Example 2. WT represents wild-type rice 'Kitaake', and OE-1 and OE-4 represent transgenic rice lines (i.e., transgenic plants) overexpressing the LOC_Os03g43100.2 gene.

[0023] Figure 3 This is a real-time quantitative PCR result of detecting the expression level of the LOC_Os03g43100.2 gene in transgenic plants in Example 2. WT represents wild-type rice 'Kitaake', and OE-1 and OE-4 represent transgenic rice lines (i.e., transgenic plants) that overexpress LOC_Os03g43100.2.

[0024] Figure 4 This is a comparison diagram of the grain shape traits of the transgenic rice plants in Example 3 with the WT; where figure a shows the grain length of mature grains, figure b shows the grain width of mature grains, figure c shows the grain length of brown rice, and figure d shows the grain width of brown rice.

[0025] Figure 5 This is a comparison and statistical analysis chart of the grain shape traits and thousand-grain weight of the transgenic rice plants in Example 3 with the WT.

[0026] Figure 3 and Figure 5 In the diagram, asterisks represent the differences between each sample and WT, with * indicating a significant difference (p<0.05), ** indicating an extremely significant difference (p<0.01), and *** indicating an extremely significant difference (p<0.001). Detailed Implementation

[0027] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0028] The first aspect of this invention provides any of the following applications of the LOC_Os03g43100.2 gene or related biological materials:

[0029] 1) Regulating rice yield;

[0030] 2) Regulating the traits of rice grains;

[0031] 3) Rice breeding.

[0032] The sequence composition of the LOC_Os03g43100.2 gene is shown in Sequence 1 of the sequence listing. The related biological materials include expression cassettes, recombinant vectors, recombinant bacteria, or transgenic cell lines containing the LOC_Os03g43100.2 gene sequence. For example, recombinant plasmids LOC_Os03g43100.2-pCambia1390 and LOC_Os03g43100.2-GFP-pCambia1390, or recombinant Agrobacterium containing either LOC_Os03g43100.2-pCambia1390.

[0033] The rice yield includes the thousand-grain weight and / or the harvested amount of rice grains (total weight of grains).

[0034] The characteristics of the rice grains include: grain length and width, brown rice grain length and width, and thousand-grain weight.

[0035] The rice breeding can be, for example, for breeding to increase rice yield; more specifically, for breeding to increase the grain length and width of rice grains, the grain length and width of brown rice grains, and the thousand-grain weight of rice grains.

[0036] In this invention, the LOC_Os03g43100.2 gene is the CDS sequence (shown in Sequence 1) of the second transcript of the LOC_Os03g43100 gene recorded in the Rice Genome Annotation Project database.

[0037] The second aspect of this invention provides any of the following applications of the protein encoded by the LOC_Os03g43100.2 gene:

[0038] 1) Regulating rice yield;

[0039] 2) Regulating the traits of rice grains;

[0040] 3) Rice breeding.

[0041] The sequence composition of the protein encoded by the LOC_Os03g43100.2 gene is shown in Sequence 2 of the sequence listing.

[0042] The rice yield mentioned includes the amount of rice grains harvested.

[0043] The characteristics of the rice grains include: grain length and width, brown rice grain length and width, and thousand-grain weight.

[0044] The rice breeding can be, for example, for breeding to increase rice yield; more specifically, for breeding to increase the grain length and width of rice grains, the grain length and width of brown rice grains, and the thousand-grain weight of rice grains.

[0045] A third aspect of the present invention provides a method for cultivating transgenic rice with larger grains and / or increased yield, the method comprising transferring the LOC_Os03g43100.2 gene or related biological material into the target rice.

[0046] The relevant biological materials are as described above.

[0047] According to a third aspect of the invention, the method further includes the operation of increasing the expression level of the LOC_Os03g43100.2 gene in the target rice.

[0048] According to a third aspect of the present invention, the expression level of the LOC_Os03g43100.2 gene can be determined by detecting the amount of mRNA transcribed from the LOC_Os03g43100.2 gene and / or the amount of protein encoded by the LOC_Os03g43100.2 gene.

[0049] According to a third aspect of the invention, the rice grains become larger and / or the yield increases, which is manifested by at least one of the following traits of the rice grains: grain length, grain width, brown rice grain length, grain width, and thousand-grain weight.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are products that can be obtained from commercial channels; unless otherwise specified, all testing and detection methods used in the following examples are conventional testing and detection methods in the field and can be obtained from textbooks, reference books or academic journals.

[0051] Example 1

[0052] This embodiment describes the acquisition of transgenic rice with the LOC_Os03g43100.2 gene.

[0053] I. Obtaining the LOC_Os03g43100.2 gene.

[0054] 1. Retrieve the LOC_Os03g43100 gene from the Rice Genome Annotation Project database, and design PCR amplification primer pairs based on the CDS sequence of its second transcript, LOC_Os03g43100.2:

[0055] Forward primer F:

[0056] 5'-CACTAGGTACCTGCAGATGAATCGACGGAAGGAGCTGG-3';

[0057] Reverse primer R:

[0058] 5'-ATCCGTCGACCTGCAGGCAAGAGATGTCCACAGAGTT-3'.

[0059] 2. Total RNA was extracted from wild Nipponbare rice using the RNAprep pure plant total RNA extraction kit (catalog number DP432) purchased from Tiangen Biotech (Beijing) Co., Ltd., following the instructions. cDNA was obtained using the StarScript III All-in-one RT Mixwith gDNA Remover reverse transcription kit (catalog number A230-10) purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd., following its instructions. Using the obtained cDNA as a template, the forward primer F and reverse primer R from step 1 were used as primers to obtain the CDS sequence of LOC_Os03g43100.2 (shown in Sequence 1). The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 15 s, 56℃ annealing for 30 s, 68℃ extension for 1 min, for a total of 30 cycles; and finally, 68℃ for 10 min to ensure complete DNA double-strand extension. The PCR reaction system consisted of: 1 μL template (cDNA), 0.5 μL KOD FX, and KOD FX buffer. 10 μL, 2 mM dNTPs 2 μL, forward primer F 1 μL, reverse primer R 1 μL, ddH2O 4.5 μL.

[0060] II. Construction of plant expression vectors.

[0061] 1. Using pCambia1390 as the starting vector, the pCambia1390 vector was double-digested with restriction endonucleases BamHI and SpeI, and the digested pCambia1390 vector backbone was recovered.

[0062] 2. Amplify the CDS of GFP (green fluorescent protein) via PCR reaction. PCR amplification primer pair:

[0063] Forward primer F:

[0064] 5'-GTCGACGGATCCATGGTGAGCAAGGGCGAGGAGCTGT-3';

[0065] Reverse primer R:

[0066] 5'-GCGTTAACACTAGTTTACTTGTACAGCTCGTCCATGCCG-3'.

[0067] A BamHI restriction site was introduced into the forward primer F, and a SpeI restriction site was introduced into the reverse primer R. The GFP (green fluorescent protein) coding sequence was introduced into the pCambia1390 vector via infusion to obtain the GFP-pCambia1390 recombinant vector (i.e., a plant binary expression vector driven by Ubiquitin).

[0068] 3. The recombinant vector GFP-pCambia1390 obtained in step 2 above was double-digested with restriction endonucleases Pst I and Sal I, and the digested GFP-pCambia1390 vector backbone was recovered.

[0069] 4. The CDS of LOC_Os03g43100.2 was amplified by PCR. The PCR amplification primer pair is shown in step 1 of "Obtaining the LOC_Os03g43100.2 gene". A Pst I restriction site was introduced into the forward primer F, and a Sal I restriction site was introduced into the reverse primer R. After infusion, the CDS of LOC_Os03g43100.2 was introduced into the GFP-pCambia1390 vector to obtain the LOC_Os03g43100.2-GFP-pCambia1390 recombinant vector (also known as Ubiquitin::LOC_Os03g43100.2-GFP recombinant plasmid).

[0070] 5. The obtained recombinant plasmid LOC_Os03g43100.2-GFP-pCambia1390 was transformed into Escherichia coli DH5α, positive clones were screened, and a large number of constitutive plant expression vectors LOC_Os03g43100.2-GFP-pCambia1390 were obtained by sequencing.

[0071] The plant expression vector LOC_Os03g43100.2-GFP-pCambia1390 fused the Ubiquitin promoter, the CDS sequence of the LOC_Os03g43100.2 gene, and the GFP coding sequence. By transforming this fused gene expression vector into rice, rice grain shape was improved, which helps to clarify the relationship between grain shape and rice yield and lays a theoretical foundation for breeding high-yield rice varieties.

[0072] III. Obtaining rice with the LOC_Os03g43100.2 gene.

[0073] The recombinant plasmid LOC_Os03g43100.2-GFP-pCambia1390 obtained in step two was amplified (e.g., by culturing in E. coli) and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant plasmid that was completely correctly sequenced (i.e. contained the complete LOC_Os03g43100.2 sequence shown in sequence 1) was sent to Wuhan Boyuan Biotechnology Co., Ltd., which was entrusted to perform genetic transformation using Agrobacterium-mediated transformation with "Kitaake" as the background to obtain rice transgenic with the LOC_Os03g43100.2 gene.

[0074] Example 2

[0075] This embodiment is used to illustrate the identification of the transgenic rice of LOC_Os03g43100.2 obtained in Example 1, in order to determine whether the LOC_Os03g43100.2 gene was successfully transferred into the rice plant.

[0076] DNA was extracted from the leaves of transgenic rice plants, and the presence of the hygromycin resistance gene in the pCambia1390 vector was used to determine whether the LOC_Os03g43100.2 gene was successfully transferred into the rice plants.

[0077] The following specific primers were designed based on the hygromycin resistance gene:

[0078] Forward primer F: 5'-AAAAAGCCTGAACTCACCGC-3';

[0079] Reverse primer R: 5'-TCCACTATCGGCGAGTACTT-3'.

[0080] PCR was performed using this primer pair. The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 15 s; 56℃ annealing for 30 s; 68℃ extension for 1 min, for a total of 30 cycles; and finally 68℃ for 10 min to ensure complete DNA double-strand extension. The PCR reaction system consisted of: 1 μL template (cDNA), 0.5 μL KOD FX, 10 μL KOD FX Buffer, 2 μL 2 mM dNTPs, 1 μL forward primer F, 1 μL reverse primer R, and 4.5 μL ddH2O.

[0081] The PCR products were then detected by electrophoresis, and the results were as follows: Figure 2 As shown, transgenic rice varieties can all amplify the target band of the hygromycin resistance gene at 900 bp (e.g., Figure 2 (OE1 and OE4 in the sample). Sequencing of the PCR product revealed that it was the correct target sequence of the hygromycin resistance gene, thus confirming that the LOC_Os03g43100.2 gene, which is driven by Ubiquitin and encodes a fusion GFP sequence, has been inserted into the rice genome via Agrobacterium-mediated transformation.

[0082] Total RNA was extracted from the leaves of transgenic rice plants using the RNAprep pure plant total RNA extraction kit (product catalog number DP432) purchased from Tiangen Biotech (Beijing) Co., Ltd., following the instructions. cDNA was obtained using the StarScript III All-in-one RTMix with gDNA Remover reverse transcription kit (product catalog number A230-10) purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd., following its instructions. Using the obtained cDNA as a template, real-time quantitative PCR (RT-qPCR) was performed on the transgenic rice plants using the 2×RealStarFast SYBR qPCR Mix enzyme (product catalog number A301-10) purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd., following the instructions, to identify the expression level of the LOC_Os03g43100.2 gene in different lines.

[0083] Design RT-qPCR primer pairs based on the CDS sequence of LOC_Os03g43100.2:

[0084] Forward primer F: 5'-CCGTAAGTCGATCCAAGAA-3';

[0085] Reverse primer R: 5'-ACCCGAACAACAAACCCT-3'.

[0086] Using the ACTIN1 gene of wild-type Nipponbare rice as an internal control, a primer pair for RT-qPCR reaction was designed based on its CDS sequence:

[0087] Internal reference primer F: 5'-CCTGGCAGTATGAAGGTAGTTG-3';

[0088] Internal reference primer R: 5'-GAAGCACTTCATGTGGACGAT-3'.

[0089] RT-qPCR was performed using these two primer pairs, following a two-step amplification program. The first step consisted of 35 cycles: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s; 60℃ annealing for 30 s. The second step consisted of 95℃ for 60 s; 55℃ for 30 s; and 95℃ for 30 s. The reaction mixture consisted of: 10 μL of 2×RealStar Fast SYBR qPCR Mix, 0.5 μL of upstream primer F, 0.5 μL of downstream primer R, 1 μL of template (cDNA), and 8 μL of ddH2O.

[0090] Use 2 -△△Ct The method processes the obtained data. The results are as follows: Figure 3 As shown, the expression level of the LOC_Os03g43100.2 gene in the LOC_Os03g43100.2 overexpression transgenic lines (OE-1, OE-2, OE-3, OE-4) was significantly higher than that in the wild type. This confirms that the LOC_Os03g43100.2 gene, which encodes a fusion GFP sequence driven by Ubiquitin, is overexpressed in the transgenic lines.

[0091] Example 3

[0092] This embodiment is used to illustrate the detection of grain shape in rice transgenic LOC_Os03g43100.2 positive rice verified in Example 2, to prove that the LOC_Os03g43100.2 gene does indeed have the function of improving rice grain shape.

[0093] According to production specifications, rice plants that were positive for the LOC_Os03g43100.2 gene, as verified in Example 2, and rice "Kitaake" plants (WT) were simultaneously planted in the experimental field. After the plants had set and the grains matured, the grains were harvested, and the grain length, grain width, brown rice grain length, grain width, and thousand-grain weight of the grains harvested from different plants were compared. Twenty plants were selected from each line.

[0094] In the grain shape survey and analysis, the grain length and width of mature rice grains and brown rice grains were measured using vernier calipers, and the thousand-grain weight of mature grains was measured using an electronic balance. Fifty grains each from the WT and LOC_Os03g43100.2 overexpression transgenic lines (OE-1 and OE-4) were measured, and the thousand-grain weight determination was performed in triplicate.

[0095] The results are as follows Figure 4 and Figure 5 As shown, the average length of mature grains of wild-type rice "Kitaake" (WT) is 7.08 mm, the average grain width is 3.48 mm, the average brown rice grain length is 4.88 mm, and the average brown rice grain width is 2.97 mm. The average length of mature grains of the overexpressing transgenic line OE-1 is 7.28 mm, the average grain width is 3.63 mm, the average brown rice grain length is 5.08 mm, and the average brown rice grain width is 3.04 mm. The average length of mature grains of the overexpressing transgenic line OE-4 is 7.43 mm, the average grain width is 3.70 mm, the average brown rice grain length is 5.23 mm, and the average brown rice grain width is 3.12 mm. It is evident that the grain length, grain width, brown rice grain length, and brown rice grain width of the overexpressing transgenic lines OE-1 and OE-4 are significantly longer than those of WT. The average thousand-grain weight of mature grains in wild-type rice “Kitaake” (WT) was 24.49 g, while the average thousand-grain weight of mature grains in the overexpressing transgenic line OE-1 was 25.75 g, and the average thousand-grain weight of mature grains in the overexpressing transgenic line OE-4 was 27.39 g. It can be seen that the thousand-grain weight of mature grains in OE-1 and OE-4 is also significantly greater than that in WT. The increase in thousand-grain weight of OE-1 and OE-4 reached 5.14% and 11.18%, respectively, indicating that the LOC_Os03g43100.2 gene has great application value in improving rice yield.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of biological materials overexpressing the LOC_Os03g43100.2 gene in breeding to improve rice yield, including: The sequence composition of the LOC_Os03g43100.2 gene is shown in Sequence 1 of the sequence listing; The biological materials include expression cassettes, recombinant vectors, or recombinant bacteria containing the LOC_Os03g43100.2 gene sequence; The breeding program aimed at increasing rice yield is characterized by at least one of the following traits: The length and width of the grains, the length and width of brown rice grains, and the thousand-grain weight of the grains.

2. The application according to claim 1, characterized in that: The biomaterial is any one of the following: Recombinant plasmids LOC_Os03g43100.2-pCambia1390 and LOC_Os03g43100.2-GFP-pCambia1390, and recombinant Agrobacterium containing either LOC_Os03g43100.2-pCambia1390 or LOC_Os03g43100.2-GFP-pCambia1390.

3. A method for cultivating transgenic rice with increased yield, characterized in that: The method includes the operation of transferring the LOC_Os03g43100.2 gene or biological material overexpressing the LOC_Os03g43100.2 gene into the target rice; The sequence composition of the LOC_Os03g43100.2 gene is shown in Sequence 1 of the sequence listing; The biological materials include expression cassettes, recombinant vectors, or recombinant bacteria containing the LOC_Os03g43100.2 gene sequence; The increased rice yield is manifested by at least one of the following traits: The length and width of the grains, the length and width of brown rice grains, and the thousand-grain weight of the grains.

4. The method according to claim 3, characterized in that: The biomaterial is any one of the following: Recombinant plasmids LOC_Os03g43100.2-pCambia1390 and LOC_Os03g43100.2-GFP-pCambia1390, and recombinant Agrobacterium containing either LOC_Os03g43100.2-pCambia1390.

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