Genetic engineering application of rice cellulose synthesis gene OsCesA9

Abstract

The invention discloses genetic engineering application of a rice cellulose synthesis gene OsCesA9, belongs to the field of genetic engineering, and discloses cloning of the gene OsCesA9 participating in regulation and control of rice grain length and application of the gene OsCesA9 in regulation and control of rice grain length. The CDS sequence of the gene is as shown in SEQ ID NO.1, and the cDNA sequence is as shown in SEQ ID NO.9. Overexpression of the rice gene OsCesA9 can increase the grain length of rice without influencing the panicle number of the whole plant, and is beneficial to genetic improvement of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and relates to the cloning and application of the rice cell wall synthesis gene OsCesA9. Background Technology

[0002] Rice is one of the world's most important crops. Increasing rice yield is an urgent need driven by the rapid growth of the global population. Rice yield components include panicle number, grain number per panicle, and grain shape, with grain shape having a significant impact on yield. Therefore, grain shape has always been an important goal in rice breeding. Rice grain shape traits mainly include grain length, grain width, grain thickness, and length-to-width ratio, which are quantitative traits controlled by different genetic systems, including the embryo, endosperm, and maternal plant. Rice grain shape also affects the appearance quality and commercial value of rice. Therefore, cloning genes that regulate grain shape and conducting in-depth research and elucidation of the gene regulatory network of rice grain shape can provide an important theoretical basis and genetic resources for high-yield and high-quality molecular breeding of rice.

[0003] Currently, G protein signaling pathways, ubiquitin-proteasome pathways, MAPK signaling pathways, epigenetic modifications, and plant hormone signaling pathways have been reported to participate in the regulation of rice grain shape, and most of the genes involved have been cloned and their functions revealed (Li et al., 2023). Among them, GS3 is the first rice grain shape QTL to be identified. Using near-isogenic lines obtained by crossing Minghui 63 (large grain) and Chuan 7 (small grain), GS3 was located using map-based cloning. This gene encodes a G protein γ subunit, and the loss of GS3 function promotes cell elongation, thereby producing long grains (Fan et al., 2006). The rice G protein α subunit D1 loss-of-function mutant d-1 exhibits a dwarf and small grain phenotype and reduced sensitivity to BR, indicating that D1-mediated heterotrimeric G protein and BR signaling pathway jointly regulate rice grain shape (Miura K et al., 2009). Decreased expression of the G protein β subunit (RGB1) also leads to a smaller grain phenotype in rice (Mao H et al., 2010). GW2 is a major-effect QTL controlling grain width. This gene encodes an E3 ubiquitin ligase containing a ring loop. Loss-of-function mutants exhibit enhanced spikelet outer cell proliferation, accelerating grain filling and thus increasing grain width, weight, and yield (Song et al., 2007). Loss of function of the deubiquitinase OsOTUB1 / WTG1 increases grain width, grain thickness, and the number of grains per spike (Huang et al., 2017). OsOTUB1 primarily regulates grain type by influencing cell proliferation. OsOTUB1 interacts with OsSPL14 / IPA1, a key regulator of ideal plant type. This interaction restricts K63-linked ubiquitination of OsSPL14, thereby promoting K48-linked ubiquitination-dependent proteasome degradation (Wang et al., 2017). BR-deficient mutants dwarf1 and dwarf2 produce small, short grains, indicating that BR can promote grain growth (Hong et al., 2005; Fang et al., 2016). qGL3 is a major-effect QTL controlling grain length, encoding a serine / threonine phosphatase (OsPPKL1) containing two Kelch domains. Rice also has two homologues of OsPPKL1, OsPPKL2 and OsPPKL3. OsPPKL1 and OsPPKL3 inhibit grain length, while OsPPKL2, as a homologue of Arabidopsis AtBSU1 and AtBSL1, promotes grain elongation. Recent studies have found that loss of OsMKK4 function leads to smaller grains (Duan et al., 2014). OsMAPK6 also plays a positive regulatory role in grain shape (Liu et al., 2015).OsMKK4 can interact with and phosphorylate OsMAPK6 (Liu et al., 2015), and these two proteins can affect the response of brassinolide (BR) and regulate the expression of BR-related genes. This indicates that different grain shape regulatory pathways are interconnected. Studies have also shown that rice grain shape is related to tiller number, specifically, an increase in tiller number leads to smaller grains (Che et al., 2015). In rice, OsCKX2 and OsCKX9 both encode cytokinin oxidases; mutations in these enzymes increase tiller number and decrease grain length (Rong et al., 2022). Furthermore, a significant negative correlation between grain length and tiller number has been reported in other species (Chen et al., 2023). Therefore, cloning superior genes that can improve rice grain shape without affecting panicle number is of great significance.

[0004] The rice cellulose synthase family gene OsCesAs plays a crucial role in cellulose synthesis and alteration of stem strength. This gene family contains eight transmembrane domains and a central cytoplasmic domain containing the D,D,D,QXXRW motif (Li F et al., 2017). The CesA family genes found in Arabidopsis thaliana can be divided into two main groups: the first group includes AtCESA1 / 3 / 6 (or one of AtCESA2 / 5 / 9, which participate in primary cell wall cellulose synthesis), while AtCESA4, AtCESA7, and AtCESA8 are cellulose synthases for secondary cell wall formation (Manoj Kumar et al., 2015). Studies have shown that in rice, OsCesA4, OsCesA7, and OsCesA9 correspond to AtCesA8, AtCesA4, and AtCesA7, respectively (Kotake T et al., 2011). Studies have shown that missense mutations in the OsCesA4 transmembrane domain affect its cellular localization and lead to abnormal cell wall structures (Zhang Be et al., 2009). The FC19 gene, co-expressed with rice cellulose synthase genes, exhibits reduced cellulose and hemicellulose content in its mutant form, resulting in increased fragility but enhanced rigidity and resistance to bending, ultimately leading to increased lodging resistance in rice (Dang et al., 2023). Notably, the CesA family also plays a regulatory role in conjunction with plant hormones. Exogenous application of GA increases the expression of CesA genes in both wild-type and mutant varieties; simultaneously, GA can also activate the expression of downstream transcription factors and CesA genes by regulating SLR1 degradation and releasing NACs, thereby promoting cellulose synthesis (Huang D et al., 2015). MYB-type transcription factors in rice can also bind to OsCesA7 through AC-II and SMRE3 elements, activating its expression (Noda S et al., 2015). Exogenous application of BL can induce the expression of the CesA gene in Arabidopsis thaliana BR deletion mutants. BES1 can bind to the CesA gene promoter through the E-box element to regulate the expression of this type of gene (Xie L et al, 2011).

[0005] This patent utilizes m-qgl3 phosphoproteomics data to screen for OsCesA9, which is involved in rice cellulose synthesis. It was found that OsCesA9 plays an important role in the regulation of rice grain length. Overexpression of OsCesA9 can significantly increase rice grain length without affecting the number of panicles per plant, indicating that overexpression of this gene can increase the yield per rice plant, providing theoretical value for rice molecular breeding. Summary of the Invention

[0006] The purpose of this invention is to disclose the cloning of the rice cellulose synthase gene OsCesA9 and its genetic engineering application in rice grain shape and lodging resistance. Overexpression of the rice gene OsCesA9 can increase rice grain length and stem strength, which is beneficial to the genetic improvement of rice yield and lodging resistance, without affecting the number of panicles per plant, thus avoiding the problem of a significant negative correlation between grain length and tiller number that is easily encountered in grain shape regulation.

[0007] The first objective of this invention is to provide a rice gene OsCesA9, the CDS sequence of which is shown in SEQ ID NO.1, located at 94bp to 3261bp of the cDNA sequence shown in SEQ ID NO.9, with a full length of 3168bp; the cDNA sequence of which is shown in SEQ ID NO.9.

[0008] The amino acid sequence encoded by the rice gene OsCesA9 is shown in SEQ ID NO.2.

[0009] A second objective of this invention is to provide an overexpression vector containing the aforementioned coding region of the rice gene OsCesA9.

[0010] The coding region of the rice gene OsCesA9 is located at 94bp to 3261bp of the cDNA sequence shown in SEQ ID NO.9, which is the sequence shown in SEQ ID NO.1.

[0011] Furthermore, the overexpression vector is obtained by inserting the coding region of the aforementioned rice OsCesA9 gene, as shown in SEQ ID NO.1, between the KpnⅠ and SalⅠ restriction sites of the plant binary expression vector pCAMBIA1300s.

[0012] A third objective of this invention is to provide the genetic engineering application of the aforementioned rice gene OsCesA9 or its overexpression vector in regulating rice grain length.

[0013] Furthermore, overexpression of the rice gene OsCesA9 can increase the grain length of rice.

[0014] Furthermore, overexpression of the rice gene OsCesA9 can increase the grain length of rice without affecting the number of panicles per plant.

[0015] Furthermore, introducing an overexpression vector of the rice gene OsCesA9 into rice can increase the expression of the rice gene OsCesA9, thereby increasing the grain length of rice without affecting the number of panicles per plant.

[0016] Furthermore, this includes the following steps:

[0017] (1) Total RNA was extracted from the rice variety “ZH11” and reverse transcribed to synthesize the first strand of cDNA;

[0018] (2) Cloning of rice gene OsCesA9: Using the first strand of cDNA synthesized by reverse transcription in step (1) as a template, PCR amplification was performed using primer pairs and the product was recovered. Sequencing and screening were performed to obtain the cDNA sequence containing the CDS of the OsCesA9 gene shown in SEQ ID NO.9. The CDS sequence of the rice OsCesA9 gene is shown in SEQ ID NO.1.

[0019] (3) Construction of overexpression vector: Using the DNA fragment obtained in step (2) as a template, PCR amplification was performed using primer pairs to obtain the coding region of the rice OsCesA9 gene shown in SEQ ID NO.1. The amplification product was inserted between the KpnⅠ and SalⅠ restriction sites of the plant binary expression vector pCAMBIA1300s to obtain the overexpression vector pCAMBIA1300s-OsCesA9.

[0020] (4) Obtaining transgenic plants: The obtained overexpression vector pCAMBIA1300s-OsCesA9 was transformed into Agrobacterium strain EHA105. The T-DNA region of the overexpression vector was integrated into the genome of the japonica rice variety Nipponbare using Agrobacterium-mediated rice genetic transformation, thereby obtaining transgenic rice with OsCesA9 overexpression.

[0021] Furthermore, the primer pair described in step (2) is:

[0022] OsCesA9F: 5'-ATGGAGGCGAGCGCC-3' (SEQ ID NO.3),

[0023] OsCesA9R: 5'-TCAGCAGTTGATG-3' (SEQ ID NO. 4).

[0024] Furthermore, the primer pair described in step (3) is:

[0025] OsCesA9OEF: 5'-AGCTTTCGCGAGCTCGGTACCATGGAGGCGAGCGCC-3' (SEQ ID NO.5) contains a KpnⅠ restriction endonuclease site.

[0026] OsCesA9OER: 5'-CTTGCATGCCTGCAGGTCGACTCAGCAGTTGATG-3' (SEQ ID NO.6) contains the SalⅠ restriction endonuclease site.

[0027] Beneficial effects

[0028] 1. This invention discloses a genetic engineering application of overexpressing the rice OsCesA9 gene to improve grain shape and lodging traits. This gene is derived from rice (Oryza sativa L.), and overexpression of this gene can increase rice grain length without affecting the number of panicles, which is beneficial for the genetic improvement of rice.

[0029] 2. The OsCesA9 gene cloned in this invention provides a new resource for high-yield and high-quality rice breeding. Attached Figure Description

[0030] Figure 1 Schematic diagram of the restriction enzyme sites of the overexpression vector pCAMBIA1300s;

[0031] Figure 2 qRT-PCR identification of OsCesA9 overexpression transgenic lines;

[0032] Figure 3 Phenotypic characteristics of OsCesA9 overexpression transgenic lines

[0033] Figure 3 A comparison of the plant type of A.OsCesA9 overexpression transgenic lines with wild type;

[0034] Figure 3 A comparison of plant height between B.OsCesA9 overexpression transgenic lines and wild-type lines;

[0035] Figure 3 A comparison of tiller number between C.OsCesA9 overexpression transgenic lines and wild-type lines;

[0036] Figure 3 A comparison of grain shape between the D.OsCesA9 overexpression transgenic lines and the wild type;

[0037] Figure 3 Statistical graph of grain length in transgenic lines overexpressing E.OsCesA9. Detailed Implementation

[0038] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0039] Example 1

[0040] The rice variety “ZH11” was selected and hydroponically grown in an artificial climate incubator (16h light / 8h darkness, 30℃ during the day and 26℃ at night). The nutrient solution used was the standard nutrient solution formula from the International Rice Research Institute. When the seedlings reached the 3-4 leaf stage, they were quick-frozen in liquid nitrogen and stored at -80℃ for later use. Total RNA was extracted from the stored rice seedling samples according to the Invitrogen Trizol method. The quality and concentration of total RNA were analyzed by 1% agarose gel electrophoresis. Total RNA meeting the quality requirements was further used for the synthesis of cDNA first strand. The synthesis of cDNA first strand was performed according to the Vazyme reverse transcription system operating manual.

[0041] Primers were designed: upstream OsCesA9F: 5'-ATGGAGGCGAGCGCC-3' (SEQ ID NO.3); downstream OsCesA9R: 5'-TCAGCAGTTGATG-3' (SEQ ID NO.4). Using the first strand of cDNA synthesized by reverse transcription as a template, the CDS sequence of the OsCesA9 gene was cloned by PCR.

[0042] PCR amplification was performed using PrimeStar GXL DNA polymerase (Takara, Dalian, China). The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min 40 s, 35 cycles, followed by a final extension at 72℃ for 5 min. The PCR products were then subjected to agarose gel electrophoresis, and the target fragment was recovered using a DNA gel recovery kit from Vazyme. Sequencing yielded the OsCesA9 CDS sequence with a complete coding region, SEQ ID NO. 1.

[0043] The CDS sequence of OsCesA9 is shown in SEQ ID NO.1. The full-length ORF is 3168 bp. BioXM software (version 2.6) analysis shows that OsCesA9 encodes a total of 1056 amino acids (SEQ ID NO.2). Its isoelectric point pI = 6.84 and molecular weight MW = 118.71 kDa are estimated.

[0044] Example 2

[0045] Based on the cDNA sequence of the rice OsCesA9 gene, primers were designed to amplify the complete coding reading frame. The upstream primer OsCesA9OEF: 5'-AGCTTTCGCGAGCTCGGTACCATGGAGGCGAGCGCC-3' (SEQ ID NO.5) contains the KpnⅠ restriction endonuclease site. The downstream primer OsCesA9OER:

[0046] 5'-CTTGCATGCCTGCAGGTCGACTCAGCAGTTGATG-3' (SEQ ID NO.6) contains the SalⅠ restriction endonuclease site.

[0047] Using the correctly sequenced DNA fragment OsCesA9 (SEQ ID NO. 1) obtained in Example 1 as a template, PCR amplification was performed. The resulting product was further cloned into the KpnI and SalI restriction sites of the plant binary expression vector pCAMBIA1300s using the Vazyme CloneExpress kit. Figure 1 ), to obtain overexpression vectors

[0048] pCAMBIA1300s-OsCesA9.

[0049] The obtained recombinant expression vector pCAMBIA1300s-OsCesA9 was transformed into Agrobacterium strain EHA105 by heat shock. The T-DNA region of the recombinant vector was integrated into the genome of the japonica rice variety Nipponbare using Agrobacterium-mediated rice genetic transformation, thereby obtaining transgenic rice OE-OsCesA9-1 and OE-OsCesA9-2 with OsCesA9 overexpression.

[0050] After the wild-type “ZH11” and transgenic rice overexpressing OsCesA9 reached seedling stage, qRT-PCR was performed to identify each line. A small amount of transgenic rice leaves were taken, and total RNA was extracted using the Invitrogen Trizol method, then reverse-engineered into cDNA. OsCesA9 gene qRT-PCR specific primers were designed: qOsCesA9F: 5'-GTTCATCATGCCTCCGATTAGCAC-3' (SEQ ID NO.7) and qOsCesA9R: 5'-GTCGCGAAGATGGAGATGAAGAG-3' (SEQ ID NO.8). Using rice actin rRNA expression as an internal control, qRT-PCR verification was performed on transgenic lines OE-OsCesA9-1 and OE-OsCesA9-2 according to the Roche Fast Start Universal SYBR Green Master (ROX) instructions. The validation results showed that the expression level of OsCesA9 in the two OsCesA9 overexpression lines, OE-OsCesA9-1 and OE-OsCesA9-2, was significantly increased compared with the wild type "ZH11". Figure 2 ).

[0051] Example 3

[0052] Example 2 yielded overexpression transgenic lines (OE-OsCesA9-1, OE-OsCesA9-2) and a wild-type (ZH11). Seeds were soaked until they showed signs of sprouting and then sown at the Nanjing Baima Field Experimental Base for agronomic trait assessment at the mature plant stage. Each line was planted in 3 rows with a row spacing of 20 cm, with 8 plants per row and a plant spacing of 15 cm, and conventional soil and fertilizer management was applied. After grain filling, the plant height, tiller number, and lodging index of the middle 6 plants in each row of both transgenic and wild-type rice were measured and statistically analyzed.

[0053] After the seeds of the OsCesA9 overexpression line and the wild-type line (ZH11) matured, the panicle nodes of each group of rice were cut off, and the seeds were dried at 42℃ for 5 days before grain length analysis was performed.

[0054] Statistical results showed that the rice grains of the OsCesA9 overexpression lines obtained in Example 2 were significantly longer than those of the wild-type ZH11. Figure 3 (D, E) Rice plant height decreased, while the number of panicles showed no significant difference. Figure 3 A, B, C). This indicates that overexpression of OsCesA9 can increase rice grain length without affecting the number of panicles per plant, which is beneficial for the genetic improvement of rice.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rice gene, OsCesA9, characterized in that, The CDS sequence of the OsCesA9 gene is shown as SEQ ID NO. 1, and the cDNA sequence of the OsCesA9 gene is shown as SEQ ID NO.

9.

2. The amino acid sequence encoded by the rice gene OsCesA9 shown as SEQ ID NO.

2.

3. An overexpression vector containing the rice gene OsCesA9 shown in claim 1.

4. The overexpression vector of claim 3, wherein, The overexpression vector is obtained by inserting the CDS sequence of the rice OsCesA9 gene shown in claim 1 into the Kpn I and Sal I enzyme cutting sites of the plant binary expression vector pCAMBIA1300s.

5. The genetic engineering application of the rice gene OsCesA9 shown in claim 1 or the overexpression vector of the rice gene OsCesA9 shown in claim 3 in regulating the grain length of rice. Overexpression of the rice gene OsCesA9 can increase the grain length of rice.

6. Use according to claim 5, characterized in that, Overexpression of the rice gene OsCesA9 can increase the grain length of rice without affecting the number of ears of the whole plant.

7. Use according to claim 5 or 6, characterized in that, Introducing the overexpression vector of the rice gene OsCesA9 into rice to improve the expression of the rice gene OsCesA9 can increase the grain length of rice without affecting the number of ears of the whole plant.

8. Use according to claim 5 or 6, characterized in that, The method comprises the following steps:

9. Use according to any one of claims 6 to 8, characterized in that, (1) Extracting total RNA from rice variety "ZH11" and reverse transcribing to synthesize cDNA first strand; (2) Cloning of the rice gene OsCesA9: using the cDNA first strand synthesized by reverse transcription in step (1) as template, performing PCR amplification with a primer pair and recovering the product, and performing sequencing screening to obtain the cDNA sequence shown as SEQ ID NO. 9; (3) Construction of overexpression vector: using the DNA fragment obtained in step (2) as template, performing PCR amplification with a primer pair to obtain the coding region of the rice OsCesA9 gene shown as SEQ ID NO. 1, and inserting the amplification product into the Kpn I and Sal I enzyme cutting sites of the plant binary expression vector pCAMBIA1300s to obtain the overexpression vector pCAMBIA1300s-OsCesA9; (4) Obtaining transgenic plants: transforming the obtained overexpression vector pCAMBIA1300s-OsCesA9 into Agrobacterium strain EHA105, and using Agrobacterium-mediated genetic transformation of rice to integrate the T-DNA region of the overexpression vector into the genome of rice japonica variety Nipponbare, thereby obtaining transgenic rice overexpressing OsCesA9. The primer pair in step (2) is: OsCesA9F: 5'-ATGGAGGCGAGCGCC-3' (SEQ ID NO. 3), 10. Use according to claim 9, characterized in that, OsCesA9R: 5'-TCAGCAGTTGATG-3' (SEQ ID NO. 4) The primer pair in step (3) is: OsCesA9OEF: 5'-AGCTTTCGCGAGCTCGGTACCATGGAGGCGAGCGCC-3' (SEQ ID NO. 5), OsCesA9OER: 5'-TCAGCAGTTGATG-3' (SEQ ID NO. 4) ​ OsCesA9 OER: 5'-CTTGCATGCCTGCAGGTCGACTCAGCAGTTGATG-3' (SEQ ID NO. 6). OsCesA9 OER: 5'-CTTGCATGCCTGCAGGTCGACTCAGCAGTTGATG-3' (SEQ ID NO. 6). OsC

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