Application of WRD1 gene in regulating rice growth and development

Abstract

The present invention discloses a rice growth and development regulating gene, its encoded protein and application, and belongs to the field of molecular biology technology. The rice growth and development related encoding gene is named WRD1, its nucleotide sequence is shown in SEQ ID NO.1, and its encoded protein amino acid sequence is shown in SEQ ID NO.2. The WRD1 gene plays an important role in the growth and development of rice plant type. The deletion of the WRD1 gene causes the rice plant type to be dwarfed, the root system to be weakened, and the tillering to be reduced. The present invention also provides the application of the rice growth and development regulating gene WRD1 in regulating the development of rice plant type. By regulating the expression of the WRD1 gene, the growth and development characteristics of rice can be changed. The gene or the encoded protein of the present invention can further explain the molecular mechanism of rice growth and development regulation, and has important application value in genetically modifying the development of rice plant type.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to the application of WRD1 gene in regulating rice growth and development, and in particular to a key gene WRD1 for regulating rice growth and development, and its encoded protein and application. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops for mankind. For a long time, people have been committed to studying how to improve the production capacity of rice and other food crops, and have achieved a significant increase in rice yield. However, in the past decade, due to factors such as reduced genetic diversity, rice yield levels have remained stagnant. Therefore, how to increase the unit yield of staple crops such as rice and achieve sustained stable yield increases has become an important challenge facing current agricultural scientific research.

[0003] Crop plant type includes plant height, tiller number, root morphology and panicle morphology, which are closely related to crop yield formation. Improving rice yield by improving agronomic traits related to rice plant type has always been the focus of breeders. However, the development of rice plant type is regulated by a complex network composed of genetics, endogenous hormones and environmental factors. Although some major QTLs / genes have been isolated and cloned, and breeding practices have been carried out through molecular breeding methods, we still do not understand the mechanism of rice plant type development and the intrinsic relationship between related agronomic traits, and there are few plant type genes available for breeding applications. Therefore, it is necessary to further explore and identify the key genes of new resources for plant type development, and deeply analyze the molecular network of rice plant type development regulation, which will provide theoretical and technical support for rice breeders to improve rice plant type and cultivate new high-yield rice varieties through molecular breeding methods. Summary of the invention

[0004] The technical problem solved by the present invention is to provide an application of a rice growth and development regulatory gene WRD1 and a protein encoded by the gene in transforming rice growth and development regulation.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention isolated and identified a rice growth-weak mutant through EMS mutagenesis. The mutant showed a stunted phenotype throughout the growth period, with a shortened taproot, fewer adventitious roots, and no root hairs. The plant height, tiller number, and panicle length were all weaker than the wild type of the same period, and the yield per plant was significantly reduced. The WRD1 (weakness and roothair defective 1) gene was cloned through map-based cloning technology, and the gene was located on chromosome 12 of rice.

[0007] In a first aspect, the present invention provides an application of a WRD1 gene in regulating the growth and development of rice. The nucleotide sequence of the WRD1 gene is shown in SEQ ID NO.1.

[0008] Furthermore, by up-regulating or increasing the expression of the WRD1 gene in rice, the growth and development performance of rice is improved.

[0009] Furthermore, inhibiting or reducing the expression of the WRD1 gene in rice will reduce the growth and development performance of rice.

[0010] Furthermore, regulating the growth and development of rice includes regulating the root growth, plant height, tillering number, panicle shape and / or yield of the rice plant.

[0011] In a second aspect, the present invention provides a preparation for regulating rice growth and development, including an agent for increasing or inhibiting the expression of the WRD1 gene, wherein the nucleotide sequence of the WRD1 gene is shown in SEQ ID NO.1.

[0012] In a third aspect, the present invention provides a method for regulating rice growth and development, the method comprising regulating rice growth and development by regulating the expression of the rice WRD1 gene or regulating the activity or content of the protein encoded by the rice WRD1 gene; the nucleotide sequence of the WRD1 gene is shown in SEQ ID NO.1.

[0013] In a fourth aspect, the present invention provides a method for improving the growth and development performance of rice, the method comprising upregulating or increasing the expression level of the WRD1 gene in rice so that the growth and development performance of the rice is higher than that of the recipient rice; the nucleotide sequence of the WRD1 gene is shown in SEQ ID NO.1.

[0014] In a fifth aspect, the present invention provides a method for reducing plant growth and development performance, the method comprising inhibiting or reducing the expression level of the WRD1 gene in rice, so that the growth and development performance of the rice is lower than that of the recipient rice; the nucleotide sequence of the WRD1 gene is shown in SEQ ID NO.1.

[0015] In a sixth aspect, the present invention provides a method for cultivating transgenic plants, wherein a plant recombinant expression vector comprising the WRD1 gene shown in SEQ ID NO.1 is transformed into a recipient plant material using an Agrobacterium-mediated method to cultivate transgenic plants.

[0016] Furthermore, the recombinant expression vector adopts vector pCAMBIA1300 or AHLG.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention obtains the rice growth and development regulatory gene WRD1 through the map-based cloning technology, and verifies the function of the gene through the transgenic functional complementation experiment. The gene WRD1 or the protein encoded by it is necessary for the regulation of rice growth and development. The mutation of the gene or protein will significantly affect the normal formation of plant cell walls, causing the accumulation of active oxygen in rice cells and abnormal auxin signals, resulting in some growth and development phenotypes such as weakened plant roots, lack of root hairs, shorter plant height, reduced tillers, short panicle shape, and reduced yield; and overexpression of the gene can restore the plant growth and development phenotype to the wild type level. The use of the gene or the encoded protein of the present invention can further explain the molecular genetic mechanism of rice growth and development regulation, and has important application value in genetically modifying rice growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The growth phenotypes of the wild type (WT) and the mutant (wrd1). A: Seedling and root phenotypes of the wild type and mutant grown in nutrient solution hydroponics one week after germination; B: Field growth phenotypes of the wild type and mutant at different growth and development stages; E: Plant height, tillering and yield-related traits of the wild type and mutant at maturity.

[0020] Figure 2 Results of WRD1 gene positional cloning and transgenic function verification. A, WRD1 gene positional cloning; B, schematic diagram of WRD1 gene structure and mutation site; C, molecular identification results of wild type (WT), mutant (wrd1) and functional complementary transgenic lines (Com#1, Com#2); D, hydroponic seedling phenotypes of wild type, mutant and transgenic complementary lines; EF, statistics of taproot (E) and root hair (F) lengths of hydroponic seedlings of wild type, mutant and transgenic complementary lines; G, field phenotypes of wild type, mutant and transgenic complementary lines at maturity; HI, statistics of plant height (H) and tiller number (I) of wild type, mutant and transgenic complementary lines at maturity.

[0021] Figure 3 The expression pattern and subcellular localization analysis results of WRD1. A. The expression level detection results of WRD1 gene in different growth stages and different tissues of rice; B. The subcellular localization analysis results of WRD1.

[0022] Figure 4 The results of enzyme activity detection of WRD1 protein. A, protein electrophoresis diagram of WRD1 and its Arabidopsis homologous protein AtUGD2; B, WRD1-induced protease activity detection, with AtUGD2 and His protein as positive and negative controls respectively; C, WRD1 enzyme activity detection results in wild type (WT), mutant (wrd1) and transgenic complementation line (Com#1) plants.

[0023] Figure 5 The following are analysis diagrams of WRD1 gene overexpression transgenic plants. A, hydroponic seedlings and root hair phenotypes of wild type (WT), mutant (wrd1) and transgenic overexpression lines (OE-1, OE-2); B, real-time fluorescence quantitative PCR detection of the expression of WRD1 gene in wild type, mutant and transgenic overexpression lines; C, statistics of taproot (C) and root hair (D) lengths of hydroponic seedlings of wild type, mutant and transgenic overexpression lines; E, field phenotypes of wild type, mutant and transgenic overexpression lines at maturity; FG, statistics of plant height (F) and tiller number (G) of wild type, mutant and transgenic overexpression lines.

[0024] Figure 6 The following are the analysis diagrams of WRD1 knockout transgenic plants. A, molecular identification of WRD1 knockout lines (wrd1-2-1, wrd1-2-2); B, hydroponic seedlings and root hair phenotypes of wild type (Nipponbare, NIP) and WRD1 knockout lines; C, statistics of taproot length of hydroponic seedlings of wild type and WRD1 knockout lines; D, field growth phenotypes of wild type and WRD1 knockout lines; EF, statistical results of plant height (E) and tiller number (F) of wild type and WRD1 knockout lines. DETAILED DESCRIPTION

[0025] The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0026] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0027] Example 1: Isolation and identification of rice WRD1 gene

[0028] 1. Obtaining rice wrd1 mutant

[0029] Rice (Oryza sativa L.) wrd1 mutant plants (WRD1 gene mutation) were obtained by mutagenesis of the indica rice variety "Shuhui 527" (wild type; Oryza sativa subsp. Indica) with EMS (ethyl methanesulfonate). Compared with the wild type, the wrd1 mutant exhibits a phenotype of plant growth and development defects. Under normal field growth conditions, the mutant exhibits a phenotype of stunted growth throughout the growth period, with a weaker root system, reduced root length and adventitious roots, and no root hairs. The aboveground part shows a shorter plant height, and the number of tillers, panicle length, number of grains per panicle, and single plant yield are significantly reduced ( Figure 1).

[0030] 2. Positioning group creation

[0031] Using F 2 The segregating population was cloned by crossing the homozygous wrd1 mutant with the japonica rice variety “Nipponbare” (Oryza sativa subsp. japonica cv. Nipponbare) to generate the F 1 Generation, F 1 F 2 3048 mutant phenotype strains were screened from the F2 population and used for positional cloning of the WRD1 gene.

[0032] 3. WRD1 gene localization

[0033] Initial positioning of the WRD1 gene: The CTAB method was used to extract the genomic DNA of the wrd1 mutant phenotype individual plants isolated from the above F2 population. The DNA of 20 individuals was randomly selected to form a mixed pool, and PCR amplification was performed using 200 polymorphic SSR (simple sequence repeats) and STS (sequence tag sites) markers evenly distributed on the 12 chromosomes of rice. The total volume of the PCR reaction system was 20μL, including 2μL of DNA template (< 500ng), 10μL of 2×Taq Master Mix, 1μL of upstream and downstream primers (10mM), and ddH 2 O was added to 20 µL. The PCR reaction conditions were: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 32 cycles; 72°C extension for 5 min; 16°C constant temperature. The PCR product was detected by agarose gel electrophoresis (gel concentration was 5wt%), and linkage analysis was performed, and the WRD1 gene was preliminarily located between the molecular markers YP1 and YP2 on both sides of the centromere of rice chromosome 12 ( Figure 2 A in ).

[0034] Fine positioning of the WRD1 gene: Based on the initial positioning results, the rice genome BAC sequence within the WRD1 locus interval was obtained using a public database (https: / / rgp.dna.affrc.go.jp / E / index.html). By comparing the sequences between indica and japonica rice (http: / / www.ncbi.nlm.nih.gov / BLAST), primers were designed on both sides of the differential segment sequence, and the amplified fragment size was between 100-250bp. The marker polymorphism was detected by PCR amplification and agarose gel electrophoresis, and 9 new polymorphic STS markers (YP3~YP11) were successfully developed within the initial positioning interval. These new STS markers were used to perform marker linkage analysis on recessive mutant plants isolated from the remaining 3028 F2 populations, and the WRD1 gene positioning interval was finally narrowed down to the 594-kb region between YP9 and YP10 on chromosome 12 ( Figure 2 A in ).

[0035] Table 1. Primers used for WRD1 map-based cloning

[0036] Primer name Upstream primer sequence (5' - 3') Downstream primer sequence (5' - 3') YP1 ATCAGCAGCAGATTGGTGC TACCCTTAGTCTCCTATGTGTCC YP2 CTCGATCCCCTAGCTCTC TCACCTCGTTCTCGATCC YP3 GCATGTAAGGTGAATGGTCAAG AAAGTAGTGGTCGAAGTGTG YP4 AGGTTCAAATGAGACCCAAT GGTGTTATGTATACGTGGGT YP5 AGGTTGCTGTTTCGATGCGT TGGAGAGGCGTGAATCTAAC YP6 CGAGGAGCCACCTGATCC AGGCGATGGAGGATTTGAC YP7 TTGTGACCAACCGAATTGAA TATGGGCTGCCATAGTTCCT YP8 TTGTTGATGGCACATGGTCT CTTTGGAGACTTGAGCAGCAT YP9 AGTCCGTAAAACGCGATGAG GTTGGAAGGCACCAGGAATA YP10 GGGTGCGTATTATTGCGAGT CTTACACTCGGCGCTCCTT YP11 ATGACGTGTGGATATGGGGT GCATCACAGCAAAGCTGAAA

[0037] 4. Candidate gene prediction and sequence analysis

[0038] Since the WRD1 locus is located near the centromere, the target interval could not be further narrowed using the expanded F2 population, so we used whole genome resequencing to analyze the sequence differences within the target interval. The genomic DNA of 20 recombinant plants was mixed to form a mutant pool, and the wild-type "Shuhui 527" genomic DNA was used as a reference for whole genome resequencing analysis. According to the results of SNP analysis, we detected a SNP located in the exon segment of the gene (LOC_Os12g25690) within the finely mapped target interval. Sequencing primers WRD1-F1: 5'-TCAGGCTCTCAAGGAGGTGT-3' and WRD1-R1: 5'-GTCCTTCTTGAACGCAAAGC-3' were further designed on both sides of the mutation site of the target gene. The genomic sequence of the candidate gene was amplified from the wild-type and mutant plants by PCR (reaction system and conditions are the same as above) and sequenced and analyzed. The results showed that ( Figure 2 In Figure (B), the wrd1 mutant had a single base mutation at the 826 bp position (1699th position in the genome sequence) of the CDS sequence of the LOC_Os12g25690 gene, from G to A, resulting in the conversion of the encoded amino acid from aspartic acid (Asp) to asparagine (Asn).

[0039] The CDS sequence of the gene is 1443 bp in length, and the nucleotide sequence is shown in SEQ ID NO.1; the protein encoded by the gene contains 480 amino acids, and the sequence is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041]

[0042] SEQ ID NO.2:

[0043] MVKICCIGAGYVGGPTTMAVIALKCPAIEVVVVDISKPRIDAWNSEQLPIYEPGLDEVVKECRGRNLFFSTDVEKHVAEANIIFVSVNTPTKTRGLGAGKAADLTYWESAARMIADVSKSD KIVVEKSTVPVKTAEAIEKILTHNSKGINYQILSNPEFLAEGTAIEDLFKPDRVLIGGRETPEGKKAVQALKEVYAHWVPEDRIITTNLWSAELSKLAANAFLAQRISSVNAISALCEAT GANVAEVAYSVGKDSRIGPKFLNASVGFGGSCFQKDILNLVYICECNGLPEVANYWKQVIKINDYQKSRFVNRVVSSMFNTVSGKKIAVLGFAFKKDTGDTRETPAIDVCHGLLGDKAQI SIYDPQVTEDQIQRDLAMSKFDWDHPMHLQPTSPTAFKQVSVVWDAYEATKGAHGVCILTEWDEFKTLDYQKIFDNMQKPAFVFDGRNVVDAEKLREIGFIVYSIGKPLDAWLKDMPAVA

[0044] Example 2

[0045] Transformation of WRD1 functional complementation transgenic plants:

[0046] Design primers based on the target gene (LOC_Os12g25690):

[0047] pC-WRD1-F: 5'- ggtaccTGCCAGAAGAGAGGAAAGGA-3';

[0048] pC-WRD1-R: 5'-gtcgacCGCTGGATTGTGAGACAGAA-3'.

[0049] Using the genomic DNA of the japonica rice variety Nipponbare as a template, the above-mentioned pCWRD1-F and pCWRD1-R primers were used to perform PCR amplification using Q5® Ultra-Fidelity DNA Polymerase (NEB, M0492S). The amplified fragment was 5.4 kb in length, including 2466 bp upstream of the 5'-end of the gene, the full length of the target gene, and 1492 bp downstream of the 3'-end. The PCR reaction system was 20 µL, including: DNA template (<500 ng) 2 µL, Q5® High-Fidelity 2X Master Mix 10µL, upstream and downstream primers (10mM) 1µL each, ddHO 2 O was added to 20 µL. The PCR reaction conditions were: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 66°C annealing for 30 s, 72°C extension for 3 min, 30 cycles; 72°C extension for 2 min; 4°C constant temperature. The target size fragment of the PCR product was recovered after electrophoresis and cloned into the T5-zero vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.; CT501). The transformation method was carried out according to the instructions of the kit. After the transformation was completed, a single clone was picked to extract the plasmid. After sequencing verification, the WRD1 gene fragment on the T5-zero vector was double-digested with KpnⅠ and SalⅠ and transferred into the binary vector pCAMBIA1300 (from the Institute of Genetics, Chinese Academy of Sciences) to obtain the fusion vector pCAMBIA1300-WRD1. The constructed vector was transferred into the Agrobacterium tumefaciens strain EHA105, and the wrd1 mutant was transformed using the Agrobacterium-mediated method (the transgenic method can be found in the following literature: Toki S, et al. 2006. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J, 47: 969-976). The genomic DNA of the obtained transgenic strain was extracted, and PCR amplification was performed using primers WRD1-F2: 5'-CTCTACGCTTTCTCCCATCG-3' and WRD1-R2: 5'-GGTCTTCTGAGACTGTATCTTTG-3'. The PCR product was digested with restriction endonuclease MboⅡ to identify transgenic positive plants ( Figure 2 The positive transgenic lines were subcultured to obtain T3 homozygous lines for phenotypic analysis. The phenotypic analysis results showed that ( Figure 2 DI in ), compared with the wild-type and wrd1 mutant plants of the same period, the growth and development defects of the two WRD1 complementary transgenic homozygous lines were restored to the wild-type level under nutrient solution hydroponic and field growth conditions.

[0050] Example 3

[0051] 1. Analysis of WRD1 gene expression pattern

[0052] Samples were collected from the wild-type indica rice variety Shuhui 527 at different growth stages: roots and leaves at the seedling stage; roots, stems, stem bases, and leaves at the tillering stage; roots, stems, leaves, leaf sheaths, and panicles at the booting stage. Total RNA from the above rice samples was extracted using the TRIzol™ (Invitrogen) method, and cDNA was generated by reverse transcription using the TaKaRa quantitative PCR dedicated reverse transcription kit (PrimeScript™ RT reagent Kitwith gDNA Eraser; RR047). Primers were designed based on the WRD1 gene sequence: WRD1-qF: 5'-CCATTGATGTGTGCCACGGT-3' and WRD1-qR: 5'-TGGTCCCAGTCGAACTTGCT-3'. Then, fluorescence quantitative PCR (qRT-PCR) detection was performed using a CFX96 fluorescence quantitative PCR instrument (Bio-Rad). The qRT-PCR reaction used the TaKaRa TB Green qPCR kit (TB Green® Premix Ex Taq™ II; RR820). All methods involving the kit were performed according to the relevant instructions. Figure 3 As shown in A, the WRD1 gene was expressed in all tested tissues at all stages, with the highest expression levels in roots and stems.

[0053] 2. Subcellular localization of WRD1 protein

[0054] Primers were designed based on the CDS sequence of the WRD1 gene (LOC_Os12g25690):

[0055] pG-WRD1-F: 5'-aagcttATGGTGAAGATCTGCTGCATT-3';

[0056] pG-WRD1-R: 5'-actagtTGCAACCGCGGGCATGTC-3'.

[0057] The amplified fragment contains the full-length sequence of the gene CDS. Using the cDNA of the japonica rice variety Nipponbare as a template, PCR amplification was performed using the Q5 high-fidelity enzyme system of NEB, and the reaction system and conditions were the same as in Example 2. After electrophoresis of the PCR product, the target fragment was recovered and cloned into the T5-zero vector (same as in Example 2). After sequencing verification, it was cloned into the pGA3427 vector (derived from the following reference: Kim SR, 2006. Cloning vectors for rice. J Plant Biol, 52:73-78) to obtain the fusion vector CaMV 35S::WRD1-GFP. The 35S::WRD1-GFP fusion vector was transferred into freshly prepared rice protoplasts (extracted from the japonica rice variety Nipponbare) by the PEG transformation method and observed by laser confocal microscopy, as shown in FIG. Figure 3 As shown in B, the fusion protein GFP signal appears in the cytoplasm, indicating that WRD1 is a cytoplasmic-localized protein in rice.

[0058] 3. WRD1 protease activity detection

[0059] Primers were designed based on the CDS sequence of the WRD1 gene (LOC_Os12g25690):

[0060] pE-WRD1-F: 5'-gaattcATGGTGAAGATCTGCTGCATT-3';

[0061] pE-WRD1-R: 5'-gtcgacTGCAACCGCGGGCATGTC-3'.

[0062] The amplified fragment contains the full-length sequence of the gene CDS. Using the cDNA of the japonica rice variety Nipponbare as a template, PCR amplification was performed using the Q5 high-fidelity enzyme system of NEB, and the reaction system and conditions were the same as in Example 2. After electrophoresis of the PCR product, the target fragment was recovered and cloned into the T5-zero vector (same as in Example 2). After sequencing verification, it was cloned into the pET30a vector to obtain the WRD1-His fusion expression vector. In order to induce the expression of WRD1 protein, the constructed WRD1-His fusion expression vector was transformed into Escherichia coli BL21 (DE3) competent cells for protein induced expression. The steps of protein induction expression and purification are as follows: the transformed BL21 (DE3) was streaked in LB solid medium (containing 50 mg / L ampicillin), and a single colony was picked in LB liquid medium (containing 50 mg / L ampicillin), and cultured overnight at 37°C in a shaker (200 rpm); the above bacterial solution was transferred to 500 mL of the same liquid LB medium, and cultured at 37°C in a shaker (180 rpm) until OD600 = 0.6; the bacterial solution was allowed to stand at room temperature for 1 hour, 10 μL of 1M IPTG solution was added, and the solution was induced overnight at 22°C in a shaker (150 rpm); the bacterial cells were collected by centrifugation (6000 rpm) and 50 ml centrifuge tubes were placed in a -20°C freezer overnight; 30 mL of pH = 7.2 phosphate buffer (PBS; containing 30 μL of protease inhibitor) was added to the centrifuge tube to resuspend the bacterial cells, and the cells were broken by an ultrasonic cell disruptor (35 Hz); 4°C Centrifuge at 10000rpm for 60 minutes, take the supernatant into a new 50ml centrifuge tube, and centrifuge again under the same conditions; mix the supernatant obtained from the second centrifugation with 500μL Beads pre-equilibrated with PBS, and rotate at 4℃ overnight to combine; let the centrifuge tube stand on ice, wait for the beads to precipitate, discard the supernatant, wash the beads with 25mL pre-cooled PBS, discard the supernatant, repeat the washing once, then transfer the beads to a 1.5mL centrifuge tube, and remove the supernatant by centrifugation; add 1mL 400mM imidazole solution for elution overnight (4℃), and collect the supernatant by centrifugation; finally, identify the induced protein by SDS-PAGE and Coomassie Brilliant Blue staining.

[0063] At the same time, aerial samples were taken at the seedling stage of the wild type, mutant and complementary transgenic lines, and the total rice protein was extracted using Tris-HCl buffer (50 mM, pH 6.8; containing 150 mM NaCl and 0.5 wt% SDS) as follows: take an appropriate amount of rice tissue, grind it into powder with liquid nitrogen, weigh 0.1 g of the powder into a 2.0 ml centrifuge tube, add 300 μL of the above Tris-HCl buffer, and vortex to mix thoroughly; centrifuge at 4°C and 12,000 rpm for 10 min, and take the supernatant, which is the crude protein extract. The purified protein and total plant protein obtained above were quantified by Coomassie Brilliant Blue, and then the UDP-glucose dehydrogenase activity was detected (the enzyme activity detection method refers to the following literature: Klinghammer and Tenhaken, 2007. Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls. J Exp Bot, 58:3609-3621). The results are as follows Figure 4 As shown, WRD1 has UDP-glucose dehydrogenase activity both in vitro and in vivo in rice.

[0064] Example 4

[0065] 1. Transformation of WRD1 overexpressing plants

[0066] Primers were designed based on the CDS sequence of the WRD1 gene (LOC_Os12g25690):

[0067] pA-WRD1-F: 5'-TAGACGATAAGCTTGGGCCATGGTGAAGATCTGCTGCA-3';

[0068] pA-WRD1-R: 5'-TGGCGGCCGCTCTAGATGCAACCGCGGGCATGTC-3'.

[0069] According to Example 3, pA-WRD1-F and pA-WRD1-R were used to amplify the full-length CDS sequence of the WRD1 gene, and the amplified fragment was integrated into the AHLG vector (from the Institute of Genetics, Chinese Academy of Sciences) using In-Fusion HD Cloning kits (TaKaRa; 639649) to construct a CaMV 35S::WRD1 expression vector. After sequencing was correct, it was transferred into the Agrobacterium strain EHA105 and transformed into the callus induced by the mature embryos of the wrd1 mutant (the transgenic method is the same as that in Example 2). Genomic DNA was extracted from the transformed T0 generation plants (the method is the same as that in Example 2), and PCR amplification was performed using primers WRD1-F3: 5'-TATGCAGCAGCTATATGTGG-3' and WRD1-R3: 5'-TGGTCCCAGTCGAACTTGCT-3' to identify positive transgenic strains. The above positive transgenic strains were subcultured to obtain T3 generation homozygous strains. The expression level of WRD1 gene in the homozygous overexpression strains was detected by qRT-PCR method, and the method was referred to Example 3. The results showed that the transcription level of WRD1 in the overexpression transgenic strains of WRD1-OE-1 and WRD1-OE-2 was significantly upregulated compared with the wild type. Through hydroponic and field phenotypic observations, it was found that the growth and development phenotypes of the two overexpression plants were restored to the wild type level, and the root length of the hydroponic seedlings was significantly increased compared with the wild type ( Figure 5 ).

[0070] 2. Creation and phenotypic analysis of WRD1 gene knockout mutants

[0071] The experimental variety used in the present invention is the japonica rice variety Nipponbare, and the WRD1 gene knockout mutant was created using CRISPR / Cas9 editing technology. According to the WRD1 gene sequence, a Cas9 recognition site was designed at 1525 bp and 1540 bp of the gene gDNA, respectively. Figure 6As shown in A in the figure, the target sequences of its sgRNA are GGCCGAGGTTGCTTACTCTGTGG and CCAGGATTGGCCCCAAGTTCCT. A CRISPR / Cas9-WRD1 knockout vector was constructed, and after sequencing verification, it was transferred into the Agrobacterium strain EHA105 and transformed into callus induced by mature embryos of Nipponbare (the transgenic method was the same as in Example 2). The genomic DNA was extracted from the T0 generation transformed plants (see Example 2 for the method), and the primers WRD1-F4: 5'-AAGACCAATGCGGAGCATATAC-3' and WRD1-R4: 5'-GTGATATCTCCACTGACGTA-3' were used to perform PCR amplification to identify the transgenic positive plants, and the primers WRD1-F5: 5'-CAAGGAGGTGTACGCCCAC-3' and WRD1-R5: 5'-GTCGAACTTGCTCATGGCG-3' were further used to amplify and sequence the positive transgenic plants (DNA sequencing was performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd.). Through sequence analysis, two homozygous mutants wrd1-2-1 and wrd1-2-2 were identified. Compared with the wild-type WRD1 sequence, wrd1-2-1 had a T insertion at 1521bp and a T deletion at 1543bp in the gDNA sequence of the target gene (LOC_Os12g25690); wrd1-2-2 had a 21bp deletion (gDNA 1522bp-1542bp), resulting in changes in the corresponding encoded amino acid sequence ( Figure 6 A in ).

[0072] The above homozygous mutant transgenic lines were subcultured to obtain T3 homozygous lines. The growth phenotypes of the homozygous mutant plants wrd1-2-1 and wrd1-2-2 showed that compared with the wild-type Nipponbare, both plants showed shortened root length, lack of root hairs, shorter plant height, and reduced tillering, which were similar to the phenotypes of the wrd1 mutant, such as Figure 6 As shown in BF.

[0073] In summary, the present invention discloses for the first time that the WRD1 gene can regulate the growth and development of rice. The mutation of the WRD1 gene will significantly affect the normal formation of the plant cell wall, resulting in some growth and development phenotypes such as weakened root system, lack of root hairs, shorter plant height, reduced tillering, short panicle shape, and decreased yield; while overexpression of the WRD1 gene can restore the plant growth and development phenotype to the wild-type level.

[0074] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined in the present invention may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed in the present invention.

Claims

1. WRD1 The application of genes in regulating rice growth and development is characterized in that: Said WRD1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. WRD1 The amino acid sequence of the gene encoding protein is shown in SEQ ID NO.2, which upregulates rice WRD1 Gene expression or increase rice WRD1 The activity of gene-encoded protein is increased, and the growth and development performance of rice is improved.

2. The use according to claim 1, characterized in that: The regulating rice growth and development includes regulating the root growth, plant height, tillering number, panicle shape and / or yield of the rice plant.

3. A method for improving the growth and development performance of rice, characterized in that: The method comprises upregulating or increasing WRD1 Gene expression may increase rice WRD1 The activity of the protein encoded by the gene makes the rice grow and develop better than the recipient rice; Said WRD1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. WRD1 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

Images