Growth-promoting gene EoBr2 from Ervatamia oxyphylla and its plant expression vector and application

Abstract

The present invention belongs to the field of plant genetic engineering and transgenic breeding, and discloses a growth-promoting gene EoBr2 of centipedegrass and a plant expression vector and application thereof. The nucleotide sequence of the growth-promoting gene EoBr2 of centipedegrass is shown in SEQ ID NO.1. A plant expression vector pCAMBIA1305.2M‑EoBr2 containing the growth-promoting EoBr2 gene is constructed and introduced into plants. Phenotypic observation and statistical analysis of transgenic plants and wild-type plants show that the plant height and total root length of transgenic plants are significantly increased compared with wild-type plants. The present invention regulates rice growth by transforming exogenous auxin-related genes and expressing them normally through transcription, thereby obtaining new germplasm with a relatively strong growth rate, which is of great significance for the cultivation of excellent lawn varieties and their wide application in production.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to an Ervatamia fruticosa growth-promoting gene EoBr2 and a plant expression vector and application thereof. Background Art

[0002] Eremochloa ophiuroides (Munro.) Hack. belongs to the genus Munroe of the subfamily Panicideae of the Poaceae family. It is the only excellent warm-season C4 herbaceous plant in the genus that can be used as a lawn grass. Eremochloa ophiuroides is mainly distributed in the Yangtze River Basin and the areas to the south of it. It is currently recognized as the best lawn grass originating from my country. It has beautiful leaves and uniform appearance, and the lawn it forms is flat and beautiful. It also has the advantages of strong soil fixation ability, acid and barrenness resistance, few diseases and pests, and extremely low maintenance level (the number of mowing times per year can be as low as 1-2 times) (Liu Mingxi et al., 2012; Islam and Hirata, 2005). Under the increasingly severe situation of water resources and energy resources in my country and even the world, and under the guidelines of energy conservation and emission reduction in my country, Eremochloa ophiuroides has a significant advantage with its characteristics of barrenness resistance, few diseases and pests, and low management intensity, and has a wide range of application prospects in my country's ecological environment construction. However, compared with the two most widely used warm-season lawn grasses, Bermuda grass and Zoysia, Eremochloa ophiuroides is slightly inferior. It grows slowly and forms a lawn slowly, which makes it vulnerable to weed invasion during the formation process (Liu Jianxiu and He Shan'an, 1996; Liu Jianxiu, 2011, Zhu Zhaohua et al., 2017), increasing its maintenance cost during the formation period and seriously affecting its widespread promotion and application.

[0003] Auxin is one of the important plant hormones that can regulate various morphological development and organ formation processes of plants (Adamowski and Friml, 2015). Auxin is mainly synthesized in the stem and root tips, and is transported to the effector site through polar transport to the basipetal. It is the only known plant hormone with polar transport. The polar transport of auxin determines its distribution gradient in different parts of the plant, thereby determining multiple plant growth and development processes such as embryo development, organ formation, apical dominance, vascular differentiation and elongation growth (Friml and Wísniewska, 2005). Current studies have shown that there are three major protein families involved in the polar transport of auxin: AUX1 / LIKE AUX1 (auxinresistant 1 / like aux1, AUX1 / LAX) family, PIN (pin-formed, PINs) family and ABC (ATP-binding cassette, ABC) / MDR (multidrug resistance, MDR) / PGP (P-Glycoprotein, PGP) family members (Friml, 2003). Existing studies have shown that auxin transport-related genes can affect plant growth rate and root development. Arabidopsis Aux1 mutations cause a reduction in lateral roots (Marchant et al., 2002), while LAX3 mutations cause a delay in the appearance of lateral roots (Swarup et al., 2008). Rice OSPIN9 overexpression lines have more tillers and taller plants (Hou et al., 2021). Mutation of atpgp1 in Arabidopsis causes elongation of the hypocotyl under low light (Sidler et al., 1998), mutation of atpgpg19 causes semi-dwarf phenotype in Arabidopsis (Noh et al., 2001), and atpgp1 / atpgp19 double mutants show a more obvious semi-dwarf phenotype (Geisler et al., 2003). Mutation of maize ZmBr2 (ZmPGP1) and sorghum SbDw3 causes shortened internodes, reduced plant height, and smaller leaf angle. An 882bp repeat fragment in the fifth exon of sorghum SbDw3 is found in 73 of the 159 sorghum varieties worldwide, proving that the insertion of this fragment has been widely used in sorghum (Xing et al., 2015; Wei et al., 2018). Resequencing analysis of 68 maize local varieties, 349 maize inbred lines and 32 teosinte materials revealed the presence of multiple ZmBr2 (ZmPGP1) haplotypes in the above materials, proving that during the maize domestication process, ZmBr2 (ZmPGP1) was domesticated and selected as density increased (Li et al., 2019).

[0004] The breeding of centipedegrass is mainly based on traditional breeding methods, which have a long breeding cycle and low efficiency. Using modern biological methods to express specific genes through genetic transformation has become an important way to quickly and efficiently obtain high-quality germplasm resources. Genes that affect plant growth have become the top priority of this process. The centipedegrass auxin transport-related gene EoBr2 was transformed into rice using Agrobacterium-mediated genetic transformation methods, and new resources with increased plant height and longer root systems were obtained, which is of great significance for the cultivation of new and excellent centipedegrass germplasm resources.

[0005] References:

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[0011] Schulz,B.(2003)TWISTED DWARF1,a unique plasma membrane-anchoredimmunophilin-like protein,interacts with Arabidopsis multidrugresistance-like transportersAtPGP1 and AtPGP19.Mol.Biol.Cell 14:4238–4249.

[0012] Hou,M.,Luo,F.,Wu,D.,Zhang,X.,Lou,M.,Shen,D.,Yan,M.,Mao,C.,Fan,X.,Xu,G.andZhang,Y.(2021),OsPIN9,an auxin efflux carrier,is required for theregulation of rice tillerbud outgrowth by ammonium.New Phytol,229:935-949.https: / / doi.org / 10.1111 / nph.16901Islam M.Anowarul and Hirata Masahiko.(2005).Centipedegrass(Eremochloa ophiuroides(Munro)

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[0014] Li Pengcheng,Wei Jie,Wang Houmiao,Fang Yuan,Yin Shuangyi,Xu Yang,LiuJun,YangZefeng,and Xu Chenwu.(2019).Natural variation and domesticationselectionof ZmPGP1 affects plant architecture and yield-related traits inMaize.Genes(Basel),10(9):

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[0018] Noh,B.,Murphy,A.S.and Spalding,E.P.(2001)Multidrug resistance-likegenes of Arabidopsis required for auxin transport and auxin-mediateddevelopment.Plant Cell 13:2441–2454.Swarup K,BenkováE,Swarup R,Casimiro I,Péret B,Yang Y,Parry G,Nielsen E,De Smet I,

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[0021] Wei Lai, Zhang Xuan, Zhang Zhihai, Liu Huanhuan, and Lin Zhongwei. (2018). A new allele of the brachytic2 gene in maize can efficiently modify plant architecture. Heredity, 121(1): 75-86. Xing Anqi, Gao Yufeng, Ye Lingfeng, Zhang Weiping, Cai Lichun, Ching Ada, Llaca Victor, Johnson Blaine, Liu Lin, Yang Xiaohong, Kang Dingming, Yan Jianbing, and Li Jiansheng.

[0022] (2015). A rare SNP mutation in Brachytic2 moderately reduces plantheight and increases yieldpotential in maize. J Exp Bot., 66(13):3791-3802.

[0023] Liu Mingxi, Lu Shaoyun and Guo Zhenfei. (2012). Screening and physiological identification of drought-resistant variants of Centipedegrass. Acta Prataculturae Sinica,

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[0025] Liu Jianxiu and He Shanan. (1996). Research and improvement of warm-season turfgrass germplasm resources. Grassland and Turf, (3): 12-21.

[0026] Liu Jianxiu, Zhu Xuehua, Guo Aigui and Guo Hailin. (2004). Variation of main characters and morphological types of Eremochloa chinensis germplasm resources in China.

[0027] Acta Grassland Sinica, 12(3):183-188.

[0028] Zhu Zhaohua, Bai Shiqi, Guan Zhaoying, Chen Xiaorong and Xu Guogang (2017). Characteristics and application of native centipedegrass in my country.

[0029] Art, (10):80-86. Summary of the invention

[0030] The purpose of the present invention is to provide a new auxin polar transport related gene EoBr2 to address the problem of slow turf formation of Ervatamia fruticosa in actual production. The sequence of the gene is SEQ ID NO.1.

[0031] Another object of the present invention is to provide a plant expression vector of the EoBr2 gene and its application. Construction of the plant expression vector of the EoBr2 gene can determine the promotion of plant height and root growth of EoBr2, and provide an excellent gene for plant germplasm improvement.

[0032] The purpose of the present invention can be achieved by the following technical solutions:

[0033] The centipedegrass growth-promoting gene EoBr2 is a gene related to polar auxin transport, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0034] The biological material contains the centipedegrass growth-promoting gene EoBr2, which is a recombinant vector, an expression cassette, a transgenic cell line or a transgenic recombinant bacterium.

[0035] As a preferred technical solution, the recombinant vector is a recombinant expression vector, which is obtained by transferring the above-mentioned centipede grass growth-promoting gene EoBr2 into a plant expression vector. Preferably, the recombinant expression vector is obtained by homologous recombination reaction between the EoBr2 gene fragment with the pCAMBIA1305.2M homology arm and the pCAMBIA1305.2M expression vector that has been digested. The pCAMBIA1305.2M is transformed from the commercially available pCAMBIA1305.2, and the 35S promoter upstream of the pCAMBIA1305.2 GUS gene is replaced with the corn Ubiquitin promoter to obtain pCAMBIA1305.2M. The vector is purchased from Nanjing Yibais Biotechnology Co., Ltd.

[0036] A method for constructing a plant expression vector of an EoBr2 growth-promoting gene of Centipedegrass, comprising the following steps:

[0037] (1) Cloning of the growth-promoting gene EoBr2 from Centipedegrass

[0038] Using 'Ganbei' Eremochloa chinensis as material, we extracted total RNA from leaves, reverse transcribed it into cDNA, and designed primers to amplify the EoBr2 gene.

[0039] Upstream primer EoBr2-F: 5′-ATGCTCGTCGGCACCCTC-3′ (SEQ ID NO. 2),

[0040] Downstream primer EoBr2-R: 5′-CGCCGCGGCCCCGTTGGAT-3′ (SEQ ID NO. 3),

[0041] The reverse transcribed cDNA was used as a template, and EoBr2-F and EoBr2-R were used as primers to perform PCR reaction. The PCR product was connected to the pCE2-T vector, and DH5α competent cells were transformed. The positive plasmid pCE2-EoBr2 was extracted, and the sequence was determined to be SEQ ID NO.1;

[0042] (2) Construction of plant expression vector pCAMBIA1305.2M-EoBr2

[0043] Primers were designed using the positive plasmid pCE2-EoBr2 sequenced correctly in (1) as a template, and homology arms at the EcoR I and Hind III sites of pCAMBIA1305.2M were introduced into the upstream and downstream of the EoBr2 gene, respectively.

[0044] Upstream primer EoBr2-EcoRI-F: 5′-acctctagacccggggaattcATGCTCGTCGGCACCCTC-3′, (SEQ ID NO. 4),

[0045] Downstream primer EoBr2-Hind III-R: 5′-cttgtcgacactagtaagcttCGCCGCGGCCCCGTTGGAT-3′, (SEQ ID NO. 5);

[0046] PCR amplification was carried out using the extracted positive plasmid pCE2-EoBr2 as a template. The PCR product was recovered through gel and then subjected to homologous recombination reaction with the pCAMBIA1305.2M linearized vector that had been digested with EcoR I and Hind III and recovered and purified. The recombinant product was transformed into Escherichia coli competent cells DH5α. After single clones were identified by PCR, the positive plasmid was extracted and sequenced to verify that it was SEQ ID NO.1. The plant expression vector pCAMBIA1305.2M-EoBr2 was successfully constructed.

[0047] (3) The pCAMBIA1305.2M-EoBr2 plant expression vector obtained in step (2) is introduced into rice by Agrobacterium-mediated method, and positive transformed plants are obtained by hygromycin resistance screening. The positive transformed plants are tested by qPCR to verify that the exogenous gene has been successfully inserted into the genome of the transgenic plant, and transcription and its expression level have increased; thus, transgenic rice plants with more vigorous growth are obtained.

[0048] The process of performing qPCR detection on positive transformed plants is as follows:

[0049] Take the young leaves of the positive transformed plants and the young leaves of the untransformed plants obtained by hygromycin resistance screening, extract the total RNA, and reverse transcribe it into cDNA. Design the upstream and downstream primers on the two adjacent exons respectively. The fragment length is 202bp. The primer sequence is:

[0050] Upstream primer EoBr2-q6F: 5′-TTCATCATCAAGCTCCCCGA-3′ (SEQ ID NO. 6),

[0051] Downstream primer EoBr2-q6R: 5′-CGATCATGAAGCGGTCCAG-3′ (SEQ ID NO. 7).

[0052] qPCR detection was performed using cDNA of positive transformed plants and untransformed plants as templates and EoBr2-q6F and EoBr2-q6R as primers, and the changes in their expression levels were analyzed;

[0053] The gene fragment amplified by rice OsUbiquitin was used as the internal standard. The fragment length was 117 bp. The primer sequence was:

[0054] Upstream primer Ubq-F: 5′-GCTCCGTGGCGGTATCAT-3′ (SEQ ID NO. 8),

[0055] Downstream primer Ubq-R: 5′-CGGCAGTTGACAGCCCTAG-3′ (SEQ ID NO. 9).

[0056] The technical solution described in the present invention utilizes the Agrobacterium tumefaciens-mediated method to introduce the EoBr2 gene into rice, and screens the callus tissue with hygromycin; the resistant callus tissue differentiates into positive transformed plants, and qPCR detection is performed on the plants to verify that the exogenous gene has been transferred into the genomic DNA of the transgenic plants and transcription has occurred and the expression level has increased, and phenotypic observation is performed on the offspring of the transgenic plants to determine the function of the EoBr2 gene of Ervatamia oxyphylla in promoting plant growth.

[0057] The detailed process of introducing the EoBr2 gene into rice by Agrobacterium-mediated method and cultivating transgenic plants is as follows:

[0058] Take 10 μL of the pCAMBIA1305.2M-EoBr2 plant expression vector obtained in step (2), add 100 μL of Agrobacterium competent EHA105, ice bath for 30 min, freeze in liquid nitrogen for 5 min, 37°C for 5 min, add 800 μL of YEB liquid culture medium, pre-culture at 28°C 200 rpm for 4 h, spread on YEB (50 μg / mL rifampicin + 50 μg / mL kanamycin) plates, and culture at 28°C for about 24h-36h. Single EHA105 colonies were picked from the plate and positive colonies were detected by PCR. The PCR system was 10 μL: 2×Rapid Taq Master Mix 5 μL, upstream primer EoBr2-KZF 0.3 μL, downstream primer EoBr2-KZR 0.3 μL, ddH2O 4.4 μL. A few plaques were picked with a sterilized pipette tip and mixed in the above PCR system by blowing; the reaction procedure was: 95°C pre-denaturation for 3 min, then 95°C melting for 15 sec, 60°C annealing for 15 sec, 72°C extension for 20 sec, reaction for 35 cycles, and 72°C extension for 5 min; the product size was about 434 bp, and the PCR product was detected by 1% agarose gel electrophoresis ( Figure 3 ), and the positive clones were used for subsequent transformation of rice callus. Rice transformation was completed by Biotech Co., Ltd.

[0059] Plants differentiated from resistant callus were selected and transferred to soil for cultivation after 7 days of seedling training. Total RNA was extracted from fresh leaves of transformed plants and wild-type plants and reversely transcribed into cDNA. Upstream and downstream primers were designed on two adjacent exons, with a fragment length of 202 bp. The primer sequences were:

[0060] Upstream primer EoBr2-q6F: 5′-TTCATCATCAAGCTCCCCGA-3′ (SEQ ID NO. 6),

[0061] Downstream primer EoBr2-q6R: 5′-CGATCATGAAGCGGTCCAG-3′ (SEQ ID NO. 7).

[0062] qPCR detection was performed using cDNA of positive transformed plants and untransformed plants as templates and EoBr2-q6F and EoBr2-q6R as primers, and the changes in their expression levels were analyzed;

[0063] The gene fragment amplified by rice OsUbiquitin was used as the internal standard. The fragment length was 117 bp. The primer sequence was:

[0064] Upstream primer Ubq-F: 5′-GCTCCGTGGCGGTATCAT-3′ (SEQ ID NO. 8),

[0065] Downstream primer Ubq-R: 5′-CGGCAGTTGACAGCCCTAG-3′ (SEQ ID NO. 9).

[0066] The seeds of the wild-type seeds and the seeds of the positive plants were soaked and germinated, and the positive plants were identified by single-plant PCR. Then they were transferred to nutrient solution hydroponics for about 25 days, and the growth of the aboveground and underground roots of the plants was observed. It was found that compared with the wild-type rice, the height and root system of the transgenic rice were significantly elongated ( Figure 4 Figure 5 Figure 6 ).

[0067] The application of the growth-promoting gene EoBr2 of Ervatamia fruticosa in improving plant growth ability or cultivating new germplasm with stronger growth ability also falls within the protection scope of the present invention.

[0068] The application of biological materials containing the above-mentioned centipedegrass growth-promoting gene EoBr2 in improving plant growth ability or cultivating new germplasm with stronger growth ability.

[0069] The above application overexpresses the above-mentioned centipede grass growth-promoting gene EoBr2 in the target plant.

[0070] During the research process of the present invention, the EoBr2 gene plant expression vector was introduced into rice to promote the growth of rice plants. It was determined that EoBr2 has the function of promoting plant growth and can be used for the improvement of Ervatamia fruticosa germplasm.

[0071] The centipedegrass growth-promoting gene EoBr2 and its plant expression vector and application provided by the present invention have the following advantages:

[0072] The present invention screened a growth-promoting gene EoBr2 of Centipedegrass, which is a gene related to polar transport of auxin, constructed a plant expression vector pCAMBIA1305.2M-EoBr2 containing the growth-promoting EoBr2 gene, introduced it into plants, and promoted plant growth. This provides an effective solution and idea and potential utilization genes for the problem of slow growth during the establishment of Centipedegrass lawns. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Diagram of the construction of the pCAMBIA1305.2M-EoBr2 plant vector.

[0074] Figure 2 Agarose gel electrophoresis of the plant expression vector pCAMBIA1305.2M-EoBr2 transformed into EHA105 Agrobacterium for identification;

[0075] M1: Marker 5000; 1-3: EHA105 monoclonal; 4-5: water negative control; 6: pCAMBIA1305.2M-EoBr2 plasmid.

[0076] Figure 3 Identification of the expression level of transgenic EoBr2 rice;

[0077] WT: wild-type plant Nipponbare; OE-EoBr2-n: EoBr2-transgenic rice line.

[0078] Figure 4 Phenotypic observation of transgenic rice with EoBr2 gene;

[0079] WT: wild-type plant Nipponbare; OE-Br2-n: rice line transformed with EoBr2. Scale bar = 3 cm.

[0080] Figure 5 Statistics of transgenic rice plant height;

[0081] WT: wild-type plant Nipponbare; OE-Br2-n: rice line transformed with EoBr2.

[0082] Figure 6 Statistics of total root length of transgenic rice;

[0083] WT: wild-type plant Nipponbare; OE-Br2-n: rice line transformed with EoBr2. DETAILED DESCRIPTION

[0084] The specific embodiments of the invention are described in detail below: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given. The specific implementation method is as follows:

[0085] Example 1. Cloning of EoBr2 gene

[0086] Using Ervatamia oxyphylla ‘Ganbei’ as the material, 0.1 g of leaves were taken, and the total RNA of the leaves was extracted according to the instructions of the Trizol RNA extraction kit (TaKaRa). Reverse transcription was performed using the M-MLV reverse transcription kit (TaKaRa) to obtain cDNA. According to the sequence information of the gene in the sorghum and maize genomes, specific primers were designed using the primer 3 software (https: / / primer3.ut.ee / ) to amplify EoBr2.

[0087] Upstream primer EoBr2-F: 5′-ATGCTCGTCGGCACCCTC-3′, (SEQ ID NO. 2),

[0088] Downstream primer EoBr2-R: 5′-CGCCGCGGCCCCGTTGGAT-3′, (SEQ ID NO. 3),

[0089] Using leaf cDNA as template, PCR was performed using High-Fidelity DNA Polymerases (M0491V, NEB). The reaction system for 50 μL was as follows: 10.0 μL 5×Q5 Reaction Buffer, 2.5 μL each of EoBr2-F and EoBr2-R primers (20 μmol·L -1 ), 2mM dNTP mix 5.0μL, 5×Q5 High GC Buffer 10.0μL, Q5 High-Fidelity DNA Polymerase 0.5μL, cDNA template 3μL, ddH2O 16.5μL; reaction procedure: 98℃ pre-denaturation for 30sec, then 98℃ melting for 10sec, 71℃ annealing for 30sec, 72℃ extension for 2min, reaction for 35 cycles, 72℃ extension for 2min; the product was recovered with a gel recovery kit (AXYGEN, USA), ligated to the pCE2-T vector (Vazyme) with T4 DNA ligase (TaKaRa), transformed into DH5α competent cells, and the sequence was determined to be SEQ ID NO.1 to obtain the positive plasmid pCE2-EoBr2.

[0090] Example 2. Construction of plant expression vector pCAMBIA1305.2M-EoBr2

[0091] Primers were designed based on the full-length gene sequence of EoBr2 for PCR reaction. The positive plasmid pCE2-EoBr2 extracted from the positive plasmid in Example 1 was used as a template for PCR reaction. The homology arms at the MEcoR I and Hind III sites of pCAMBIA1305.2 were introduced into the upstream and downstream of the EoBr2 gene, respectively.

[0092] Upstream primer EoBr2-EcoRI-F: 5′-acctctagacccggggaattcATGCTCGTCGGCACCCTC-3′, (SEQ ID NO.4)

[0093] Downstream primer EoBr2-HindIII-R: 5′-cttgtcgacactagtaagcttCGCCGCGGCCCCGTTGGAT-3′, (SEQ ID NO.5)

[0094] use PCR was performed using High-Fidelity DNA Polymerases (M0491V, NEB). The reaction system for 50 μL was as follows: 10.0 μL 5×Q5 Reaction Buffer, 2.5 μL each of EoBr2-EcoRI-F and EoBr2-HindIII-R primers (20 μmol·L -1 ), 2mM dNTP mix 5.0μL, 5×Q5 High GC Buffer 10.0μL, Q5 High-Fidelity DNA Polymerase 0.5μL, pCE2-EoBr2 positive plasmid 0.2μL, ddH2O 19.3μL; reaction procedure: 98℃ pre-denaturation for 30sec, then 98℃ melting for 10sec, 71℃ annealing for 30sec, 72℃ extension for 2min, reaction for 35 cycles, 72℃ extension for 2min; the product was recovered using a gel recovery kit (AXYGEN, USA).

[0095] pCAMBIA1305.2M was digested with EcoRI and Hind III, the digestion system was 50μL: 10×CutSmartbuffer 5.0μL, Hind III-HF 1μL, EcoRI-HF 1.0μL, pCAMBIA1305.2M vector 5.0μL (about 1μg), ddH2O was added to 50.0μL; digestion was carried out at 37℃ overnight, and the digestion product was purified. The EoBr2 gel recovery product and the pCAMBIA1305.2M digestion purification product were homologously recombined with recombinase (Vazyme), 10μL reaction system: 5×CE IIBuffer 2.0μL, Exnase II 1.0μL, EoBr2 recovery product 3μL, pCAMBIA1305.2M digestion purification product 2.0μL, ddH2O 2.0μL, 37℃30min. The recombinant product was transformed into E. coli competent cells DH5α, cultured at 37°C overnight, and single clones were picked for PCR detection.

[0096] Upstream primer EoBr2-KZF: 5′-TCATCGTCCAGAACTCGGC-3′, (SEQ ID NO.10)

[0097] Downstream primer: EoBr2-KZR: 5′-CATGAAGACGCGGATGGTG-3′, (SEQ ID NO.11)

[0098] The positive colonies identified by PCR were expanded and cultured to extract the positive plasmid, which was sequenced and verified to be SEQ ID NO.1. The plant expression vector pCAMBIA1305.2M-EoBr2 was successfully constructed ( Figure 1 , Figure 2 ).

[0099] Example 3. Transformation of rice by Agrobacterium EHA105-mediated infection

[0100] Take 10 μL of pCAMBIA1305.2M-EoBr2 plasmid and add 100 μL of Agrobacterium competent EHA105, ice bath for 30 min, freeze in liquid nitrogen for 5 min, 37°C for 5 min, add 800 μL of YEB liquid culture medium, pre-culture at 28°C 200 rpm for 4 h, spread on YEB (50 μg / mL rifampicin + 50 μg / mL kanamycin) plates, and culture at 28°C for about 24h-36h. Single EHA105 colonies were picked from the plate and positive colonies were detected by PCR. 10 μL of PCR (Vazyme) system: 5 μL of 2× Rapid Taq Master Mix, 0.3 μL of upstream primer EoBr2-KZF, 0.3 μL of downstream primer EoBr2-KZR, 4.4 μL of ddH2O. A few plaques were picked with a sterilized pipette tip and mixed in the above PCR system by blowing; reaction procedure: pre-denaturation at 95°C for 3 min, then melt at 95°C for 15 sec, anneal at 60°C for 15 sec, extend at 72°C for 20 sec, react for 35 cycles, and extend at 72°C for 5 min; the product size was about 434 bp, and the PCR product was detected by 1% agarose gel electrophoresis ( Figure 2 ), and the positive clones were used for subsequent transformation of rice callus. Rice transformation was completed by Biotech Co., Ltd.

[0101] Example 4. qPCR identification of transgenic rice lines by transgenic EoBr2 resistant plants

[0102] Plants differentiated from resistant callus were selected and transferred to soil for cultivation after 7 days of seedling training. Total RNA was extracted from fresh leaves of transformed plants and wild-type plants and reversely transcribed into cDNA. Upstream and downstream primers were designed on two adjacent exons, with a fragment length of 202 bp. The primer sequences were:

[0103] Upstream primer EoBr2-q6F: 5′-TTCATCATCAAGCTCCCCGA-3′ (SEQ ID NO. 6),

[0104] Downstream primer EoBr2-q6R: 5′-CGATCATGAAGCGGTCCAG-3′ (SEQ ID NO. 7).

[0105] qPCR (Vazyme) was performed using cDNA of positively transformed plants and untransformed plants as templates and EoBr2-q6F and EoBr2-q6R as primers, and the changes in expression levels were analyzed;

[0106] The gene fragment amplified by rice OsUbiquitin was used as the internal standard. The fragment length was 117 bp. The primer sequence was:

[0107] Upstream primer Ubq-F: 5′-GCTCCGTGGCGGTATCAT-3′ (SEQ ID NO. 8),

[0108] Downstream primer Ubq-R: 5′-CGGCAGTTGACAGCCCTAG-3′ (SEQ ID NO. 9).

[0109] Reaction system 20 μL: 2×ChamQ Universal SYBR qPCR Master Mix 10.0 μL, EoBr2-q6F / Ubq-F (10 μM) 0.4 μL, EoBr2-q6R / Ubq-R (10 μM) 0.4 μL, cDNA 1.0 μL, ddH2O 8.2 μL;

[0110] The amplification conditions were: 95°C pre-denaturation for 30 sec, 95°C for 10 sec, 60°C for 30 sec, 40 cycles, melting curve 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec. The data were obtained by 2 –ΔΔCt Method analysis and making a bar graph ( Figure 3 ). As can be seen in the figure, the expression level of EoBr2 in the EoBr2-transfected plants is greatly increased compared with that in the wild-type plants.

[0111] Example 5. Phenotypic observation of transgenic plant offspring

[0112] The seeds of the positive plants that grew up were collected. The wild-type seeds and the seeds collected from the positive plants were soaked and germinated, and the positive plants were identified by single-plant PCR, and then transferred to nutrient solution hydroponics. Wild-type rice (Nipponbare) and transgenic rice (OE-EoBr2-n) that had been hydroponically cultivated for 25 days were selected as materials for phenotypic observation. The aboveground and underground root growth of the transgenic line OE-EoBr2-n was observed ( Figure 4 ). The plant height and total root length of the transgenic lines and wild-type lines were statistically analyzed and found that compared with the wild-type plants, the plant height and total root length of the transgenic lines OE-EoBr2-6, OE-EoBr2-9, and OE-EoBr2-22 were significantly increased, with significant differences (P<0.01) ( Figure 5 Figure 6 ).

[0113] The phenotypic observation experiment of transgenic plants showed that the plant height of transgenic plants was significantly increased and the total root length of the root system was significantly increased, indicating that the EoBr2 gene in Ervatamia oxyphylla can significantly promote the growth of the aboveground and underground parts of the plant.

[0114] Sequence Listing

[0115] SEQ ID NO.1 ('Ganbei' EoBr2 gene nucleotide sequence)

[0116]

[0117] SEQ ID NO.2 (PCR amplification of the full-length sequence of the EoBr2 gene using upstream primer EoBr2-F):

[0118] 5′-ATGCTCGTCGGCACCCTC-3′

[0119] SEQ ID NO.3 (PCR amplification of the full-length sequence of the EoBr2 gene using downstream primer EoBr2-R):

[0120] 5′-CGCCGCGGCCCCGTTGGAT-3′

[0121] SEQ ID NO.4 (PCR amplification of EoBr2 gene and introduction of upstream primer EoBr2-EcoRI-F of homologous recombination arm):

[0122] 5′-acctctagacccggggaattcATGCTCGTCGGCACCCTC-3′

[0123] SEQ ID NO.5 (PCR amplification of EoBr2 gene and introduction of downstream primers of homologous recombination arm EoBr2-Hind III-R):

[0124] 5′-cttgtcgacactagtaagcttCGCCGCGGCCCCGTTGGAT-3′

[0125] SEQ ID NO.6 (upstream primer EoBr2-q6F for qPCR detection of EoBr2 gene in transgenic plants):

[0126] 5′-TTCATCATCAAGCTCCCCG-3′

[0127] SEQ ID NO.7 (qPCR detection of the downstream primer EoBr2-q6R of the EoBr2 gene in transgenic plants):

[0128] 5′-CGATCATGAAGCGGTCCAG-3′

[0129] SEQ ID NO.8 (Ubq-F, internal reference upstream primer for qPCR detection of transgenic plants):

[0130] 5′-GCTCCGTGGCGGTATCAT-3′

[0131] SEQ ID NO.9 (Ubq-R, an internal reference downstream primer for qPCR detection of transgenic plants):

[0132] 5′-CGGCAGTTGACAGCCCTAG-3′

[0133] SEQ ID NO.10 (pCAMBIA1305.2M-EoBr2 vector construction monoclonal detection upstream primer EoBr2-KZF):

[0134] 5′-TCATCGTCCAGAACTCGGC-3′

[0135] SEQ ID NO.11 (pCAMBIA1305.2M-EoBr2 vector construction monoclonal detection downstream primer):

[0136] EoBr2-KZR: 5′-CATGAAGACGCGGATGGTG-3′.

Claims

1. A growth-promoting gene of Centipedegrass EoBr2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Containing the growth-promoting gene of Centipedegrass according to claim 1 EoBr2 The biomaterial is characterized in that The biological material is a recombinant vector, an expression box or a transgenic recombinant bacterium.

3. The biomaterial according to claim 2, characterized in that The recombinant vector is a recombinant expression vector, which is a recombinant expression vector containing the centipedegrass growth-promoting gene of claim 1. EoBr2 Transformed into a plant expression vector.

4. The biomaterial according to claim 3, characterized in that The recombinant expression vector is a vector containing pCAMBIA1305.2M homology arms. EoBr2 The gene fragment was obtained by homologous recombination reaction with the enzyme-cut pCAMBIA1305.2M expression vector.

5. A growth-promoting gene in Centipedegrass EoBr2 A method for constructing a plant expression vector, characterized in that: The steps include: (1) Centipedegrass growth-promoting gene EoBr2 Clone Using the leaves of 'Ganbei' Eremochloa as materials, total RNA was extracted, reverse transcribed into cDNA, and primers were designed for amplification. EoBr2 Gene, Upstream primer EoBr2-F: 5′-ATGCTCGTCGGCACCCTC-3′, Downstream primer EoBr2-R: 5′-CGCCGCGGCCCCGTTGGAT-3′, The reverse transcribed cDNA was used as a template and EoBr2-F and EoBr2-R were used as primers for PCR reaction. The product was connected to the pCE2-T vector and transformed into DH5α Competent cells, extract positive plasmid pCE2- EoBr2 , the determined sequence is SEQ ID NO.1; (2) Plant expression vector pCAMBIA1305.2M- EoBr2 Construction Primers were designed using the positive plasmid pCE2-EoBr2 sequenced correctly in (1) as a template. EoBr2 The upstream and downstream of the gene were introduced into the homology arms at the EcoR I and Hind III sites of pCAMBIA1305.2M, respectively. Upstream primer EoBr2-EcoRI-F: 5′-acctctagacccggggaattcATGCTCGTCGGCACCCTC-3′, Downstream primer EoBr2- Hind III-R: 5′-cttgtcgacactagtaagcttCGCCGCGGCCCCGTTGGAT-3′; pCE2- EoBr2 The plasmid was used as a template for PCR amplification; the PCR product was recovered through gel and then subjected to homologous recombination reaction with the pCAMBIA1305.2M linearized vector that was digested and purified by EcoR I and HindIII, and the recombinant product was transformed into Escherichia coli competent cells DH5α After PCR identification of single clones, the positive plasmid was extracted and sequenced to verify SEQ ID NO.1, plant expression vector pCAMBIA1305.2M- EoBr2 Build successful.

6. The centipedegrass growth-promoting gene according to claim 1 EoBr2 The application of the method in improving the growth ability of rice or cultivating new rice germplasm with strong growth ability is characterized in that: Overexpression of the centipedegrass growth-promoting gene of claim 1 in rice EoBr2 .

7. Use of the biomaterial according to any one of claims 2 to 4 in improving the growth ability of rice or cultivating new rice germplasm with strong growth ability, characterized in that: Overexpression of the centipedegrass growth-promoting gene of claim 1 in rice EoBr2 .

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