CN116751273B

CN116751273B - Application of OsbHLH002 protein or its encoding gene in regulating plant cellulose synthesis or secondary wall development

Info

Publication number: CN116751273B
Application number: CN202310735186.4A
Authority: CN
Inventors: 宋士勇; 吴俣; 李艾蓬; 陈颖; 祁皓月; 杨丽佳; 许亮; 郭佳卓
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-06-20
Filing date: 2023-06-20
Publication date: 2025-01-10
Anticipated expiration: 2043-06-20
Also published as: CN116751273A

Abstract

The present invention provides an application of OsbHLH002 protein or its encoding gene in regulating plant cellulose synthesis or secondary wall development, and relates to the technical field of plant molecular biology. The amino acid sequence of the OsbHLH002 protein of the present invention is shown in SEQ ID No. 2. The present invention obtains mutants of the OsbHLH002 gene by means of genetic engineering, and the results show that the four mutant types of Osbhlh002 show a lodging phenotype compared with wild-type plants, and the complementary strain can restore the phenotype of secondary wall development defects of the mutant. The present invention provides an important application direction for increasing the cellulose content of plants and increasing the thickness of secondary walls, and also provides an important reference for breeding lodging-resistant excellent varieties.

Description

Use of OsbHLH002 protein or its coding gene in regulating plant cellulose synthesis or secondary wall development

Technical Field

The invention relates to the technical field of plant molecular biology, in particular to an application of OsbHLH002 protein or a coding gene thereof in regulating and controlling plant cellulose synthesis or secondary wall development.

Background

Cell walls are a characteristic structure of plant cells that is distinguished from animal cells and play a vital role in the growth and development of plants. Depending on the component of cell wall biosynthesis and the cell location, the plant cell wall is divided into primary and secondary walls (Underwood, 2012; barros et al, 2015). Secondary walls are produced by thickened specific plant cell types that are involved in many critical biological processes in plants, such as mechanical support, transport of moisture and nutrients, dehiscence of anthers and pods, and various stress responses, etc. (Zhong and Ye,2015; zhang et al, 2021). The secondary wall is mainly composed of cellulose, hemicellulose and lignin, which account for the majority of plant biomass. Cellulose in primary walls and secondary walls is the most main component, accounts for about 1/3 of dry weight of plants, is the main determinant of mechanical strength of plant tissues, is the most abundant natural organic macromolecules on the earth, and has important application prospect in aspects of agricultural production, industrial manufacturing, new energy development and utilization and the like. Therefore, the method has important theoretical value and scientific significance for researching and controlling molecular mechanisms of cellulose deposition and secondary wall formation.

In 1986, researchers observed a cellulose complex enzyme structure CSC (Cellulose synthase complex) in the cell membrane of higher plants, as a "factory" for the catalytic synthesis of cellulose, assembled from cellulose synthase subunits (cellulose synthase, CESA) (Haigler and Brown,1986; pear et al, 1996). At least 10 CESA genes are currently identified in arabidopsis, of which AtCESA, atCESA, 7, atCESA are key genes controlling the synthesis of secondary wall cellulose in arabidopsis (Han Xiao et al, 2014). 11 CESA genes have been identified in rice, and OsCESA.sup.4/7/9 of the co-participation in the synthesis of secondary wall cellulose is required. OsCESA4/7/9 is a AtCESA/7/8 homologous gene in rice (Tanaka et al, 2003), demonstrating that secondary wall cellulose synthesis is conserved among different species.

Transcriptional regulation is a large scale control mechanism of cellulose synthesis because it affects multiple metabolic pathways at the same time and regulates the transcription of many genes involved in cellulose formation. In dicotyledonous mode plant Arabidopsis, a precise model of secondary wall cellulose synthesis regulatory networks has been established. In this transcriptional regulatory network, a range of NAC and MYB transcription factors are included, as well as small amounts of bHLH and WRKY transcription factors (Zhong et al, 2010; zhang et al, 2021). Some NAC proteins function as primary layer-one regulatory factors, including VND1-7 (VASCULAR-RELATED NACDOMAIN 1-7), NST1, and SND1/NST3 and NST2.VND1-7 is involved in the development of xylem vessel cells, where VND6 and VND7 act as the main switching genes for xylem vessel formation, and overexpression causes thickening of the metawood and native xylem, respectively (Zhou et al, 2014; kubo et al, 2005), NST1/2 and SND1 are reported to regulate the synthesis of secondary cell walls together (Zhong and Ye, 2015). These secondary wall-associated NACs have been shown to regulate the expression of a range of downstream transcription factors, such as MYBs, and thus regulate the biosynthesis of secondary wall cellulose deposits. MYB46 and MYB83 are regulated by direct transcription of the first layer main switches NST1/2, SND1 and VND6/7 in a regulation network, and are specifically expressed in stem duct cells and vascular tissue cells (Zhong et al, 2007; yamaguchi et al, 2011), and are key node genes for regulating and controlling the formation of the secondary cell walls of Arabidopsis thaliana. OsMYB61 and OsMYB103L regulate secondary wall cellulose synthesis by direct binding to OsCesAs promoter. Some known NAC-like transcription factors, including rice OsSND, osNAC9, and OsNAC31, act as first layer regulators, directly mediating expression of other MYB-like genes and promoting cellulose biosynthesis.

BHLH transcription factors are a widely existing class of proteins in plants, and play an important role in plant development regulation, stress response, hormone synthesis, optical signal transduction and other pathways. Classical ICE1 (Inducer ofCBF Expression 1) encodes a bHLH transcription factor, and plants overexpressing the ICE1 gene have significantly improved cold tolerance (Chinnusamy et al, 2003). Another bHLH transcription factor, ZHOUPI (ZOU), was reported to mediate the Arabidopsis seed development process (Yang et al, 2008), and in addition it regulates the seed dormancy process with ICE1 by regulating the accumulation of ABA during seed maturation (MacGregor et al, 2019). Arabidopsis PHYTOCHROME B mutant phyB deposited with thinner secondary cell walls in stem fiber cells, but thicker cell walls were observed in PHYTOCHROME INTERACTING FACTOR (PIF) quadruple mutant PIF1PIF3PIF4PIF5 (pifq), and furthermore PIF4 interacted with MYC2/4, inhibiting their transduction activity on primary regulatory transcription factor NST1, and thus inhibiting thickening of secondary walls of Arabidopsis stem fiber cells (Luo et al 2022). However, currently there are few studies on the role of bHLH-like transcription factors in secondary wall formation. Despite the tremendous advances in Arabidopsis transcriptional regulatory networks, our understanding of the regulatory mechanisms of secondary wall cellulose development in monocot rice remains quite limited. Rice is one of the most important staple food crops, which produces huge agricultural biomass residues. Thus, there is a need to discover and find a more relevant gene involved in the development of secondary walls of rice.

In view of this, the present invention has been made.

Disclosure of Invention

Aiming at the defects of the research on the plant secondary wall development regulation mechanism in the prior art, the invention provides the application of OsbHLH002 protein or the coding gene thereof in regulating and controlling the synthesis of plant cellulose or the development of secondary walls.

The technical scheme provided by the invention is as follows:

In one aspect, the invention provides an application of OsbHLH002 protein or its coding gene in regulating plant cellulose synthesis or secondary wall development, and the amino acid sequence of OsbHLH002 protein is shown in SEQ ID No. 2.

The invention obtains OsbHLH002 gene mutant by means of genetic engineering, and the result shows that Osbhlh002 four mutation types show lodging phenotype compared with wild type plants, and the complementary strain can restore phenotype of defective secondary wall development of the mutant. The OsbHLH protein or the coding gene thereof can be used for regulating and controlling the development of secondary walls of plants, in particular cellulose synthesis and deposition of the secondary walls.

The "OsbHLH protein" in the present invention encompasses fusion proteins obtained by linking tags to the N-terminal and/or C-terminal of the protein shown in SEQ ID No.2 and proteins having the same functions obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the nucleotide sequence of the OsbHLH002,002 protein encoding gene is shown in SEQ ID No. 1.

The present invention encompasses sequences having more than 90%, preferably more than 95%, more preferably more than 99% similarity to the nucleotide sequence shown in SEQ ID No.1 and having the same function. The invention also covers sequences having one or several base substitutions, deletions and/or additions to the nucleotide sequence shown in SEQ ID No.1 and having the same function.

In another aspect, the present invention provides the use of a biomaterial comprising a gene encoding OsbHLH a 002 protein in modulating plant cellulose synthesis or secondary wall development, wherein the biomaterial comprises:

(A) An expression cassette comprising a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID No. 1;

(B) A recombinant vector comprising the expression cassette of (a);

(C) A recombinant microorganism comprising the expression cassette of (A) or comprising the recombinant vector of (B);

(D) A recombinant cell comprising the expression cassette of (A) or comprising the recombinant vector of (B).

In one embodiment, the recombinant vector is an over-expression vector of OsbHLH002 genes.

In one embodiment, the recombinant expression vector comprises a transcript that initiates transcription of the gene of interest. In order to obtain overexpression of the target gene, the promoter contained in the recombinant expression vector comprises, but is not limited to, constitutive promoters, tissue, organ and development specific promoters and inducible promoters.

In one embodiment, the overexpression vector contains a Ubiquitin promoter or a CaMV 35S promoter.

In one embodiment, the recombinant vector includes binary Agrobacterium vectors and vectors useful for plant microprojectile bombardment and the like, including, for example, but not limited to, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb vectors and the like.

In one embodiment, the recombinant vector is a pENTR-3FLAG vector.

In one embodiment, the recombinant microorganism is agrobacterium, preferably the recombinant microorganism is agrobacterium EHA105.

In one embodiment, the modulation is promotion of secondary wall cellulose synthesis by increasing the expression level of OsbHLH002 gene or activity of OsbHLH002 protein in the plant.

In one embodiment, the plant is a monocot or dicot.

In one embodiment, the plant comprises rice.

In a specific embodiment, the plant is wild-type rice (Nippon-Qing).

In another aspect, the invention provides a method for cultivating lodging-resistant transgenic rice, which uses a gene OsbHLH002 as a target gene, and improves the expression level of OsbHLH002 gene or the activity of OsbHLH protein in the rice by a genetic engineering method to obtain the transgenic rice.

In one embodiment, the method of genetic engineering comprises introducing the OsbHLH gene into the recipient rice.

In one embodiment, the nucleotide sequence of OsbHLH002,002 gene is shown in SEQ ID No. 1.

In the present invention, the transgenic rice includes seeds, calli, whole plants and cells. The transgenic rice not only comprises first-generation transgenic rice obtained by transforming the target plant with the gene, but also comprises offspring thereof.

The invention has the beneficial effects that:

The invention clearly verifies the important role of OsbHLH002 protein or the coding gene in regulating and controlling the synthesis of plant cellulose or the development of secondary wall for the first time. When the gene function is lost, the plant develops secondary wall development defects. This shows that the gene plays an important role in secondary wall development, and is favorable for promoting plant secondary wall biosynthesis, increasing plant cellulose content and increasing secondary wall thickness. The invention provides an important theoretical basis for researching and cultivating new lodging-resistant varieties for the development mechanism of secondary walls.

Drawings

FIG. 1 is a schematic diagram of four mutant mutation sites of OsbHLH002 gene provided by the invention;

FIG. 2 shows that Osbhlh002,002 mutant types provided by the present invention exhibit a lodging phenotype compared to wild type plants.

FIG. 3 shows that Osbhlh002-1g OsbHLH002 and Osbhlh002-1gOsbHLH002-3FLAG complementation lines provided by the invention are capable of restoring the phenotype of Osbhlh002-1 secondary wall development defects;

FIG. 4 is a graph showing the difference in secondary wall thickness between wild-type and Osbhlh-002 mutant leaf sheaths provided by the present invention;

FIG. 5 shows the measurement results of cell wall thickness and cellulose content of wild type Osbhlh mutant and its complementary line.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The following embodiments and features of the embodiments may be combined with each other without conflict. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The discovery and verification of more genes involved in plant cell wall regulation are of great significance to crop breeding, yield improvement and the like. At present, genes related to plant secondary wall development are gradually discovered and functionally identified, but the research on the role of bHLH transcription factors in secondary wall formation is less, and the functions and the role modes of OsbHLH002 in the molecular regulation network mediated by the bHLH transcription factors in rice secondary wall development or cellulose synthesis are not reported yet.

The experimental result of OsbHLH gene mutation shows that the OsbHLH002 function deletion mutant strain has secondary wall development defect, and is particularly expressed as a phenotype that the leaf sheath supporting force is weakened, the secondary wall thickness is obviously reduced and the cellulose content is obviously reduced. This indicates that OsbHLH002 gene has important function in the synthesis of secondary rice wall. In addition, the invention further verifies the effect of the gene in plant secondary wall development by constructing a complementary strain and finding that the complementary strain can recover the phenotype of Osbhlh002-1 secondary wall development defect. The invention provides new gene resources for synthesizing rice secondary wall cellulose and theoretical basis for crop molecular design breeding.

The full length of OsbHLH gene sequence related by the invention is 1575bp, the nucleotide sequence is shown as SEQ ID NO.1, and 524 amino acids are encoded.

In one example, the invention provides the application of OsbHLH protein or its coding gene in regulating plant cellulose synthesis or secondary wall development, the amino acid sequence of OsbHLH protein is shown as SEQ ID No.2 ：MLPRFHGAMWMQDDGGGDQEHGQAAPPGQEQHHHDQHLMALAAAAAGGAGFGAAQAPAPLLDEDWYFDAAGGGGGGAHGSMMLGLSSVHGGIGAGTSGGGHGQQFSLLNMGAAAAPFDVSGFDLGIACGGVGGGGDVVSFLGGGNASNTALLPVGNAGFLGTFGGFGTAASQMPEFGGLAGFDMFDAGAVNTGGSSSSSSAAAAAASASAHVSNTAPFSGRGKAAVLRPLDIVPPVGAQPTLFQKRALRRNAGEDDDDKKRKAAAGAGAGALSADGADMVLDDGDDDGLSIDASGGLNYDSEDARGGEDSGAKKESNANSTVTGDGKGKKKGMPAKNLMAERRRRKKLNDRLYMLRSVVPKISKMDRASILGDAIEYLKELLQKINDLQNELESSPATSSLPPTPTSFHPLTPTLPTLPSRIKEEICPSALPSPTGQQPRVEVRLREGRAVNIHMFCARRPGLLLSAMRAVEGLGLDVQQAVISCFNGFTLDIFKAEQCKDGPGLLPEEIKAVLMQSAGFHTMI.

In one example, the cDNA sequence of the OsbHLH002,002 protein encoding gene is shown in SEQ ID No.1 ：ATGCTGCCGCGGTTTCACGGCGCCATGTGGATGCAGGACGACGGCGGCGGCGACCAAGAACACGGGCAGGCGGCGCCGCCTGGGCAGGAGCAGCACCACCACGACCAGCATCTCATGGCGTTGGCGGCCGCGGCCGCGGGCGGCGCCGGGTTCGGCGCGGCGCAGGCGCCGGCGCCGCTGCTCGATGAGGACTGGTACTTCGACGCGGCGGGTGGTGGTGGTGGTGGCGCGCATGGGTCCATGATGCTGGGTTTGTCGTCCGTCCATGGCGGGATTGGGGCGGGGACGTCTGGTGGTGGGCATGGGCAGCAGTTCTCGCTGCTCAACATGGGCGCCGCGGCCGCGCCGTTCGACGTCTCCGGGTTCGACCTCGGGATCGCCTGCGGCGGCGTTGGCGGCGGCGGCGACGTGGTGTCGTTTCTTGGCGGCGGGAACGCGTCGAACACCGCGCTGCTCCCCGTCGGGAACGCGGGGTTCCTCGGCACGTTCGGCGGGTTCGGCACCGCGGCGTCCCAAATGCCGGAGTTCGGCGGGCTCGCCGGGTTCGACATGTTCGACGCGGGCGCCGTGAACACCGGGGGCAGCTCCTCCTCCTCGTCGGCGGCGGCGGCGGCGGCGTCCGCCTCGGCGCACGTGAGCAACACCGCGCCGTTCTCCGGGCGCGGCAAGGCGGCGGTGCTGCGGCCGCTGGATATCGTCCCGCCCGTGGGCGCGCAGCCGACGCTGTTCCAGAAGCGCGCGCTCCGCCGCAACGCCGGCGAGGACGACGACGACAAGAAGCGCAAGGCCGCCGCGGGCGCGGGCGCGGGCGCGCTGTCCGCCGACGGCGCCGACATGGTGCTCGACGACGGCGACGACGACGGCCTCAGCATCGACGCGTCGGGCGGCCTCAACTACGACTCCGAGGACGCCAGGGGCGGCGAGGACAGCGGCGCCAAGAAGGAGTCGAACGCCAACAGCACGGTCACCGGCGACGGGAAGGGGAAGAAGAAGGGGATGCCGGCCAAGAACCTCATGGCGGAGCGCCGCCGCCGGAAGAAGCTCAACGACCGCCTCTACATGCTCCGCTCCGTCGTGCCCAAGATCAGCAAGATGGACAGGGCTTCCATTCTCGGCGACGCGATTGAGTACCTGAAGGAGCTGCTGCAGAAGATCAATGATCTTCAGAATGAGCTCGAGTCGTCCCCCGCGACGTCGTCATTGCCTCCAACACCCACAAGCTTCCATCCCCTGACACCGACGCTGCCCACATTGCCGTCCCGCATCAAGGAAGAGATCTGCCCAAGTGCATTGCCAAGCCCCACTGGACAACAGCCAAGGGTTGAGGTTAGGCTGAGGGAAGGCCGGGCTGTCAATATCCACATGTTCTGTGCTCGGAGGCCCGGTCTACTGCTCTCTGCCATGAGGGCCGTCGAAGGCCTTGGTCTCGATGTCCAGCAAGCTGTAATCAGTTGCTTCAATGGCTTTACGTTGGATATTTTTAAGGCTGAGCAATGCAAGGACGGCCCTGGGCTGTTGCCTGAAGAAATCAAGGCCGTTCTGATGCAATCCGCCGGGTTCCATACCATGATCTAG.

In one embodiment, upregulation of the activity or expression level of OsbHLH002 protein or a gene encoding the same may regulate plant secondary wall development or secondary wall cellulose synthesis.

In a specific example, the OsbHLH002 gene was introduced into a plant by using an over-expression vector to perform over-expression, and the expression level of OsbHLH002 gene was controlled (the expression level was increased) to promote the deposition of plant secondary wall cellulose.

In a specific embodiment, increasing the amount of OsbHLH gene expression increases the secondary wall cellulose content, increases the secondary wall thickness, and increases leaf sheath support.

The invention defines OsbHLH and the function of biological material containing the same for promoting the deposition of plant secondary wall cellulose, and as OsbHLH002 mutation can cause the lodging character of plants, lodging-resistant transgenic plants can be cultivated by increasing or improving the expression of draft genes.

In one embodiment, the invention provides a method for cultivating lodging-resistant transgenic rice, which improves the expression level of OsbHLH002 genes or the activity of OsbHLH002 proteins in the rice by a genetic engineering method to obtain lodging-resistant transgenic rice.

Definition of terms in connection with the present invention

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form.

In the present invention, the term "identity" or "similarity" refers to sequence similarity to a native nucleic acid sequence. Identity or similarity can be assessed by means of computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the present invention, the term "expression" or "gene expression" means the transcription of a particular gene or genes or a particular gene construct into structural RNA (rRNA, tRNA) or mRNA, which RNA is subsequently translated or not translated into protein. This process involves transcription of DNA and processing of the resulting mRNA product.

In the present invention, the term "increased expression/overexpression" means any form of expression that is increased relative to the original wild-type expression level. Methods for increasing expression of a gene or gene product have been described in the art and include, for example, overexpression driven by a suitable promoter, the use of transcriptional enhancers or translational enhancers.

In the present invention, the terms "increase", "improvement" or "enhancement" are interchangeable and shall mean a yield and/or growth and/or variation of at least 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more compared to a control plant as defined herein.

In the present invention, the term "transformation" refers to a method of introducing a heterologous DNA sequence or vector containing a DNA sequence into a host cell or organism.

In the present invention, the term "recombinant expression vector" means one or more DNA vectors used to effect transformation of plants, and these vectors are often referred to in the art as binary vectors.

In the present invention, the term "host cell" or "recombinant host cell line" means a cell comprising a polynucleotide of the present invention, regardless of the method used to insert to produce a recombinant host cell. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

In the present invention, the term "complementary vector" means "functional complementary vector" which is a functional complementary experiment for mutants in genetic engineering to verify whether the mutant phenotype can be restored after complementation, and the complementary vector usually contains a gene of interest and may optionally use a constitutively strong promoter or a promoter of the gene itself.

Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.

EXAMPLE 1 cloning of the OsbHLH002 Gene nucleotide sequence

RNA was extracted from rice using the Omega plant extraction kit. Then, the first strand cDNA was synthesized using 1. Mu.g of RNA as a template according to the instructions of the cDNA synthesis kit (Yeasen). According to the (http:// price. Plant biology. Msu. Edu/expression. Shtml) website, the complete ORF of OsbHLH002 was obtained, and specific primers were designed, 5 'end primer ATGCTGCCGCGGTTTCA (SEQ ID No. 3) and 3' end primer CTAGATCATGGTATGGAAC (SEQ ID No. 4).

The PCR reaction system was 2X PhantaMax MasterMix. Mu.L, 10. Mu.M forward/reverse primer each was 1. Mu.L, 5. Mu.L template (cDNA), and sterilized water was filled to 50. Mu.L, and the reaction procedure was 95℃pre-denatured for 3min,95℃denatured for 30s, tm annealed for 30s,72℃extended for 2kb/min, 35-40 cycles, 72℃extended for 5min. . Finally, the full-length cDNA sequence of 1575bp (containing a stop codon) of OsbHLH (shown as SEQ ID No. 1) is obtained through amplification, and 524 amino acids are encoded (shown as SEQ ID No. 2).

EXAMPLE 2 construction of OsbHLH002 Gene mutant and complementary Strain vector

The present study utilizes the CRISPR/Cas9 genome editing system to edit OsbHLH002 gene in wild type rice (japan) to obtain mutant plants. The exon sequence of the target gene OsbHLH was analyzed using the CRISPR-GE website (http:// skl. Scau. Edu. Cn /), and two specific target sequences were selected and designated target site 2 (Cas 9-2) GGCGACCAAGAACACGGGCAGG (SEQ ID No. 5) and target site 3 (Cas 9-3) CCATGTGGATGCAGGACGACGG (SEQ ID No. 6). And synthesizing sgRNA-2 (GGCGACCAAGAACACGGC, SEQ ID No. 7) and sgRNA-3 (CCATGTGGATGCAGGACGA, SEQ ID No. 8), performing enzyme digestion on the gene editing vector psgR-CAS9-Os by using BsaI, annealing the primer, namely uniformly mixing target sites 2,10μLF(5'-TGTGTGGGCGACCAAGAACACGGGC-3',SEQ ID No.9)+10μLR(5'-AAACGCCCGTGTTCTTGGTCGCCCA-3',SEQ ID No.10)+80μL ddH₂O, uniformly mixing target sites 3,10μLF(5'-TGTGTGCCATGTGGATGCAGGACGA-3',SEQ IDNo.11)+10μL R(5'-AAACTCGTCCTGCATCCACATGGCA-3',SEQ IDNo.12)+80μL ddH₂O, annealing at 95 ℃ for 10min, and connecting the mixture with the digested vector psgR-CAS 9-Os. The ligation system was as follows, 2. Mu.L of annealed product (containing sgRNA) +2. Mu.L of recovered digested vector+0.5. Mu.L of 10 XT 4 buffer+0.5. Mu.LT 4 ligase was ligated for 15min at room temperature to obtain psgR-CAS9-OsbHLH002 vector containing OsbHLH002 specific target, which was transformed into E.coli competent DH 5. Alpha. The transformation system was as follows, ligation product 5. Mu.L was added to E.coli competence, on ice for 30min, heat shock at 42℃for 90s, on ice for 2min, 400. Mu.L of antibiotic-free LB was added, resuscitated at 37℃for 1h, centrifuged at 5000rpm for 1min, most of the supernatant was aspirated, 100. Mu.L of liquid was left to mix well, plated on LB plates (50 mg/LKan) for cultivation, and 37℃overnight for cultivation. The positive clone is sent to a company for sequencing after plasmid extraction, and the psgR-CAS9-OsbHLH002 plasmid with correct selection result is used for obtaining mutant plants by a method of infecting rice callus by agrobacterium.

To construct gOsbHLH002-3FLAG, the amplified 4.94kb genomic sequence contained the 2.12kb 5' upstream sequence and the 2.82kb coding sequence was purified according to the Omega gel recovery kit procedure. The recovered product and ECORI digested vector pENTR-3FLAG were ligated by homologous recombination in a reaction system of 2. Mu.L of linearized vector, 3. Mu.L of insert, 4. Mu.L of 5 XCell buffer, exnase II. Mu.L, and sterilized water to 20. Mu.L at 37℃for 30min. Ligation products transformed DH 5. Alpha. Competent cells were cultured overnight at 37 ℃. The positive monoclonal was picked the next day and sent for testing.

EXAMPLE 3 construction of OsbHLH002 Gene mutants and complementary plants

Obtaining of OsbHLH002 Gene mutant plants

Rice (Japanese) callus is used as an experimental material. The OsbHLH002 gene mutant vector obtained in example 2 was transformed into Agrobacterium EHA105 by freeze thawing. Selecting agrobacterium (containing OsbHLH-002 gene mutant vector) monoclonal in LB liquid medium of 2mL of Rif+Spe, shaking at 28 ℃ and 200rpm for overnight, then taking 1mL of bacterial liquid, culturing in 10mL of resistant LB for 5h, centrifuging at 4000rpm at room temperature for 10min, discarding supernatant, resuspending thallus with 50mLAAM-As heavy suspension, picking out rice callus with a certain size, placing into agrobacterium suspension for dip-dying for 30min, pre-filling a layer of sterile filter paper soaked with AAM on the culture medium, spreading the callus on the culture medium by a spoon, and culturing at 28 ℃ in dark for 2 days. Washing the callus with sterile water after 2 days until the washing liquid is clear, oscillating with Cef sterile water containing 500mg/L for 30min, spreading the washed callus on sterile filter paper, airing for 2h, and transferring to a screening culture medium for screening for about two weeks. The newly grown resistant calli were transferred to differentiation medium containing 50mg/L hygromycin for cultivation. Transferring the green-turning rice callus to a rooting culture medium for inducing rooting after 2-3 weeks. For mutant positive plants, gDNA from T0 generation plants needs to be extracted, identified by PCR and sequenced.

And (3) identifying Osbhlh002 mutant, namely extracting leaf genome DNA of T0 generation transgenic plant, taking the leaf genome DNA as a template, designing a specific primer F2 (AGGAAAAACGGCTTGTGGGA, SEQ ID No. 13) and a primer R2 (GGACGACAAACCCAGCATCA, SEQ ID No. 14) according to OsbHLH002 target information, carrying out PCR amplification on the specific primer F3 (GTAGAGTGCTCTCCCCTCCA, SEQ ID No. 15) and the primer R3 (GGACGACAAACCCAGCATCA, SEQ ID No. 16), recovering a single and clear amplification product of a target band (the size of a positive plant PCR amplification product is 477bp and 369 bp), and sending the positive plant PCR amplification product to a company for sequencing, and screening mutant strains. And (5) carrying out continuous selfing on the generation T0 to obtain the generation T2. Hygromycin screening and PCR identification are carried out on the T2 generation plants again, independent strains which do not contain vectors and have homozygous mutation are screened out, and as shown in figure 1, four mutant strains which are named Osbhlh-1, osbhlh002-2, osbhlh002-3 and Osbhlh002-4 are finally obtained. Wherein two mutations were made at target sequence 1, osbhlh002-1 for the 1 base (+T) added, osbhlh002-2 for the 1 base (-G) deleted, osbhlh002-3 for the 1 base (+A) added, osbhlh002-4 for the 1 base (-A) deleted, and frame shift mutations in the encoded protein were caused by all four mutants.

Obtaining of OsbHLH002 Gene complementation plant line plant

The methods of Agrobacterium-mediated genetic transformation of rice were used mainly with reference to the methods reported by Hiei et al (see Agrobacterium-mediated transformation of rice using immature embryos or calli induce from mature seed,2008,Nature protocol.Doi:10.1038/nprot.2008.46) introducing the complementing vector gOsbHLH002 or gOsbHLH002-3FLAG into the homozygous callus of the OsbhLh002-1 mutant, germinating on a hygromycin-containing medium to obtain homozygous transgenic plants, we randomly selected four strains #5, #6, #8, #11, each of which is capable of restoring the phenotype of Osbhlh-1 secondary wall development defects (see FIG. 3).

EXAMPLE 4 phenotypic analysis of related genetic Material

Seeds of rice (wild type, mutant and complementation lines) were sown after soaking, and the phenotype of the plant material was observed for about 2-3 weeks (see FIGS. 2, 3). The leaf sheath was transected for about 2-3 weeks, the change in thick-walled cells was observed using methylene blue staining, the secondary wall thickness of thick-walled cells was measured using a transmission electron microscope (FIG. 4), the cellulose content in the secondary wall of leaf sheath of the relevant material was measured using Solarbio kit (FIG. 5), and the phenotype of the relevant genetic material was determined from a number of angles.

Although the present disclosure is described above, the scope of protection of the present disclosure is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the scope of the invention.

Claims

1. Application of OsbHLH002 protein or its encoding gene in regulating rice cellulose synthesis or rice secondary wall development, characterized in that the amino acid sequence of the OsbHLH002 protein is shown in SEQ ID No.2.

2. The use according to claim 1, characterized in that the nucleotide sequence of the gene encoding the OsbHLH002 protein is shown in SEQ ID No.1.

3. Use of a biomaterial comprising a gene encoding an OsbHLH002 protein in regulating rice cellulose synthesis or rice secondary wall development, characterized in that the biomaterial comprises:

(A) an expression cassette containing a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 1;

(B) a recombinant vector containing the expression cassette described in (A);

(C) a recombinant microorganism containing the expression cassette described in (A) or the recombinant vector described in (B);

(D) A recombinant cell containing the expression cassette described in (A) or containing the recombinant vector described in (B).

4 . The use according to claim 3 , characterized in that the recombinant vector is an overexpression vector of the OsbHLH002 gene.

5. The use according to claim 4, characterized in that the overexpression vector contains Ubiquitin promoter or CaMV 35S promoter.

6. The use according to any one of claims 1 to 5, characterized in that the regulation is to promote the synthesis of secondary wall cellulose by increasing the expression level of the OsbHLH002 gene or the activity of the OsbHLH002 protein in the rice.

7. A method for cultivating lodging-resistant transgenic rice, characterized in that the gene OsbHLH002 is used as a target gene, and the expression level of the OsbHLH002 gene or the activity of the OsbHLH002 protein in rice is increased by a genetic engineering method to obtain transgenic rice, wherein the nucleotide sequence of the OsbHLH002 gene is shown in SEQ ID No.1.

8 . The method according to claim 7 , wherein the genetic engineering method comprises introducing the OsbHLH002 gene into the recipient rice.