CN113583990A - Rice full-breeding-period semi-dwarf phenotype regulation gene SD38 and application thereof - Google Patents

Abstract

The invention relates to the field of molecular biology, in particular to a rice full-growth half-dwarf phenotype regulation gene SD38 and application thereof, wherein the nucleotide sequence of the SD38 gene is shown as SEQ ID No.1, the amino acid sequence of the coded protein is shown as SEQ ID No.2, and the gene regulates the expression of OsACS3 by mediating the synthesis of C24:0VLCFA, so that the synthesis of ethylene is promoted, the normal morphological development of rice is influenced, the rice full-growth stage shows a half-dwarf phenotype, and a new tool and a new method are provided for high-yield breeding of rice.

Description

Rice full-breeding-period semi-dwarf phenotype regulation gene SD38 and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a rice full-growth-period semi-dwarf phenotype regulation gene SD38 and application thereof.

Background

Rice is an important grain crop in China, more than half of people use rice as staple food, and therefore, the rice yield directly concerns the grain safety in China. With the development of science and technology, particularly the sequencing of rice genome sequences, the functional analysis of the yield character regulation genes is accelerated, and the cognition of a molecular mechanism regulation network of rice yield is increased. The rice plant height is an important support character of an ideal plant type, and plays a decisive role in improving the rice yield. The identification and utilization of a large number of dwarf genes in the 60 th generation of the last century lead to the first green revolution of rice yield and accelerate the research work on the functional mechanism of the dwarf-related genes. Up to now, more than 60 rice dwarf mutants have been identified, but not many genes have been cloned that are associated with dwarfing (Li et al, 2010). Since the dwarf phenotype is related to the whole growth and development stage of rice and is related to numerous physiological and biochemical processes, the genetic mechanism of dwarfing is still in need of further excavation.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel rice full-term semi-dwarf phenotype regulatory gene SD38 and its encoded protein.

The invention also aims to provide the rice full-breeding half-dwarfing phenotype regulatory gene SD38 and application of the encoded protein thereof in rice breeding.

The invention also aims to provide a method for regulating the plant type of rice by using the rice full-growth half-dwarf phenotype regulating gene SD38 and the protein coded by the gene.

In order to realize the purpose, the invention discloses a rice full-growth half-dwarf phenotype regulation gene SD38, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the coding protein is shown as SEQ ID No. 2.

The inventor screens a mutant sd38(semi-dwarf 38) which shows a half-dwarf phenotype in the full-bearing period from an Ethyl Methane Sulfonate (EMS) mutant library of indica type rice restorer line red silk-10 of southwest university rice institute, and observes the phenotype of the mutant sd38, wherein the ear length and all internodes of the mutant sd38 are obviously shorter than those of a wild type, and the characters such as effective ear number, grain length, grain width and the like have no obvious difference from the wild type. Phenotypic identification and histological analysis are carried out on the mutant, and the result shows that the shortening of the cells is the reason for the half dwarfing of the sd38 mutant. SD38 gene identification is carried out through a map-based cloning strategy, and finally a gene LOC _ Os10g33370 which codes for a beta-ketoacyl-CoA synthase (beta-ketoacyl-CoA synthase, KCS) positioned on endoplasmic reticulum and involved in the synthesis of Very Long Chain Fatty Acids (VLCFAs) is confirmed to be an SD38 gene, and single base mutation of the SD38 gene is proved to be a cause of half-dwarfing of an SD38 mutant through a complementation experiment and a CRISPR/Cas9 mutant phenotype. The expression level of the SD38 gene in the caulicles, tillering buds and leaf sheaths is high as shown by phylogenetic tree analysis and gene expression pattern analysis of SD 38; compared with the wild type, the sd38 mutant has disordered lipidosome metabolism, and the content of endogenous C24:0 is greatly reduced; the heterologous expression of the SD38 gene in saccharomyces cerevisiae can promote the accumulation of C24:0 content, which indicates that SD38 is involved in the extension of C24:0VLCFA and influences the lipid change in vivo; the expression level of ethylene synthesis related genes in the sd38 mutant is reduced, and the content of an ethylene synthesis precursor ACC is also remarkably lower than that of a wild type; exogenous addition of C24:0, ACC and ethephon can promote the elongation of SD38 mutant seedlings to a certain extent, and C24:0 can induce the expression of OsACS3, which shows that SD38 regulates and controls the expression of OsACS3 by mediating the synthesis of C24:0VLCFA, thereby promoting the synthesis of ethylene and influencing the normal morphological development of rice.

The invention also discloses a protein coded by the half-dwarf phenotype regulatory gene SD38 in the rice full-growth period, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

The invention also discloses the application of the rice full-growth half-dwarf phenotype regulation gene SD38 and the coded protein thereof in rice molecular breeding.

The invention also discloses a method for regulating the plant type of rice, which regulates the plant height phenotype of the rice by regulating the content of C24:0VLCFAs in the rice.

The invention has the beneficial effects that: the invention provides a rice full-breeding half-dwarf phenotype regulation gene SD38, which regulates the expression of OsACS3 by mediating the synthesis of C24:0VLCFA, thereby promoting the synthesis of ethylene, further influencing the normal morphological development of rice and providing a new tool and a new method for high-yield breeding of rice.

Drawings

FIG. 1 is a phenotypic characterization of wild type and sd38 mutants; wherein, A: 1 month of wild type and sd38 mutant seedling plants, scale 5 cm; b: wild type and sd38 mutant plants at the jointing stage, scale 25 cm; c: wild type and sd38 mutant plants at maturity scale 30 cm; d: ear length and internode length of wild type and sd38 mutant, scale 8 cm; E-F: the bases of the upper three functional leaves of the wild type (E) and the sd38 mutant (F) were, from left to right, xiphoid leaf, inverted two leaves and inverted three leaves, respectively, and the scale was 3 cm;

FIG. 2 is a microscopic view of the bases of wild type and sd38 mutant flag leaves; wherein, A-B: flag leaves of wild type (a) and sd38 mutant (B) enveloped by leaf sheath, scale 200 μm; c: expanded sword leaves of wild type and sd38 mutant, scale 5 mm;

FIG. 3 is a histological analysis of the aerial parts of Wild Type (WT) and sd38 mutants; wherein, A: scanning electron micrographs of intrathecal epidermis of 3 weeks old wild type (left) and sd38 mutant (right) leaves, scale 200 μm; b: counting the length of the epidermal parenchyma cells in the leaf sheath, wherein the value is the average value +/-standard deviation (n is 7); c: paraffin sections from the middle of the two internodes at the heading stage of the wild type (left) and sd38 mutant (right) were cut longitudinally, and the scale is 200 μm; d: counting the length of the middle parenchyma cells between two nodes in the heading stage of the wild WT and sd38 mutant, wherein the value is the average value +/-standard deviation (n is 10); e: paraffin section longitudinal map of wild type (left) and sd38 mutant (right) at the apical meristem at the elongation Stage (SAM), red arrow representing the first and second internodal region under SAM, scale 200 μm; f: the area between the wild type and sd38 mutant SAM lower nodes was counted as mean ± standard deviation (n ═ 10). Student's t-test (, P < 0.05;, P <0.01) was used to detect the significance of the difference;

FIG. 4 is a map-based clone of the SD38 gene; wherein, A: SD38 gene location; b: arabidopsis thaliana neutralizes the phylogenetic tree analysis of the reported KCS family members of rice, and red represents the reported KCS protein in rice; c: wild type at mature stage, SD38 mutant and SD38-COM transgenic plant, scale is 25 cm; d: sequencing peak maps of the mutant sites of SD38-COM complementary plants, and the black arrows indicate SD38 mutant sites;

fig. 5 is the phenotype of SD38Cas9 mutant; wherein, A: mutation sites of the SD38Cas9-1 and SD38Cas9-2 mutants, red letters indicate sgRNA target sequences; b: morphology of 3-week-old wild-type (ZH11), SD38Cas9-1, and SD38Cas9-2 plants, scale 3 cm;

FIG. 6 shows the subcellular localization and expression pattern analysis of SD38 protein; wherein, A: subcellular localization of SD38-GFP fusion protein in rice protoplasts, scale 10 μm; B-C: qRT-PCR and RT-PCR analysis of SD38 expression at different tissue sites, mean. + -. standard deviation (n-3); D-H: detecting GUS staining in stems, leaves, leaf sheaths, young ears and mature ears, wherein the scale is 1 cm;

FIG. 7 shows complementation verification of SD38 in yeast elo3 knockout mutant; wherein, A: growth rates of Saccharomyces cerevisiae BY4741 and BY4741-elo3 yeast cells transformed with pYES2 null and pYES2-SD38 vectors, respectively, on YPG medium; b: growth curves of yeast strains in YPG liquid medium; c: GC-MS analysis of the content of saturated VLCFAs (C16-C30) in BY4741-elo3 yeast cells transformed with pYES2 empty and pYES2-SD38 vectors, respectively, as mean. + -. standard deviation (n. about.3); d: mass spectrum in GC-MS analysis. Student's t-test (, P <0.01) was used to detect significance of the difference;

FIG. 8 is statistics of lipids and their subclasses identified in lipidomics;

FIG. 9 is a lipid analysis of wild type and sd38 mutant; wherein, A: total lipid content in wild type and sd38 mutant; B-G: the content of various classes of Glycerophospholipids (GP), including Phosphatidylethanolamine (PE) (B), lysophosphatidylethanolamine (lysoPE) (C), lysophosphatidylcholine (lysophosphatidylcholine) (D) and its subclasses LysoLPC (18:1) and LysoLPC (20:4) (E), phosphatidic acid (phosphatidylcholine, PA) (F) and its subclasses (G); h: diglyceride (DG) content; i: acyl sitosterol ester (acylglcsistosterol ester, AGlcSiE) content; j: coenzyme (Co) content. Values are mean ± sd (n ═ 3), Student's t-test (, P < 0.05;, P <0.01) was used to detect significance of the difference;

FIG. 10 is C24:0VLCFA promoting elongation of sd38 seedlings; wherein, A: VLCFAs content of one month old wild type, SD38 mutant and complementary transgenic plants (SD38-COM), mean ± standard deviation (n ═ 3); b: phenotype of sd38 mutant seedlings after C16:0 and C24:0 treatments, scale 5cm, values mean ± standard deviation (n 10); c: phenotypes of wild type and sd38 mutant seedlings under treatment with the VLCFAs synthesis inhibitor, Cafenstrole (CS), were 5cm on a scale, and values were mean ± standard deviation (n 10). Student's t-test (ns, non-significant; P < 0.05; P <0.01) was used to detect the significance of the difference;

FIG. 11 shows sd38 seedling ethylene synthesis deficiency; wherein, A: expression of ethylene synthesis-related genes in wild-type and sd38 mutants, values mean ± standard deviation (n ═ 3); b: the content of 1-aminocyclopropane-1-carboxylic Acid (ACC) in the WT and sd38 mutants was as a mean value ± standard deviation (n ═ 3); c: exogenous ACC treatment of wild type and sd38 mutant seedlings, scale 3cm, values mean ± standard deviation (n 10); d: exogenous Ethephon (ETH) treatment of wild type and sd38 mutant seedlings, scale 3cm, values mean ± standard deviation (n 10). Student's t-test (ns, non-significant; P < 0.05; P <0.01) was used to detect the significance of the difference;

FIG. 12 is C24:0VLCFA induced expression of OsACS 3; wherein, A: expression of ethylene synthesis-related genes under C24:0VLCFA treatment, values mean ± standard deviation (n ═ 3); b: OsACS3 expression under C24:0 and Cafenstrole (CS) treatment, values mean. + -. standard deviation (n-3). Student's t-test (, P < 0.05;, P <0.01) was used to test the significance of the difference.

Detailed Description

The invention is further described below with reference to the figures and examples. The following examples are intended only to illustrate the invention and should not be construed as limiting it; the experimental methods in the following examples are all conventional methods; materials and reagents used in the following examples are commercially available; the instrumentation used in the examples described below is conventional in molecular biology laboratories. Modifications or substitutions to methods, steps or conditions of the invention by those skilled in the art without departing from the spirit and substance of the invention are within the scope of the invention.

Example 1

1. Materials and methods

1.1 materials

1.1.1 plant Material

In the research, the half-dwarf mutant sd38 is derived from an Ethyl Methanesulfonate (EMS) mutant library of indica type rice restorer line red silk-restorer 10 of southwest university rice institute, and is subjected to continuous multi-generation selfing, so that the half-dwarf phenotype is stably inherited. The CRISPR/Cas9 mutant was purchased from baige bio. All materials in the research, including wild type, mutant, positioning group and various transgenic plants, are planted under the natural environment conditions of the Rice research institute of Minam university of Beizhongmazhen, Chongqing, and are planted in greenhouse and south propagation base of Lingshui county, Hainan province in winter.

1.1.2 strains and vectors

Saccharomyces cerevisiae strain BY4741 and yeast knockout vector pUG6 were purchased from Hangzhou Fenghai Biotech, Inc.

1.1.3 molecular biological Agents

Aminocyclopropane carboxylic acid (1-aminocyclopropane-1-carboxylic acid, ACC), ethephon and very long chain saturated fatty acids of various carbon chain lengths were purchased from SIGMA, USA.

1.2 morphological observations

During the period of full-growth, the wild type and sd38 mutant were observed continuously for morphological changes and photographed. And (3) counting main agronomic traits of the wild type and the sd38 mutant in the mature period, such as plant height, internode length, effective spike, grain number per spike, seed setting rate, grain length, grain width, thousand seed weight and the like, wherein 10 biological single plants are counted in each group.

1.3 histological analysis

1.3.1 Paraffin section

The apical meristem, the inverted internode and the base of the xiphoid leaf of the wild type and sd38 mutants were sampled and placed in a 50ml centrifuge tube previously filled with FAA fixative (45% distilled water, 45% absolute ethanol, 5% glacial acetic acid, 5% formalin), and the samples were allowed to sink to the bottom of the tube by a vacuum pump and stored at 4 ℃.

1.3.2 scanning Electron microscope

Refer to the conventional method of scanning electron microscopy.

1.4 extraction of genomic DNA

Reference is made to the conventional method of genomic DNA extraction.

1.5 Gene mapping

Preparing a hybrid combination of Xida 1A multiplied by sd38, observing the F1 generation phenotype in the field, counting the segregation situation of each character in the F2 generation population, and adopting a chi-square method to test the segregation ratio of the normal phenotype and the mutant phenotype. All semi-dwarf phenotypic individuals in the population of generation F2 were used for gene mapping. The primer sequences used for gene mapping were as follows:

1.6 complementation verification of candidate genes

1.6.1 complementary vector construction

3 pairs of specific primers are designed according to a reference sequence provided by an NCBI website (https:// blast. NCBI. nlm. nih. gov/blast. cgi), and comprise a fragment 1683bp sequences upstream of a start codon of the SD38 gene, a whole coding frame sequence and downstream of a stop codon. Primer sequences are shown in the following table:

using genome DNA of wild red silk hui No. 10 as template, amplifying 3 corresponding fragments by using synthesized complementary primer pair, detecting and recovering target fragment by 1% agarose gel electrophoresis. EcoRI and HindIII restriction enzymes cut the plant expression vector pCAMBIA1301, EcoRI/KpnI, KpnI/BamHI and BamHI/HindIII cut 3 fragments, respectively, 1% agarose gel electrophoresis recovered the cut fragment and vector backbone and T4 ligase 16 ℃ overnight connection. The ligation products were transformed into E.coli DH 5. alpha. competence and the positive single spots screened were sequenced. Extracting the monoclone strain plasmid with correct sequencing according to the operation steps provided by the plasmid extraction kit, namely the SD38 complementary vector, and storing at-20 ℃ for later use.

1.6.2 obtaining of complementary transgenic plants

The SD38 complementary vector is transformed into the callus of the SD38 mutant in an agrobacterium-mediated mode, and an SD38 complementary transgenic plant is obtained through a series of tissue culture operation technologies such as differentiation, screening, rooting and the like. The genetic transformation process is completed by Wuhanbo Yuanzhi biological Co. The positive transgenic plant is planted in Setman base of the university of southwest rice research institute, and mutation site sequencing detection and target character observation are carried out on the positive transgenic plant.

1.7 phylogenetic Tree analysis

The full-length amino acid sequence of the SD38 gene and the reported rice KCS gene was downloaded from the NCBI database (http:// www.ncbi.nlm.nih.gov), and the homologous gene protein sequence of the SD38 gene in Arabidopsis was downloaded from Phytozome (https:// Phytozome. The phylogenetic tree was constructed using the maximum likelihood method in MEGA 5 (1000 replicates).

1.8 Gene expression analysis

The operational flow of RNA extraction, cDNA synthesis and real-time quantitative detection (qRT-PCR) is referred to 1.3.8. The quantitative primer sequences used are shown in the table below.

1.8.1 semi-quantitative (RT-PCR)

Semi-quantitative analysis was performed using cDNA obtained from reverse transcription of RNA samples from different tissue sites. The cDNA amount of each tissue part is determined by using an Action primer to ensure the consistency of the electrophoresis strip brightness. On the basis, different tissue sites with determined cDNA amount were PCR amplified with SD38-RT primer. Bands of expected size were separated by electrophoresis in a 1% agarose gel and photographed using a gel imaging analysis system.

1.8.2 promoter + GUS assay

(1) Promoter + GUS detection vector primer design

With reference to the sequence provided by NCBI website (https:// blast. NCBI. nlm. nih. gov/blast. cgi), a fragment 3502bp upstream of the initiation codon of SD38 gene was selected as a putative promoter fragment, and the designed primer sequences were as follows:

(2) promoter + GUS detection vector construction

The genomic DNA of wild type red silk-restored No. 10 was used as a template, and the fragment was amplified using a synthesized promoter plus GUS vector primer. The target fragment and the plant expression vector pCAMBIA1301 were double-digested with BamHI and NcoI restriction enzymes, respectively, and the digested fragment and vector backbone were recovered by 1% agarose gel electrophoresis and ligated with T4 ligase overnight at 16 ℃. And transforming the ligation product to the competence of escherichia coli DH5 alpha, carrying out sequencing verification on the screened positive clone containing pCAMBIA1301-PSD38-GUS, storing the positive clone bacterial liquid with correct sequencing, and extracting the plasmid and storing at-20 ℃ for later use.

(3) Obtaining promoter + GUS transgenic plant

The constructed pCAMBIA1301-PSD38-GUS expression vector plasmid is sent to Wuhan Boehringer biology company to complete genetic transformation to wild type No. red silk Hei 10, and the obtained positive plant is verified and planted in the Setman base of the Rice research institute of southwest university.

(4) GUS staining of tissue sites

And (3) taking each tissue part of the positive transgenic plant, soaking the tissue parts in GUS staining solution, vacuumizing, and standing for 2 hours at 37 ℃ in a dark place. The staining solution is poured off and decolorized with 75% ethanol for a plurality of times until the color of the decolorized solution does not change any more. And observing and photographing the decolored tissue sample.

1.9 subcellular localization

With reference to the sequence provided by NCBI website (https:// blast.ncbi.nlm.nih.gov/blast.cgi), the coding sequence of SD38 gene was selected to design primers (minus stop codon), SpeI and NcoI cleavage sites were added to both ends of the primers, and protection base CGC was added, and the primer sequence is shown in the following table. Using cDNA of wild type red silk-restored No. 10 as a template, PCR amplifies specific target fragment and recovers it. The target fragment and the expression vector pAN580 were double-digested with SpeI and NcoI restriction enzymes, respectively, and after 1% agarose gel electrophoresis, the digested fragment and vector backbone were recovered and ligated with T4 ligase overnight at 16 ℃. And transforming the ligation product to the competence of escherichia coli DH5 alpha, sending the screened positive clone containing pAN580-SD38 to sequencing verification, storing the positive clone bacterial liquid with correct sequencing, and extracting plasmids to store at-20 ℃ for later use.

1.10 hormone and fatty acid treatment

(1) Material treatment

Seeds of wild type and sd38 mutant were cut in half, glume was removed, and half containing endosperm was retained. Cleaning with sterilized double distilled water, sodium hypochlorite solution and 75% ethanol in sequence on a super clean bench, and soaking in the double distilled water. And respectively planting the cleaned seeds on a prepared MS solid medium flat plate with the endosperm upward.

(2) 1-Aminocyclopropane-1-carboxylic Acid (ACC) and ethephon treatment

MS solid medium was prepared, dispensed into sterilized flasks before coagulation, and 10. mu.M ACC (Sigma-Aldrich, USA) or 120. mu.M ethephon (Sigma-Aldrich) was added and mixed well. And after the culture medium is solidified, selecting seeds with consistent growth vigor of the embryos, transplanting the seeds into an MS solid culture medium containing ACC and ethephon, and growing for about 10 days in a sterile tissue culture room. Each treatment was repeated 3 times.

(3) Fatty acid and Cafenstrole (CS) treatment

The fatty acids (C16:0 and C24:0, Sigma-Aldrich) were dissolved in MTBE (Solarbio) and finally made up to the desired concentration with sterile double distilled water. Cafenstrole (Sigma-Aldrich) was dissolved in methyl cyanide. Seeds with consistent germ growth were selected and transplanted into MS solid medium containing 50. mu.M fatty acid (C16:0 and C24:0) and 0.05. mu.M cafenstrole, and grown for about 10 days between sterile tissue cultures. Each treatment was repeated 3 times.

Determination of the content of 1.111-Aminocyclopropane-1-carboxylic Acid (ACC)

And (3) taking aerial parts of the wild type and the sd38 mutant at the seedling stage for ACC content determination. The measurement work was performed by the New Life Biotechnology Co., Ltd. The specific process is as follows:

(1) grinding a sample by using liquid nitrogen, putting 100 +/-5 mg of the sample into a 2mL centrifuge tube, adding 30 mu L of internal standard solution, adding 1mL of acetonitrile water solution (1% FA), and shaking and uniformly mixing for 2 min; extracting at 4 deg.C in dark for 12h, centrifuging at 14000g for 20min, collecting supernatant 800 μ L, blowing with nitrogen gas, redissolving with 100 μ L acetonitrile water (1:1, v/v), centrifuging at 14000g for 20min, collecting supernatant, and sampling for analysis.

(2) Taking a standard substance, diluting the standard substance with methanol aqueous solution to obtain standard working solution with series concentration, preparing standard curve solution, and establishing a standard curve by an isotope internal standard method.

(3) The samples were separated using a Waters I-Class LC ultra performance liquid chromatography system. Mobile phase: the solution A is 0.05% FA aqueous solution, and the solution B is 0.05% FA acetonitrile. The sample was placed in an autosampler at 4 ℃ at a column temperature of 45 ℃ at a flow rate of 400. mu.L/min and a sample volume of 2. mu.L. The relevant liquid phase gradients are as follows: the liquid B is linearly changed from 2 percent to 98 percent in 0-10 min; for 10-10.1 min, the liquid B is changed from 98% to 2% linearly; 11.1-13min, and the B liquid is maintained at 2%. And setting a mixed sample (quality control sample) at intervals of a certain number of experimental samples in the sample queue for detecting and evaluating the stability and repeatability of the system.

(4) Mass spectrometry was performed using a 5500QTRAP mass spectrometer (AB SCIEX) in positive/negative ion mode. 5500 QTRAPESI source conditions are as follows: source temperature 500 ℃, ion Source Gas1(Gas 1): 45, Ion Source Gas2(Gas 2): 45, Curtain gas (CUR): 30, ionsaparaty Voltage flowing (ISVF) -4500V; and detecting ions to be detected by adopting an MRM mode.

(5) Chromatographic peak area and retention time were extracted using Multiquant software. The measured ACC content of the sample was calculated according to the standard curve.

1.12 Liposomal analysis

5 biological replicates of the aerial parts of the wild type and sd38 mutants at seedling stage were taken and subjected to lipidomic analysis by Shanghai Kagaku New Life Biotech Co. The specific process is as follows:

(1) grinding the sample in liquid nitrogen, after slowly thawing at 4 ℃, respectively weighing about 50mg of the sample, sequentially adding 100 mu L of water, 200 mu L of precooled methanol, 400 mu L of dichloromethane and 120 mu L of water, mixing by vortex, standing at room temperature for 20min, centrifuging at 8000g and 10 ℃ for 15min, taking a lower-layer organic phase, drying by blowing with nitrogen, adding 100 mu L of isopropanol solution for redissolving during mass spectrometry, vortex, centrifuging at 8000g and 10 ℃ for 15min, taking a supernatant, and analyzing by sample injection.

(2) The sample was separated using a UHPLC Nexera LC-30A ultra performance liquid chromatography system. The column temperature is 45 ℃; the flow rate is 300 mu L/min; the sample size was 5. mu.L. Mobile phase composition A: 10mM aqueous ammonium formate acetonitrile (acetonitrile: water ═ 6:4, v/v), B: 10mM ammonium formate acetonitrile isopropanol solution (acetonitrile: isopropanol ═ 1:9, v/v). The gradient elution procedure was as follows: 0-7min, keeping B at 30%; 2-25min, B changes linearly from 30% to 100%; b is maintained at 30% for 25.1-30 min. The samples were placed in a 10 ℃ autosampler throughout the analysis. In order to avoid the influence caused by the fluctuation of the detection signal of the instrument, the samples are continuously analyzed in a random sequence.

(3) Detection was performed using electrospray ionization (ESI) positive and negative ion modes, respectively. Separating the sample by UHPLC and using Q active plus mass spectrometer (Thermo Scientific)^TM) Mass spectrometry was performed. The mass-to-charge ratio of the lipid molecules and the lipid fragments was collected as follows: 10 fragment patterns (MS2 scan, HCD) were acquired after each full scan (full scan). MS1 at M/Z200The resolution was 70000, and the resolution of MS2 was 17500 at M/Z200.

(4) LipidSearch software version 4.1(Thermo Scientific) was used^TM) Performing peak identification, lipid identification (secondary identification), peak extraction, peak alignment, quantification and other treatment. The main parameters are as follows: 5ppm for precursor tolerance, 5ppm for product ion threshold: 5 percent. Extracting the obtained data, and deleting RSD>30% lipid molecules. Deleting deletion values in the group from the data extracted by the LipidSearch>50% lipid molecules, data were normalized for total peak area. The pattern recognition is carried out by using software SIMCA-P14.1 (Umetrics, Umea, Sweden), and multidimensional statistical analysis is carried out after data is preprocessed by Pareto-scaling.

1.13 measurement of saturated fatty acids

(1) 80-100mg of rice sample is added into a 15ml centrifuge tube, and 2ml of 5% hydrochloric acid methanol solution, 3ml of chloroform methanol solution (volume ratio is 1:1) and 100 microliter of methyl nonadecanoate internal standard are added continuously. Water bath is carried out in a water bath kettle at the temperature of 85 ℃ for 1 hour. And (3) after the water bath is finished, waiting for the temperature to be reduced to the room temperature, adding 1ml of n-hexane into a centrifugal tube, shaking and extracting for 2min, standing for one hour, and waiting for layering. Taking 100 microliter of supernatant, and using normal hexane to fix the volume to 1 ml. And (4) performing machine test after the membrane is filtered by a 0.45 micron filter membrane.

(2) The samples were subjected to compositional measurements according to the following chromatographic and mass spectrometry procedures.

Chromatographic conditions are as follows: a chromatographic column: TG-5MS (30 m.times.0.25 mm.times.0.25 μm); temperature rising procedure: keeping the temperature at 80 ℃ for 1min, heating to 200 ℃ at the speed of 10 ℃/min, continuing heating to 250 ℃ at the speed of 5 ℃/min, finally heating to 270 ℃ at the speed of 2 ℃/min, and keeping the temperature for 3 min; sample inlet temperature: 290 ℃; flow rate of carrier gas: no-flow-splitting sample injection is carried out at 1.2mL/min, and the valve opening time is 1 min.

Mass spectrum conditions: ion source temperature: 280 ℃; transmission line temperature: 280 ℃; solvent delay time: 5.00 min; scanning range: 30-400 amu; an ion source: EI source 70 eV.

1.14 Saccharomyces cerevisiae complementation test

Construction of 1.14.1 Saccharomyces cerevisiae mutant Strain BY4741-elo3

A BY4741-elo3 haploid mutant strain is constructed BY using a yeast knockout technology. Primers are designed to amplify two fragments of 2000bp respectively at the upstream and downstream of the yeast gene ELO3, and loxP sites at two ends of kanMX of a yeast knockout vector pUG6 are respectively inserted as homologous arms. The fragment containing both homology arms and kanMX was amplified, then transformed into the wild-type Saccharomyces cerevisiae BY4741 strain, and screened on YPD medium (1% yeast extract, 2% peptone, 2% D-glucose) supplemented with 200mg/mL G418. The primer sequences are as follows:

1.14.2 construction of expression vector SD38-pYES2

A pair of specific primers, comprising the coding frame of the SD38 gene, was designed based on the reference sequence provided by the NCBI website (https:// blast. NCBI. nlm. nih. gov/blast. cgi). Primer sequences are shown in the following table:

the cDNA of wild red silk-restored No. 10 is used as a template, a synthesized primer pair is used for amplifying a fragment, EcoRI and BamHI restriction enzymes are used for cutting the yeast expression vector pYES2, and the amplified fragment and the cut vector skeleton are respectively recovered by 1% agarose gel electrophoresis and are connected for 30min at 50 ℃ by using recombinase. The ligation products were transformed into E.coli DH 5. alpha. competence and the positive single spots screened were sequenced. Extracting the monoclonal strain plasmid with correct sequencing according to the operation steps provided by the plasmid extraction kit, namely SD38-pYES2, and storing at-20 ℃ for later use.

1.14.3 complementation test of saccharomyces cerevisiae

The constructed yeast expression vector SD38-pYES2 and the unloaded pYES2 were transformed into the constructed Saccharomyces cerevisiae mutant strain BY4741-elo3, respectively. The transformation method is referred to 1.3.10. Transformed yeast strains were screened on SC-Ura plates containing 2% galactose and 200mg/mL G418. Wild type strain BY4741 and BY4741-elo3 strain containing SD38-pYES2 and no load pYES2, respectively, were spread on YPG plates (1% yeast extract, 2% peptone and 2% galactose), cultured at 30 ℃ for 2 days, and the growth rate of each strain was observed. Different yeast strains are subjected to shaking table continuous culture at 30 ℃ in a YPG liquid culture medium, the light absorption value of each bacterial liquid at 600nm is measured at different time points, the cell density is measured, and a growth curve is drawn.

1.14.4 determination of saturated fatty acid content in Saccharomyces cerevisiae

(1) Sample preparation

a) 5 biological replicates of the BY4741-elo3 strain containing SD38-pYES2 and no load pYES2, respectively, were collected after 2 days of shake culture at 30 ℃ in YPG broth. And (5) quickly freezing the deposited bacteria in liquid nitrogen.

b) A200. mu.L sample was added with 2mL of 10% chloromethylated acetol and 1mL of n-hexane, and reacted at 95 ℃ for 2 hours with shaking 1 time at 5min intervals.

c) 6mL of 6% potassium carbonate solution was added and vortexed for 2 min. Centrifuging to obtain n-hexane layer, and performing rotary evaporation to remove n-hexane.

d) Add 100. mu.L of n-hexane and 5. mu.L of internal standard (C19:0,5mg/mL) and vortex for 1 min. Centrifuging at 2000rpm/min for 5min, and taking the supernatant in a sample injection vial for detection.

(2) GC-MS analysis

Fatty acid content analysis was performed on the samples in a full scan manner using a Thermo Trace1300 gas chromatography-ISQ 7000 mass spectrometer (GC-MS), with the following specific parameters: a chromatographic column: DB-5(60m 0.25mm 0.25 μm); sample introduction amount: 1 mu L of the solution; sample introduction temperature: 260 ℃; carrier gas: helium (99.999%); flow rate: 1.5 mL/min; column temperature: maintaining at 140 deg.C for 2min, heating to 180 deg.C at 10 deg.C/min, heating to 210 deg.C at 4 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 20 min; the split ratio is as follows: 20: 1; interface temperature: 260 ℃; ion source temperature: 230 ℃; an ionization mode: EI +, 70 ev; the mass range is as follows: 33-550;

(3) data analysis

Extracting characteristic peaks of GC/MS data under Thermo data software Chromeleon7.0, sorting the data according to an NIST 17 database, editing the data in an Excel 2016 software at a later stage, and organizing a final result into a two-dimensional data matrix containing information such as retentition time (Retention time), observed quantity (sample), peak intensity and the like.

2. Results and analysis of the experiments

2.1 morphological Observation of sd38 mutant

2.1.1 phenotypic characterization of sd38 mutants

In a field growing environment, the sd38 mutant shows a semi-dwarf phenotype from the seedling stage to the mature stage at each development stage (FIGS. 1A-C). Heading date statistics revealed that the sd38 mutant was significantly shorter in ear length and all internodes than the wild type (fig. 1D, table 1). Compared with the wild type, the characters such as the effective spike number, the grain length, the grain width and the like have no significant difference with the wild type (Table 1). Furthermore, the sword-like leaves of the sd38 mutant exhibited an abnormal rolling morphology, whereas the inverted two and three superior leaves developed normally, similar to the wild type (FIGS. 1E-F). Specifically, the basal expansion of sd38 xiphoid leaves was hindered, half of the leaves curled inward, and there was no curled phenotype in the middle and upper portions. Paraffin section observation shows that when the sword leaves are wrapped by the leaf sheaths, both sides of the base of the wild sword leaves are regularly arranged in a curling manner, and both sides of the sd38 mutant sword leaves are irregularly arranged in a curling manner (fig. 2A-B), when the sword leaves are completely unfolded, both sides of the base of the wild sword leaves can be normally unfolded outwards, and the leaves of the sd38 mutant are curled inwards (fig. 2C).

TABLE 1 major agronomic traits for wild type and mutant sd38

2.1.2 histological analysis of wild type and sd38

To explore the underlying cause of the semi-dwarf phenotype of the sd38 mutant, the inventors performed anatomical observations of the wild-type and the aerial part of the sd38 mutant. The length of the epidermal parenchyma cells in the leaf sheath of the sd38 mutant at the seedling stage is 20% shorter than that of the wild type (FIG. 3A-B). Longitudinal comparison of the wild type and sd38 mutant at heading stage between the poured two sections was performed using paraffin sections. Also compared to wild type, the sd38 mutant had a very significant decrease in parenchyma cell length in the middle of the inverted two internodes (fig. 3C-D). Furthermore, the first and second internode areas under the sd38 mutant SAM region were significantly reduced compared to the wild type (fig. 3E-F). These results indicate that the reduction in cell length may be the root cause of the semi-dwarfing of the sd38 mutant.

2.2 map-based cloning of the SD38 Gene

2.2.1 genetic analysis and Gene mapping

The SD38 gene was identified by using a map-based cloning strategy. The location population is formed by hybridizing indica male sterile line Xinong 1A and sd38 mutant. All individual plants of the F1 generation showed a normal phenotype similar to the wild type. Genetic analysis indicated that the segregation ratio of F2 wild-type phenotype to mutant phenotype individuals was 3:1 (a total of 886 individuals in 3706 populations exhibited mutant phenotypes; χ 2 ═ 0.86< χ 20.05 ═ 3.84). This indicates that the semi-dwarf mutant phenotype of the sd38 mutant is controlled by a pair of recessive nuclear genes. The SD38 gene was genetically mapped by 886 half-dwarf phenotypic single strains in the F2 population, and finally the SD38 gene was mapped between SSR markers RM25546 and RM25553 on the long arm of chromosome 10 at a physical interval of 99kb (FIG. 4A).

2.2.2 candidate Gene prediction

According to the gene annotation information provided by the Gramene website (http:// www.gramene.org /) and Rice genome annotation project (http:// Rice. plant biology. msu. edu /), a total of 11 annotated genes were included in the SD38 gene mapping region, of which 5 encoded putative proteins, 2 encoded transposon proteins, and 4 encoded annotated functional proteins, encoding cyclin-dependent kinase inhibitor proteins, endo-beta-N-acetylglucosaminidase, beta-ketoacyl-acetyl-CoA synthase, non-lysosomal-glucosylceramidase, respectively (Table 2). By sequencing 11 annotated genes, it was found that a single nucleotide mutation from G to a at the exon site of the LOC _ Os10G33370 gene in the SD38 mutant resulted in a mutation of the encoded amino acid from serine to asparagine, and that LOC _ Os10G33370 was initially identified as a candidate gene for SD38 (fig. 4A).

TABLE 2 functional annotation of genes within the SD38 localization interval

2.2.3 complementation verification

To confirm that LOC _ Os10g33370 is the gene of SD38, the inventors performed a complementation experiment. A9525 bp genomic fragment of LOC _ Os10g33370 in wild type, comprising 3364bp sequence upstream of the start codon and 1683bp sequence downstream of the stop codon, was transformed into the sd38 mutant in an Agrobacterium-mediated manner. In the SD38 complementation (SD38-COM) transgenic line, the half-dwarf phenotype of the mutant was completely restored (FIG. 4C), and sequencing revealed that the mutation sites in the complementation transgenic line were heterozygous (FIG. 4D). To further confirm the accuracy of the SD38 gene, the inventors obtained SD38 knockout mutants SD38Cas9-1 and SD38Cas9-2 in the background of japonica rice variety "middle flower 11" by using CRISPR/Cas9 technology, with deletion and insertion of a single base T in the coding frame of SD38 gene, respectively (fig. 5A). Both SD38Cas9 mutants showed a similar semi-dwarf phenotype as the SD38 mutant compared to the wild type "middle flower 11" (fig. 5B). These results confirmed that LOC _ Os10g33370 is the SD38 gene.

2.2.4SD38 phylogenetic Tree analysis

SD38/LOC _ Os10g33370 encodes beta-ketoacyl-CoA synthetase (KCS), involved in the biosynthesis of very-long-chain fatty acids (VLCFAs) (Haslam & Kunst, 2013). Phylogenetic tree analysis revealed that SD38 has the highest homology with two arabidopsis thaliana KCS family members AtKCS3 and AtKCS12 of unknown function (fig. 4B). Up to now, 4 KCS genes have been identified in rice, ONI1, ONI2, WSL1 and WSL4/HMS1(Ito et al, 2011; Tsuda et al, 2013; Yu et al, 2008; Wang et al, 2017; Chen et al, 2020). However, SD38 has low homology with these 4 known functional rice KCS proteins (fig. 4B). These results suggest that SD38 may play a new unknown functional role in the growth and development of rice.

2.3SD38 Gene expression Pattern analysis

2.3.1SD38 subcellular localization

To determine the subcellular localization of SD38, the inventors constructed a Green Fluorescent Protein (GFP) fusion vector containing the SD38 coding sequence and transiently expressed it in rice protoplasts. The green signal of the SD38-GFP fusion protein was able to coincide with the red signal on the endoplasmic reticulum Marker (FIG. 6A), indicating that SD38 is localized on the endoplasmic reticulum. This result is consistent with the recognition that proteins of the KCS family are localized on the endoplasmic reticulum and participate in the elongation of VLCFAs together with several other synthetases to form fatty acid elongase complexes (Haslam & Kunst, 2013).

2.3.2SD38 spatio-temporal expression Pattern

The expression of SD38 in different tissue sites was analyzed by qRT-PCR and RT-PCR methods for detection. SD38 was expressed in higher amounts in caulicles, tillering buds and leaf sheaths, in leaves, ear stems and ears, and in roots and petioles (FIGS. 6B-C). The expression pattern of SD38 was further analyzed using the SD38 promoter-GUS reporter gene. GUS signals were detected in the stem, leaf sheath and ear of promoter + GUS positive transgenic plants, consistent with the results of the qRT-PCR and RT-PCR analyses (FIGS. 6D-H).

2.4 functional verification of SD38 Gene in Saccharomyces cerevisiae

Three proteins in the cell of s.cerevisiae, ELO1, ELO2 and ELO3, are essential for the synthesis of VLCFAs and for maintaining the normal growth viability of the yeast (Pual et al, 2006). Haploid Elo3 yeast cells lack Elo3p activity, resulting in slower growth rate than wild type cells, and are ideal materials for functional identification of plant KCS genes (Qin et al, 2007 b). To verify that the SD38 gene encodes the KCS protein, the inventors performed a functional complementation test in Saccharomyces cerevisiae. An ELO3 knockout mutant BY4741-ELO3 of a saccharomyces cerevisiae haploid BY4741 strain is obtained BY adopting a homologous recombination knockout strategy. The growth rate of BY4741-elo3 cells carrying pYES2-SD38 expression vector was significantly higher than that of BY4741-elo3 cells carrying pYES2 empty load on YPG medium (FIG. 7A). The growth rate of BY4741-elo3 cells carrying pYES2-SD38 expression vector was also detected to be significantly higher than BY4741-elo3 cells carrying pYES2 empty load after continuous culture in YPG liquid medium (FIG. 7B). These results indicate that SD38 was able to partially restore the activity of ELO3 in the BY4741-ELO3 yeast mutant, demonstrating that the SD38 gene encodes the KCS protein.

In order to determine the specific function of KCS protein encoded BY SD38 gene in VLCFAs synthesis process, the content of VLCFAs in BY4741-elo3 cells carrying pYES2-SD38 expression vector and pYES2 empty was determined BY gas chromatography-mass spectrometry (GC-MS). As a result, it was found that the heterologous expression of the SD38 gene in Saccharomyces cerevisiae produced a large amount of C24:0 and C26:0, while the content of VLCFAs of other carbon chain lengths was the same as that of BY4741-elo3 cells containing pYES2 empty (FIG. 7C-D), indicating that SD38 was involved in the synthesis of C24:0 VLCFAs.

2.5 Lipomics analysis of wild type and sd38 mutants

The mutation of the KCS protein encoded by SD38, which acts as the rate-limiting enzyme in the synthetic pathway of VLCFAs, may cause the change in lipid content in vivo. To test this hypothesis, the inventors performed lipidomic analysis on wild type and sd38 mutant seedlings. In total, 26 lipids and 694 lipid subclasses were identified in the lipidomic data (fig. 8). The total lipid content of sd38 mutant was slightly reduced compared to the wild type, but did not reach a significantly different level (fig. 9A). As key components of the cell membrane, Phosphatidylethanolamine (PE), lysophosphatidylethanolamine (lysoPE), and lysophosphatidylcholine (LysoLPC) were significantly reduced in the sd38 mutant, and also included lysophosphatidylcholine subclasses LysoLPC (18:1) and LysoLPC (20:4) (fig. 9B-E). The total Phosphatidic Acid (PA) content did not differ significantly between the wild type and the sd38 mutant, but the phosphatidic acid subclass PA (34:2) and PA (37:1) content was significantly reduced in the sd38 mutant (FIGS. 9F-G). The content of other lipid components, such as Diglycerides (DG), acylsitosterol esters (AcylGlcSitocol ester, AGlcSiE and Coenzymes (Co) showed slight differences between wild type and SD38 mutant (FIGS. 9H-J). these results indicate that mutations in the SD38 gene affect changes in lipid content in rice.

2.6C24: 0VLCFA promotes elongation of sd38 seedlings

To further investigate the relationship of VLCFAs to rice plant height, the inventors examined the VLCFAs content of seedling stage wild type and sd38 mutant. Consistent with the results of the saccharomyces cerevisiae function complementation test, compared with the wild type, the SD38 mutant has the extremely significantly reduced content of C24:0VLCFA, and is recovered in SD38-COM transgenic plants, while the content of VLCFAs with other carbon chain lengths is not significantly changed (fig. 10A). Wild type and sd38 mutant seedlings were cultured in MS solid medium supplemented with 50 μ M C24:0 or C16: 0. After 10 days, the seedling length of sd38 mutant was counted and found to be significantly increased in the medium supplemented with C24:0, while the length of sd38 mutant seedlings did not change significantly under C16:0 treatment (fig. 10B). Furthermore, both wild type and sd38 mutant seedlings were reduced in length under treatment with the VLCFAs biosynthesis inhibitor, Cafenstrole (CS), but significant differences were achieved in wild type seedling length changes (fig. 10C). These results indicate that the defect in C24:0VLCFA is responsible for the semi-dwarf phenotype of the sd38 mutant.

2.7sd38 seedling ethylene Synthesis deficiency

As a gaseous phytohormone, ethylene is involved in fruit ripening, abscission and senescence, and also is involved in elongation of cotton fiber cells and various types of Arabidopsis cells (Shi et al, 2006; Qin et al, 2007a) and rice internode elongation (Iwamoto et al, 2010; Iwamoto et al, 2011) as a downstream regulatory factor of VLCFAs. To investigate the relationship between the semi-dwarfing of sd38 mutant and ethylene synthesis, the inventors examined the expression of ethylene synthesis genes (3 OsSMAS genes, 6 OsACS genes and 6 OsACO genes) in wild-type and sd38 mutant seedlings. Compared with the wild type, except that the expression levels of OsSAMS1, OsSAMS3, OsACS5 and OsACO1 are up-regulated in the sd38 mutant seedlings, the expression levels of the rest genes are down-regulated, particularly the expression levels of OsACS2, OsACS3, OsACS4 and OsACO7 are greatly and significantly down-regulated in the sd38 mutant seedlings (FIG. 11A). Consistent with the expression of the ethylene synthesis gene, the content of 1-aminocyclopropane-1-carboxylic Acid (ACC), which is an ethylene synthesis precursor, in seedlings of the sd38 mutant was very significantly reduced compared to the wild type (FIG. 11B). Exogenous ACC treatment had no significant effect on wild-type seedling growth, but was able to very significantly promote elongation of sd38 mutant seedlings (fig. 11C). Under exogenous ethylene treatment, both wild type and sd38 mutant seedlings were able to elongate, but sd38 mutant seedlings elongate more significantly (fig. 11D). These results indicate that ethylene synthesis in the sd38 mutant is hindered.

2.8C24: 0VLCFA induced expression of OsACS3

C24:0 VLFCA, ACC and ethephon can promote the elongation of sd38 mutant seedlings, and in order to further explore the relation between VLFCA and ethylene synthesis, the inventor analyzes the expression condition of ethylene synthesis related genes under the treatment of C24:0 VLFCA through qRT-PCR. As a result, the expression level of most of the ethylene biosynthesis genes was found to be up-regulated in the C24:0 VLFCA treatment, and particularly, the expression level of OsACS3 showed a very significant up-regulation (FIG. 12A). The inventors further observed that the expression level of OsACS3 reached a maximum 12 hours after C24:0 VLFCA treatment, in contrast to the VLCFAs biosynthesis inhibitor, Cafenstrole (CS), which inhibits the expression of OsACS3 (fig. 12B). These results indicate that VLCFAs may influence the morphological development of rice by regulating the expression of oscs 3 to promote ethylene synthesis.

3 discussion of results

3.1SD38 is involved in the Synthesis of C24:0VLCFA

The very long chain fatty acids VLCFAs are an important constituent of cell membranes. Mutations in genes associated with VLCFAs synthesis in plants exhibit severe developmental defects including abnormal cell division and differentiation, cell cycle defects, hormone response defects, and abnormal embryonic development (Bellec et al, 2002; Baud et al, 2003; Harrar et al, 2003; Baud et al, 2004; Da Costa et al, 2006; Bach et al, 2008; Roudier et al, 2010; Bach et al, 2011). VLCFAs are catalytically synthesized by elongase complexes localized on the endoplasmic reticulum, where KCS is thought to determine the carbon chain length of the substrate. Many KCS genes are annotated in different plants, which means that there is functional redundancy of KCS members in plants, however, each KCS protein exhibits a unique substrate specificity according to its role in the biosynthesis of VLCFAs (Haslam & Kunst, 2013). The KCS gene, FAE1(FATTY ACID elonga 1), first cloned in arabidopsis thaliana is responsible for the extension of C20:0 and C22:0 and is involved in seed Triacylglycerol (TAG) synthesis (Rossak et al, 2001). KCS1 is involved in C26:0 biosynthesis and epidermal wax formation (Todd et al, 1999). KCS2/DAISY and KCS20 are involved in C22: 0VLCFA elongation, and are essential for biosynthesis of suberin from the root under epidermal wax and osmotic stress (Franke et al, 2009; Lee et al, 2009). KCS5/CER60 and KCS6/CER6/CUT1 are involved in the synthesis of VLCFA of C24:0 and over 28 carbon chain lengths, and are precursors of epidermal waxy and pollen lipids (Millar et al, 1999; Fiebig et al, 2000; Hooker et al, 2002). KCS9 participates in the extension of C22:0 to C24:0, and is a precursor for various VLCFAs derivatives (Kim et al, 2013). The research identifies a new rice KCS gene SD38 by using a map-based cloning strategy. The complementary transgenic plant of SD38 and the CRISPR/Cas9 mutant phenotype confirmed that the single base mutation of the SD38 gene was responsible for the SD38 mutant exhibiting the semi-dwarf phenotype (FIG. 4C; FIG. 5). The SD38 mutant has reduced content of C24:0 (FIG. 10A), and the exogenous expression of SD38 in Saccharomyces cerevisiae BY4741-elo3 strain can promote the increase of content of C24:0 and C26:0 (FIG. 9C-D), indicating that SD38 is a key enzyme in the synthesis process of C24:0VLCFA in rice.

3.2C24: 0VLCFA promotes ethylene biosynthesis

An increasing number of studies have shown that VLCFAs or their derivatives mediate various biological processes in plants as signals. VLCFAs are capable of inhibiting the expression of the cytokinin biosynthesis gene ISOPENTENYL TRANSFERASE (ISOPENTENYL TRANSFERASE 3, IPT3), reducing cytokinin synthesis in vascular tissues and thus participating in the regulation of growth of arabidopsis thaliana organs (Nobusawa et al, 2013). In arabidopsis, VLCFAs and their derivatives affect the development of lateral roots by inhibiting the ability of ALF4 transcription levels to limit callus formation in the pericycle (Shang et al, 2016). Arachidonic acid (Arachidonic acid) is a VLCFA derivative that activates the arabidopsis stress response and defense signaling network by inducing the expression of stress-related genes (Savchenko et al, 2010). Saturated VLCFAs, or more precisely C24:0VLCFA, can activate the transcription of 1-aminocyclopropane-1-carboxylate oxidase (ACO), leading to the production of large amounts of ethylene, promoting the elongation of cotton fibers and arabidopsis stem cells (Qin et al, 2007 a). VLCFAs increased ethylene biosynthesis and promoted root airway formation by inducing the expression level of oscs 1 under hypoxic conditions in rice (Yamauchi et al, 2015). In rice KCS gene mutants, auxin-related genes are abnormally expressed, and the KNOX family gene specifically expressed in apical meristems is ectopically expressed in leaves (Ito et al, 2011; Takasugi & Ito, 2011; Tsuda et al, 2013). These findings indicate that VLCFAs are involved in a variety of biological processes by regulating the expression of certain key genes as a signaling factor to maintain normal plant growth. The expression of certain ethylene synthesis genes was down-regulated in sd38 mutants and ACC content was reduced (fig. 11A-B), and thus the inventors speculated that VLCFAs might also induce expression of key genes of the ethylene synthesis pathway. Further research shows that C24:0 can significantly improve the expression level of OsACS3 (figure 12). Similar effects of exogenous VLCFA (C24:0), ACC and ethephon on seedling elongation of sd38 mutant also indicate a correlation between VLCFAs and ethylene biosynthesis.

3.3 role of ethylene biosynthesis in Rice development

Ethylene is synthesized via ACC from S-adenosyl-L-methionine (S-adenosyl-L-methionine) catalyzed by ACC synthase (ACS) and ACC oxidase (ACO) (Chen et al, 2013). It has been shown that in Arabidopsis, ethylene acts synergistically with other hormones (auxins, brassinosteroids and gibberellins) to participate in the growth and development of different tissue sites, including root hair, apical hook and hypocotyls, by affecting cell elongation (Raz & Ecker, 1999; Achard et al, 2003; Seifert et al, 2004; De Grauwe et al, 2005; Stepanova et al, 2005). Ethylene also regulates cell division in the quiescent center of Arabidopsis root tip stem cells later in embryonic development (Ortega-Martinez et al, 2007). In addition, ethylene also plays an important role in promoting cotton fiber cell elongation (Shi et al, 2006; Qin et al, 2007 a). In rice, the ethylene synthesis gene, OsACO1, is highly expressed in elongated internodes, indicating that ethylene is involved in development of internode elongation during heading (Iwamoto et al, 2010). Further studies have shown that ethylene together with phytochromes and biologically active gibberellins can induce internode elongation in rice (Iwamoto et al, 2011; Iwamoto & Takano, 2011). These findings suggest that ethylene plays an important role in the signaling pathway that regulates plant cell elongation and division. In the present invention, exogenous addition of ACC and ethephon partially restored the seedling half-dwarf phenotype of sd38 mutant (FIGS. 11C-D), which also demonstrates that ethylene promotes elongation of rice.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the intention of all modifications, equivalents, improvements, and equivalents falling within the spirit and scope of the invention.

Sequence listing

<110> university of southwest

<120> rice full-growth-period semi-dwarf phenotype regulation gene SD38 and application thereof

<141> 2021-06-04

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actttatatg ctcacaagta cacaaccatg gttagtcagt gatacagctg tttagataat 4320

ggagctagtt agtactactg cttaattaag ttaggtctct ttctgttacc tttctatttt 4380

ttgtgcagaa aaaagaaaag agctcctgac caattttagc tatcacttcc cccaaaaaag 4440

cgcagtactg aaattaatta ccagtggtgg ttaggaaatt aattaagaaa ctgtgtgaaa 4500

gtaggaataa aatgaagaaa ggagccgagg taaaaaaaag agtacacacc aaattaatta 4560

gatgtttaca aacattttac aaattaatca caattctcaa ataaaaggga gttatcccaa 4620

ctatccttat taaatctact tcatatggtg catattagta aactataaag atgcttgtgc 4680

atgtactatt atcatttatc atacagcatg ctacccttat taaatctact acaagtagtg 4740

catattagta tactataagg atgtacatgc atgtaccatt accatttagg cattgtttag 4800

ttccaaactt tttcttcaaa cttccaactt ttccatcaca tcaaaacttt cctacacaca 4860

taaactttca acttttccat cacatcgttc caatttcaac caaacttcca atttttgacg 4920

tgaactaaac acacccttat tagaaggcac gaattttggt ccattatata tatatacata 4980

tagagagata ttcgttttga aaatatcgta ttagactgtt tccgatcgtc aaaatattaa 5040

gatttaatta tgggcaaatg cataatagat tttcttttca ggctttcagt ctctccacca 5100

tgttttcata aatttcataa aaacctataa taacatgaag gagaatacag tgatgattgt 5160

tgtagtagaa cagtagaagt acacactctt tggtactaca ctccaatact ccatcgctca 5220

tcgacagaga agtaaatggg atcgtgaagc tagagctgcg ctgcactgga ctgagctttc 5280

aaccacactg catgtgagcc agctgctctg ctccatactg cccccatagc ttaatactaa 5340

cagctagcta gctccttctt cttcctcctc ctccattaat ggcggctcag caagtttggt 5400

ttgcctttat aagagcagct accttacccc tagcctagcc accctgtaag cagcaagcat 5460

caatattgta ctgacgtgga tcatgtcttc ttcttcctcc tatggaacgt aggagtggta 5520

cagtacgtag taatggtaga tagacactgc ctttcaccgg tttgggcctt tgggatccat 5580

ggttgccggt ttcagtggtg aaagccagtg gcttttcagt tggagaaaaa tcggagagta 5640

tggttacccc accgaccgta gtccgacctg acaggtcagc aaaagcgggc ggtgaggtgg 5700

cgcatgcgtg gaaatggtgt gcccacgtgt ataaagcatg ggccgaggtt tggtctagga 5760

ataacaactg agatctatca attgacggag taaaaaaaat atagaggtca acagtttaga 5820

tcgattgtcc gtttgatcta gttggtcaat atatttaagt ttctaagtaa atgcaaggat 5880

ttttattttt cttgtggaga aggcattttt ctaaatttat ttacatttag acaccaatac 5940

attatgtatt ggaatactat tatgcctcta tccagtctcc atctctccga tcaattaacg 6000

gatcaaccga tattgcaaca aaacatacat ggcttgcatt ttaggattca tggtgcataa 6060

tctccatgca tggatgaagt agtaatttgg agtgtaaact ttgaacatac tctatctatt 6120

cagacatatt tgacatttag cataaatttc ggttgaacta acaataactt ttaaaatgcc 6180

tattttttta ttaaaaaaag tatatgtaga ttcaccatag aaatgtaagc agtaacatta 6240

ttataagctc attagatttt gtaaacacat tgtaataaaa aaatccacag tcaaattctt 6300

aaaaatctga ctaattaaat cttattctaa acatgagatt tgttttcttg ggaacaaaat 6360

aaaaggccca cccttttgct tattcctgga ataagcgaaa cgaagtgatc tttgaataaa 6420

cctttatata catgttctta acgatctaaa agcaaaggtt gaagaataaa cttcgatgaa 6480

aaaaacctta aaatcacctt caaatttaag gttaaaaatt taaattttgg ctgataagta 6540

gtataagcat ttgttggaat gcatatggca ggacgcacgc gaacaaggtg gcgctggtga 6600

tgacgtcgga gtcgatcgcc ccaaactggt acgccgggaa caagcgcagc ttcatgctgg 6660

gcaactgcct cttccgctcc ggcggctgcg cctacttcct caccaacgac ccccgccacc 6720

gccgccacgc caagctccgc ctccgccacc tcgtccgcac ccacaccggc gcctccgacg 6780

acgcctactc ctgcgccctc cagatggagg acgacgccgg ccgcccggga ttccacctcg 6840

gcaaggacct cccccgcgcc gccgtccacg ccttcgtcaa gaacctccgc ctcctcgccc 6900

cccgcgtcct ccccctcccc gagctcctcc gcctcgcctt cgccaccttc ctctcctccg 6960

gccgccgcag cggcggcaag aagacctcgc cgtcgcagca gccgctcacc atccgcatga 7020

aggccggcgt cgaccacttc tgcgtgcaca ccggcggcgc cgcggtgatc gacggcgtcg 7080

gcaaggggct caccctgacg gagcacgacc tcgagccgtc gcggatgacg ctgcacaggt 7140

tcgggaacac gtcggcgagc aacgtctggt acgtgctcgg ctacatggag gccaagcgcc 7200

gcctccgccc cggcgaccgc gtcctcatgc tcaccttcgg cgccggcttc aagtgcaaca 7260

gctgcgtctg gaccgtcgag aagcccgtct ccgacgccgg cgtctggaag gactgcatcg 7320

accactaccc tcccaaggag ctcgccaacc ccttcatgga gaagtacggc ttcgtcaagg 7380

acatgatgaa cttgtgatcg atccatcaaa ttccccaatt aattcctcga tcgatctctc 7440

aagaaccaat ataatcaatc gatattgcat gtgcttgctt gcgtgcatgg attaattaag 7500

cgatcggtta gttaattaat tttatcgcca tttgctgcag ctctgttgat gattaggatg 7560

aacattatat ttacagggag ggggaagaag aagtaagaac aatcaatgag ttgaacaaaa 7620

aaaaaactaa ggattcaatt cataggtaca tatagcagct tgtgattcca taattttctt 7680

ttcagcatgg tggtagtata tggattttat ttatttatct tatttttttt gttgttgttt 7740

gtaactctct gtatgttgct agctacgagt gtcgtcctag ctggtggcag gtgttgtgtc 7800

tttcattaat tgttcatggt gatggactat ttctattcaa ggatcaaatt aaattatttt 7860

tttttgaaag gaggattatt tatcagttca tggtcatgtg caagtctcat gctgcaataa 7920

atatcatata ccctgtaaag atacaaatag tatactatgt actagctact ataaggagta 7980

tatgttttat tcatatcttt tgagaaccaa ttcagacttg attaaggttt atcctttttt 8040

cattctaagt atttttccta aaacacaata tacatataga ggctcatata taaacattag 8100

cacactctta ttcagagtta gagcatccca gcaactcgtc taaatttgat cttccatata 8160

gctatataga tggtcataaa aaaaaggaaa aagtacgaat tactccctct caactattgc 8220

acgcgttcaa tttacccccc taaattacaa taccagatat ttttaaccct aaacttttca 8280

aaccggacaa attacccccc taactcacct cgaagcggtt ttgatccaac gtggcgcaca 8340

cgtggcggtc cagtcaacca aaaataatac tcctaaaaaa tagtggctcc cacttgtcag 8400

catcttctcc tctcttctac tttcttccac cttcccctcc catccatctc tctctctctc 8460

tctctctcac caaggagcaa gggccgggga acggcagcaa cggcgtcacc agcgtgggag 8520

gagacgagca gcaggcagcg cgcgtctcgc gaccgcaacg gcacgagcta ggggtggcgg 8580

aggacggcgc gacagtctag gctagggc 8608

<210> 2

<211> 465

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Glu Val Ala Thr Met Leu Thr Leu Thr Leu Leu Ala Tyr Ser Ala

1 5 10 15

Ala Met Leu Ala Arg Leu Leu Val Ala Arg Ser Arg Arg Arg Arg Cys

20 25 30

Tyr Leu Leu Asp Tyr Val Cys Tyr Lys Ala Thr Asp Asp Arg Lys Leu

35 40 45

Pro Thr Asp Leu Cys Gly Glu Ile Ile Gln Arg Asn Lys Leu Leu Gly

50 55 60

Leu Glu Glu Tyr Lys Phe Leu Leu Lys Val Ile Val Asn Ser Gly Ile

65 70 75 80

Gly Glu Glu Thr Tyr Gly Pro Arg Asn Ile Ile Gly Gly Gly Asp Ala

85 90 95

Arg Pro Asp Arg Leu Ala Glu Gly Met Glu Glu Met Asp Glu Thr Phe

100 105 110

His Ala Val Leu Asp Glu Leu Phe Ala Arg Ser Ser Ala Ala Gly Gly

115 120 125

Gly Gly Val Arg Pro Ala Asp Val Asp Leu Leu Val Val Asn Val Ser

130 135 140

Met Phe Ser Pro Ala Pro Ser Leu Ser Ala Arg Val Val Arg Arg Tyr

145 150 155 160

Asn Leu Arg Glu Asp Val Lys Val Tyr Asn Leu Thr Gly Met Gly Cys

165 170 175

Ser Ala Thr Leu Ile Ala Leu Asp Leu Val Asn Asn Phe Leu Arg Thr

180 185 190

His Ala Asn Lys Val Ala Leu Val Met Thr Ser Glu Ser Ile Ala Pro

195 200 205

Asn Trp Tyr Ala Gly Asn Lys Arg Ser Phe Met Leu Gly Asn Cys Leu

210 215 220

Phe Arg Ser Gly Gly Cys Ala Tyr Phe Leu Thr Asn Asp Pro Arg His

225 230 235 240

Arg Arg His Ala Lys Leu Arg Leu Arg His Leu Val Arg Thr His Thr

245 250 255

Gly Ala Ser Asp Asp Ala Tyr Ser Cys Ala Leu Gln Met Glu Asp Asp

260 265 270

Ala Gly Arg Pro Gly Phe His Leu Gly Lys Asp Leu Pro Arg Ala Ala

275 280 285

Val His Ala Phe Val Lys Asn Leu Arg Leu Leu Ala Pro Arg Val Leu

290 295 300

Pro Leu Pro Glu Leu Leu Arg Leu Ala Phe Ala Thr Phe Leu Ser Ser

305 310 315 320

Gly Arg Arg Ser Gly Gly Lys Lys Thr Ser Pro Ser Gln Gln Pro Leu

325 330 335

Thr Ile Arg Met Lys Ala Gly Val Asp His Phe Cys Val His Thr Gly

340 345 350

Gly Ala Ala Val Ile Asp Gly Val Gly Lys Gly Leu Thr Leu Thr Glu

355 360 365

His Asp Leu Glu Pro Ser Arg Met Thr Leu His Arg Phe Gly Asn Thr

370 375 380

Ser Ala Ser Asn Val Trp Tyr Val Leu Gly Tyr Met Glu Ala Lys Arg

385 390 395 400

Arg Leu Arg Pro Gly Asp Arg Val Leu Met Leu Thr Phe Gly Ala Gly

405 410 415

Phe Lys Cys Asn Ser Cys Val Trp Thr Val Glu Lys Pro Val Ser Asp

420 425 430

Ala Gly Val Trp Lys Asp Cys Ile Asp His Tyr Pro Pro Lys Glu Leu

435 440 445

Ala Asn Pro Phe Met Glu Lys Tyr Gly Phe Val Lys Asp Met Met Asn

450 455 460

Leu

465

Claims (4)

1. A rice full-breeding half-dwarfing phenotype regulation gene SD38 has a nucleotide sequence shown as SEQ ID No.1, and an amino acid sequence of a coded protein shown as SEQ ID No. 2.

2. The protein encoded by the rice full-breeding half-dwarf phenotype regulatory gene SD38 of claim 1.

3. The use of the rice full-term dwarf phenotype regulation gene SD38 of claim 1 or the encoded protein of claim 2 in rice molecular breeding.

4. A method for regulating the plant type of rice is disclosed, which can regulate the plant height phenotype of rice by regulating the content of C24:0VLCFAs in rice.

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