HRC1, a gene for high rubber content in rubber grass, and its application

Abstract

The present invention belongs to the field of biotechnology, and relates to a rubber grass high rubber content gene HRC1 and its application. A nucleic acid molecule is provided, which comprises any nucleotide sequence of the following (a1)-(a5): (a1) nucleotide sequence shown in SEQ ID NO:1; (a2) nucleotide sequence shown in SEQ ID NO:3; (a3) ​​a nucleotide sequence having at least 90% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO:1 and 3; (a4) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2; (a5) a nucleotide sequence encoding the amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2; wherein the nucleic acid molecule can confer a high rubber content to the rubber grass comprising the nucleic acid molecule. The present invention provides new gene resources for the cultivation of new varieties of high-rubber content rubber grass, and provides new ideas for studying the regulation of natural rubber synthesis pathways.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a rubber grass high rubber content gene HRC1 and an application thereof. Background Art

[0002] Natural rubber is a natural polymer compound with cis-1,4-polyisoprene as the main component. There are more than 2,500 different plants in the world that can produce polymers similar to natural rubber, but the one with real commercial application value is the Brazilian Hevea brasiliensis Muell.Arg. When the surface of the rubber tree is cut, the latex cells in the bark are cut off, and the latex flows out of the latex cells. After the subsequent collection and processing, the commercially available natural rubber is obtained. Natural rubber has high elasticity, plasticity, flexibility, good electrical insulation, etc. at room temperature. It has a wide range of uses and is indispensable in medical care, transportation, industry, agriculture, national defense, and people's daily lives. Since rubber trees can only grow in tropical and subtropical areas, they are affected by unsuitable climatic conditions, serious pests and diseases, and low automation, and the natural rubber produced is becoming increasingly scarce.

[0003] Rubber grass (Taraxacum kok-saghyz Rodin, TKS) is a perennial herbaceous plant of the genus Taraxacum in the family Asteraceae, originating in Kazakhstan and Xinjiang, my country. The rubber content of its perennial roots can be as high as more than 20% of dry weight, and the quality of the rubber is comparable to that produced by the Brazilian rubber tree, and can be directly used to produce rubber products. Rubber grass likes cool climates and has a wide range of adaptation, and is a promising rubber-producing plant. At present, rubber grass is mostly wild resources. By improving its agronomic traits and increasing the rubber content through genetic engineering, rapid breeding of rubber grass high-rubber germplasm can be achieved.

[0004] The biosynthesis of natural rubber is a typical plant isoprenoid secondary metabolic pathway, and its synthetic precursors are mainly synthesized by the mevalonate (MVA) pathway in the cytoplasm. First, three molecules of acetyl CoA are catalyzed by acetyl-CoA acetyltransferase (AACT) and HMG-CoA synthase (3-hydroxy-3-methyl-glutaryl-CoA synthase, HMGS) to complete a two-step condensation reaction to generate 3-hydroxy-3-methyl-glutaryl-CoA (3-hydroxy-3-methyl-glutaryl-CoA, HMG-CoA), and then MVA is generated under the action of HMG-CoA reductase (HMGR); MVA is phosphorylated under the action of mevalonate kinase (MK) and phosphomevalonate kinase (MPK), and then decarboxylated by pytophosphomevalonate decarboxylase (MDC) to generate isopentenyl pyrophosphate (IPP). IPP is catalyzed by isopentenyl pyrophosphateisomerase (IPI) to form dimethylallyl pyrophosphate (DMAPP). One molecule of IPP and one molecule of DMAPP are condensed head-to-tail under the catalysis of isopentenyl pyrophosphate transferase to form geranyl pyrophosphate (GPP). On this basis, IPP units are added successively to obtain other isopentenyl compounds: farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The MEP (2-C-methyl-D-erythritol-4-phosphate) pathway in the plastid can also form IPP and DMAPP, and there is a certain degree of overlap with the MVA pathway. Therefore, the MEP pathway may also be the source of rubber biosynthesis precursors.The synthesis of natural rubber requires one molecule of trans-configuration allyl pyrophosphate (such as DMAPP, GPP, FPP or GGPP) as the starting material. Under the catalysis of cis-prenyltransferase (CPT), the methylene group of IPP reacts with the dissociation product of allyl pyrophosphate diphosphate group to regenerate one molecule of allyl pyrophosphate terminal group, thereby starting the biosynthesis of rubber molecules. Subsequently, under the action of small rubber particle protein (SRPP) and rubber elongation factor (REF), thousands of IPPs are incorporated into the long chain of polyisopentenyl pyrophosphate to form rubber molecules of different sizes. Regulating the expression of genes in the natural rubber synthesis pathway can improve the efficiency of natural rubber synthesis. Summary of the invention

[0005] We found a mutant c112 with increased rubber content from the rubber grass SARE (Sense / antisense RNA expression) mutant library constructed earlier. This mutant plant blooms early, has an increased number of leaves, has little change in root dry weight, and has little change in the number of latex tubes in the roots. By analyzing the insertion sites in the c112 mutant, a gene related to dormancy / auxin inhibition was discovered and isolated. This gene comes from the rubber grass sequencing line 1151 and is expressed in the roots, leaves, flowers, seeds and other organs of rubber grass, with the highest expression in latex. Overexpression of this gene in rubber grass can increase the natural rubber content in the roots, so the gene was named HRC1 (High natural Rubber Content 1) gene.

[0006] Based on the above research results, the present invention provides a nucleic acid molecule comprising any one of the following nucleotide sequences (a1)-(a5):

[0007] (a1) the nucleotide sequence shown in SEQ ID NO: 1;

[0008] (a2) the nucleotide sequence shown in SEQ ID NO: 3;

[0009] (a3) a nucleotide sequence having at least 90% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NOs: 1 and 3;

[0010] (a4) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2;

[0011] (a5) a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2;

[0012] The nucleic acid molecule is capable of imparting high rubber content to the rubber grass comprising the nucleic acid molecule.

[0013] The expression cassette, vector or host bacteria containing the above nucleic acid molecule also fall within the protection scope of the present invention.

[0014] The vector may be a cloning vector, comprising the rubber grass high-rubber content gene HRC1 and other elements required for plasmid replication. The vector may also be an expression vector, comprising the rubber grass high-rubber content gene HRC1 and other elements that enable successful protein expression. In some embodiments, the expression vector is a pB7WG2D vector into which the rubber grass high-rubber content gene HRC1 is inserted.

[0015] The host bacteria can be a host bacteria containing a cloning vector, such as E. coli DH5α, by culturing the bacteria under appropriate conditions to replicate the rubber grass high rubber content gene HRC1; it can also be a host bacteria containing an expression vector, such as Agrobacterium tumefaciens AGL1, by using Agrobacterium tumefaciens-mediated genetic transformation technology to transfer the expression vector into the rubber grass receptor material to obtain a transgenic rubber grass plant.

[0016] The use of the above nucleic acid molecules in cultivating rubber grass plants with high rubber content also falls within the protection scope of the present invention.

[0017] The application of the above nucleic acid molecules in the production of natural rubber also falls within the protection scope of the present invention.

[0018] The present invention also provides a method for obtaining a rubber grass plant with a high rubber content, characterized in that it comprises: enhancing the expression of a gene encoding a protein in the rubber grass to obtain a rubber grass plant with a high rubber content; the protein is as follows (B1) or (B2):

[0019] (B1) a protein with an amino acid sequence as shown in SEQ ID NO: 2;

[0020] (B2) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2.

[0021] The coding gene of the protein is the above-mentioned nucleic acid molecule.

[0022] In some embodiments of the present invention, rubber grass plants with high rubber content are obtained by introducing an overexpression vector containing the above-mentioned nucleic acid molecule into a rubber grass receptor material.

[0023] In some embodiments of the present invention, the overexpression vector is introduced into the rubber grass recipient material through Agrobacterium-mediated introduction.

[0024] The rubber grass receptor material can be the rubber grass 1151 strain.

[0025] The present invention also provides a method for producing natural rubber, comprising: adopting any of the above methods to obtain transgenic rubber grass plants with high rubber content, cultivating the transgenic rubber grass plants and extracting natural rubber from the roots.

[0026] The present invention also provides a protein, which is as follows (B1) or (B2):

[0027] (B1) a protein with an amino acid sequence as shown in SEQ ID NO: 2;

[0028] (B2) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2.

[0029] The HRC1 gene separated by the present invention belongs to the dormancy / auxin inhibitory protein gene family, has an Auxinrepress conserved domain, encodes a complete dormancy / auxin associated protein (Dormancy / auxin associated protein, DRM / ARP) of 13.3KD, and its amino acid sequence is shown in SEQ ID NO:2. The experimental results show that overexpression of the HRC1 gene in the rubber grass 1151 strain can increase the content of natural rubber in the rubber grass root, and the expression of some key enzyme genes in the natural rubber synthesis pathway in the root of the rubber content increased mutant c112 increases, indicating that the HRC1 gene is involved in the expression regulation of some key enzyme genes in the natural rubber synthesis pathway. Therefore, the HRC1 gene can be used to improve the genetic improvement of the rubber content in the rubber grass root, provide new gene resources for the cultivation of new varieties of rubber grass with high rubber content, and provide new ideas for studying the regulation of the natural rubber synthesis pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The phenotypic detection results of rubber grass c112 mutant are shown. In the figure: A is a comparison of the whole plant of rubber grass wild type (WT) and c112 mutant grown for 4 months, with a scale length of 5 cm; B is the dry root drawing phenotype of 3-month-old rubber grass wild type (WT) and c112 mutant, with a scale length of 1 mm; C is the root section staining phenotype of 3-month-old rubber grass wild type (WT) and c112 mutant, with a scale length of 500 microns; D is the statistical result of the number of leaves of 2-month-old rubber grass wild type (WT) and c112 mutant; E is the statistical result of the single plant dry root weight (g) of 3-month-old rubber grass wild type (WT) and c112 mutant; F is the statistical result of the latex cell index (%) in rubber grass wild type (WT) and c112 mutant grown in soil for 3 months; G is the detection result of natural rubber content in 3-month-old rubber grass wild type (WT) and c112 mutant. Values ​​in DF are mean ± SD, n = 12 in D, n = 20 in E, and n ≥ 9 in F. P values ​​are indicated in the figures, and significance was analyzed by two-tailed Student's t test.

[0031] Figure 2 Fourier infrared spectra of rubber content in the roots of wild-type and c112 mutant plants of rubber grass are shown. In the figure: A is a Fourier infrared spectra of rubber content in wild-type (WT) rubber grass; B is a partial enlargement of the rectangular dotted box in A; C is a Fourier infrared spectra of rubber content in mutant c112; D is a partial enlargement of the rectangular dotted box in C; 699cm -1 The peak position near the external standard indicates the narrow distribution polystyrene absorption peak, 835cm -1 The nearby peak positions indicate the natural rubber absorption peak, and the shaded portion indicates the integrated area.

[0032] Figure 3 The results of the analysis of the insertion site of the rubber grass c112 mutant are shown. In the figure: A is the mutant form of the c112 mutant, where HRC1 represents the CDS region, and its two ends are UTR regions, the dark arrows represent the vector amplification primers, and the light arrows represent the vector sequencing primers; B is the PCR detection result of the insertion site of the c112 mutant, where the left lane is the amplification product of the primers c112-Site-1D-Sense / c112-Site-1D-Anti, and the right lane is the amplification product of the primers c112-Site-2D-Sense / c112-Site-2D-Anti; C is the qRT-PCR detection result of the gene utg1288.20 expression level in the rubber grass wild type (WT) and c112 mutant; D is the qRT-PCR detection result of the HRC1 expression level in the rubber grass wild type (WT) and c112 mutant. The values ​​in C and D are mean ± sd (n = 3), P values ​​are shown in the figures, and significance was analyzed by two-tailed Student's t test.

[0033] Figure 4 The phenotypic analysis results of 35S::HRC1 transgenic rubber grass are shown. In the figure: A is a root latex tube section of the wild type (WT) and 35S::HRC1 transgenic rubber grass grown for 3 months, where the scale length is 500 microns; B is the detection result of the transcription level of the HRC1 gene in the wild type (WT) and 35S::HRC1 transgenic rubber grass grown for 3 months; C is the detection result of the natural rubber content in the wild type (WT) and 35S::HRC1 transgenic rubber grass grown for 3 months. The values ​​in B and C represent the mean ± sd (n = 3), the P value is shown in the figure, and the significance analysis adopts a two-tailed student t test. 6#, 7# and 15# represent three strains of 35S::HRC1 transgenic rubber grass.

[0034] Figure 5 The structure and expression pattern of the HRC1 gene are shown. In the figure: A is the amino acid sequence alignment of the HRC1 gene and the homologous gene AT1G56220 in Arabidopsis thaliana, and the black bottom line indicates the position of the conserved domain predicted by NCBI; B is the relative expression of the HRC1 gene in different organs of rubber grass detected by quantitative PCR, and the values ​​in the figure are mean ± sd (n = 3). DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with examples and drawings. It should be understood that the following examples are only used as explanations and illustrations of the present invention and are not intended to limit the scope of the present invention.

[0036] The experimental methods used in the following examples, unless otherwise specified, are conventional methods in the art, and reference may be made to relevant experimental manuals or manufacturer's instructions; the reagents and materials used, unless otherwise specified, can be obtained from commercial channels or prepared by conventional methods.

[0037] Plant materials used in the following examples:

[0038] Plants and seeds of rubber grass (Taraxacum kok-saghyz Rodin, TKS) 1151 strain are preserved and propagated by our laboratory. Rubber grass 1151 strain has been published in the literature Lin, T., Xu, X., Ruan, J., Liu, S., Wu, S., Shao, X., Wang, X., Gan, L., Qin, B., Yang, Y., et al. (2018). Genome analysis of Taraxacum kok-saghyz Rodin provides new insights into rubber biosynthesis. Natl Sci Rev 5, 78-87. The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0039] The strains used in the following examples are:

[0040] Escherichia coli strain DH5α is preserved in our laboratory and can be purchased commercially. Agrobacterium tumefaciens strain AGL1 is a strain preserved in our laboratory and can be purchased from Beijing Huayueyang Biotechnology Co., Ltd. with the catalog number NRR01250.

[0041] The vectors used in the following examples are:

[0042] The intermediate vector pDONR221 is a DONR vector of the Gateway system, which is preserved in our laboratory and can be purchased from Invitrogen, catalog number 12536017. The binary vector pB7WG2D (Gateway) is preserved in our laboratory and can be purchased from the BioVector NTCC Plasmid Vector Bacteria Cell Gene Collection Center.

[0043] Reagents and consumables used in the following examples:

[0044] MS medium was purchased from Beijing Qiwei Yicheng Technology Co., Ltd., catalog number: M0222.0050. MES, 6-BA, NAA, and plant gel were purchased from Sigma. Sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10021418. Oil Red O was purchased from Sigma-Aldrich, Oil Red O solution, Cat#O1391. Polystyrene standards (NIM-RM2068) were purchased from the Chinese Academy of Metrology. Natural rubber standards were purchased from Sigma-Aldrich, Cat#431257-100G. RNA rapid extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd., catalog number: 0416-50. III First-Strand Synthesis System reverse transcription kit, purchased from Invitrogen, Cat#18080051. KOD FX (Toyobo, Cat#KFX-101), KOD Plus (Toyobo, Cat#KOD-201), purchased from Beijing Bailingke Biotechnology Co., Ltd. TaKaRa Taq TM , purchased from Beijing Liuhetong Economic and Trade Co., Ltd., item number: R001A. SsoFast Supermix, purchased from Bio-Rad, Cat#1725201. Agarose gel recovery kit (Cat. No.: DH101-01) and plasmid small-scale rapid extraction kit (centrifugal column type) (Cat. No.: DP102-01) were purchased from Beijing Bomade Gene Technology Co., Ltd. BP CLONASE II ENZYME MIX (Cat. No.: 11789100) and LR CLONASE II ENZYME MIX (Cat. No.: 11791020) were products of Invitrogen. Latex RNA extraction kit was purchased from Qiagen, Cat. No.: 74804.

[0045] Example 1. Acquisition and phenotypic identification of a rubber grass mutant with increased rubber content

[0046] 1. Obtaining the rubber grass mutant c112

[0047] In the early stage, our laboratory constructed a rubber grass SARE (Sense / antisense RNA expression) mutant library using rubber grass (Taraxacum kok-saghyz Rodin, TKS) 1151 strain as material, and obtained 8117 mutant plants. SARE stands for sense / antisense RNA expression system, which mainly overexpresses full-length cDNA and truncated cDNA. The fragments can be inserted into the vector in the forward or reverse direction. Its overexpression can not only lead to gene function gain and cosuppression, but also cause antisense inhibition, thereby producing a large number of mutant phenotypes. The construction method of the rubber grass SARE mutant library mainly refers to the published literature Mou, ZL, Wang, XQ, Fu, ZM, Dai, Y., Han, C., Ouyang, J., Bao, F., Hu, YX, and Li, JY (2002). Silencing of phosphoethanolamine N-methyltransferase results in temperature-sensitive malesterility and salt hypersensitivity in Arabidopsis. Plant Cell 14, 2031-2043. The mutant library was modified according to the different plant binary expression vectors used. The construction method of the mutant library is as follows: the roots, leaves, flowers and seeds of rubber grass are taken, and the total RNA is extracted respectively, the mRNA is separated, and the cDNA library is constructed. pB7WG2D (Gateway) is used as the terminal plant binary expression vector, and the cDNA library plasmid is electrotransduced into Agrobacterium tumefaciens AGL1 competent cells, and rubber grass is transformed, and mutant plants are obtained after herbicide screening.

[0048] Since rubber grass is self-incompatible and homozygous offspring cannot be obtained through seeds, rubber grass is cloned and propagated through tissue culture. Take the leaves of wild-type and mutant plants of rubber grass 1151 strain that are growing healthily, disinfect them with 12% (v / v) Kao bleach, and inoculate them into callus induction medium. When buds emerge from the wound, inoculate them into seedling medium. After 2-3 subcultures, the buds will differentiate into seedlings. After the sterile seedlings take root, they are transplanted to the greenhouse. The transplanting medium is nutrient soil: vermiculite = 1:1 (v / v). The greenhouse culture conditions are a temperature of 21°C, long daylight (16h light / 8h dark), and a light intensity of 80-120μE·m -2 ·s -2 .

[0049] The formula of callus induction medium is: MS 4.4g / L+MES 0.5g / L+6-BA 0.5mg / L+NAA 0.01mg / L+sucrose 20g / L+phytagel 3.6g / L, pH 5.8.

[0050] The formula of seedling culture medium is: MS 2.2g / L+MES 0.5g / L+sucrose 10g / L+plant gel 3.6g / L, pH 5.8.

[0051] By observing the phenotype of the mutant plants, testing the rubber content of the mutant plants and comparing them with the wild-type plants, a mutant with increased rubber content was found, which was numbered c112. The rubber grass mutant c112 blooms earlier and has more leaves than the wild-type ( Figure 1 A, 1D).

[0052] 2. Phenotypic identification of rubber grass mutant c112

[0053] (1) Observation of rubber grass root slices and broken wire drawing

[0054] Take the roots of the wild type and mutant c112 plants of rubber grass planted in the greenhouse for 3 months, and take a small section of about 1.5 cm long. Place it in 80% (v / v) ethanol and fix it for 24 hours. Then slice it with an oscillating slicer. The slice thickness is 100 μm, and it is stained with Oil Red O for 1 min. Then it is rinsed once with 45% (v / v) glacial acetic acid, washed twice with distilled water, and observed and photographed with a stereoscope (OLYMPUS SZX16) after sealing with 60% (v / v) glycerol. The latex area is counted with ImageJ software, and the latex cell index = latex cell area / root slice area × 100%. After the rubber grass root is washed, it is placed in a 50°C oven and dried to constant weight, and then the density of rubber filaments in the root is judged by the fracture method.

[0055] The results showed that compared with the wild type, the c112 mutant had more strands in the stem roots ( Figure 1 B), dry root weight does not change much ( Figure 1 E), there are more latex tubes in the root slices, but the difference is not significant ( Figure 1 C, 1F).

[0056] (2) Detection of natural rubber content in the roots of rubber grass

[0057] Extraction of natural rubber: Take the roots of the wild type and mutant c112 plants of Rubber Grass planted in the greenhouse for 3 months, wash them and place them in a 50℃ oven to constant weight, then freeze and grind them into powder, weigh 100mg of powder and place them in a 2.0mL centrifuge tube, add 1mL of toluene to suspend all the powders and fully mediate for 1min, extract at 50℃, 750rpm for 2h, centrifuge at 10,000rpm at room temperature for 10min, take the supernatant, repeat this 3 times. Concentrate the supernatant and make it constant to 500μL, add 1mL of methanol, place at 4℃ for 30min, centrifuge to remove the supernatant, add 1mL of distilled water, wash at room temperature for 10min, centrifuge to remove the supernatant, add 1mL of acetone, wash at room temperature for 10min, centrifuge to remove the supernatant, and store the dried rubber in a -20℃ refrigerator.

[0058] Detection of natural rubber content:

[0059] Natural rubber standards (Sigma-Aldrich, Cat#431257-100G) of different concentrations (0.5, 1, 2, 4, 6, 8 mg / mL) were prepared with toluene. 150 μL of each standard solution was taken and mixed with 15 μL of 10 mg / mL narrow distribution polystyrene external standard. 70 μL was taken with a pipette and evenly applied on a potassium bromide wafer that had been pressed by a tablet press in advance. The wafer was dried in an oven and then tested with a far-infrared spectrometer (BRUKER, TENSOR 27). The wafer was scanned 32 times during the test. The infrared spectrum obtained was calculated after simple atmosphere compensation and baseline correction at 835 cm -1 (natural rubber absorption peak) and 699cm -1 The peak area ratio of (narrow distribution polystyrene absorption peak). The standard curve was drawn with the peak area ratio and natural rubber concentration as coordinates, and the standard curve equation was obtained as X = (Y-0.0698) / 0.2596, R 2 = 0.9961, where X represents the concentration of natural rubber and Y represents 835 cm -1 and 699cm -1 The peak area ratio.

[0060] The extracted root rubber of the wild type and mutant c112 plants of rubber grass was dissolved in 1 mL of toluene, and 150 μL was taken. 15 μL of narrow distribution polystyrene with a concentration of 10 mg / mL was added as an external standard. After mixing, 70 μL was taken with a pipette. Infrared spectrum detection was performed according to the above method. The 835 cm -1 (natural rubber absorption peak) and 699cm -1Substitute the peak area ratio of the natural rubber absorption peak of rubber grass to the peak area ratio of the narrow distribution polystyrene absorption peak into the above standard curve X = (Y-0.0698) / 0.2596 to calculate the concentration of natural rubber. Relative content of natural rubber = concentration of natural rubber (mg / mL) × volume of toluene used for extraction (mL) / mass of powdered root used for rubber extraction (mg) × 100%.

[0061] The results are as follows Figure 1 As shown in G, the rubber content of the c112 mutant is nearly twice that of the wild type.

[0062] Example 2: Analysis of the insertion site of the rubber grass mutant c112

[0063] 1. Extraction and PCR amplification of genomic DNA from rubber grass

[0064] Young leaves of wild-type and c112 mutant of rubber grass were taken, ground into powder with liquid nitrogen, and DNA was extracted with 2% CTAB extraction solution. The leaves were incubated in a 65°C water bath for about 1 hour, extracted with an equal volume of chloroform for about 10 minutes, centrifuged at 4°C, 8,000rpm for 10 minutes, the supernatant was aspirated into a new centrifuge tube, 80% volume of isopropanol was added, gently shaken to mix, and placed at room temperature for 10 minutes. The precipitate was rinsed with 70% ethanol and placed at room temperature to dry, and then dissolved in an appropriate volume of sterilized ddH2O to obtain leaf DNA for PCR identification and resequencing.

[0065] 2. Extraction and reverse transcription of RNA from rubber grass roots

[0066] RNA extraction: Take the roots of the wild-type plants of Rubber Grass 1151 strain and the c112 mutant plants grown for 3 months, wash them, grind them with liquid nitrogen, and extract them with the Huayueyang RNA Rapid Extraction Kit (Cat#0416-50). The specific method is carried out according to the instructions of the kit. The kit is equipped with DNase to remove DNA contamination in the total RNA. Dissolve the total RNA in the elution buffer EB in the kit. Use a UV spectrophotometer to measure the OD values ​​at 230nm, 260nm, and 280nm to detect the quality and quantify the RNA.

[0067] Reverse transcription: Use III First-Strand Synthesis System Reverse Transcription Kit (Invitrogen, Cat#18080051) was used according to the product instructions with Oligo(dT) 20 For reverse transcription, the system and conditions are as follows: Oligo(dT) 20(50μM) 1μL, 10mM dNTPs 1μL, RNA 2μg, add ddH2O to a total volume of 10μL, mix, centrifuge, heat at 65℃ for 5min, and place on ice for 5min; add 10×RT buffer 2μL, 25mM MgCl2 4μL, 0.1M DTT 2μL, RNaseOUT (40U / μL) 1μL, SuperScript III RT (200U / μL) 1μL to the above system, mix, centrifuge, heat at 50℃ for 50min, heat at 85℃ for 5min, place on ice for 5min, add 1μL RNaseH, incubate at 37℃ for 20min, and heat at 65℃ for 20min. Obtain root cDNA of wild-type plants and c112 mutant plants, dilute 10 times with ddH2O and store in a -20℃ refrigerator.

[0068] 3. Identification of the inserted fragment in the rubber grass c112 mutant

[0069] In order to identify the gene fragment inserted in the c112 mutant, PCR amplification was performed using the leaf DNA of the c112 mutant as a template and the vector sequence amplification primers P35S-F1 / T35S-R1. The nucleotide sequences of the primers are as follows:

[0070] P35S-F1: 5'-CCAACCACGTCTTCAAAGC-3' (SEQ ID NO: 4);

[0071] T35S-R1: 5'-TCGCATGCCTGCAGGTCACT-3' (SEQ ID NO: 5).

[0072] The PCR system was: 2×KOD FX Buffer 25μL, 2mM dNTPs 10μL, KOD FX (1.0U / μL) 1μL, P35S-F1 (10μM) 0.75μL, T35S-R1 (10μM) 0.75μL, DNA template 2μL, ddH2O to 50μL, mix, centrifuge. The PCR program was: 94℃3min; 98℃10s, 55℃30s, 68℃1.5min, 30 cycles; 68℃10min.

[0073] After the reaction, the PCR product was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd., and the nucleotide sequence of the inserted fragment was obtained by sequencing using the vector sequence sequencing primers P35S-F2 / T35S-R2. The nucleotide sequence of the primers is as follows:

[0074] P35S-F2: 5'-TCATTTGGAGAGGACTCCGGT-3' (SEQ ID NO: 6);

[0075] T35S-R2: 5'-GAGAGAGATAGATTTGTAGAGAG-3' (SEQ ID NO: 7).

[0076] PCR sequencing results showed that a 640 bp fragment was inserted in the c112 mutant. The nucleotide sequence of the fragment is shown in SEQ ID NO: 3, which includes a 32 bp 5'UTR region, a 372 bp coding region and a 236 bp 3'UTR region. The coding region of the fragment was subjected to blastx sequence alignment on the NCBI website. The results showed that the coding region encodes a complete dormancy / auxin associated protein (DRM / ARP) with a size of 13.3 KD. The gene was named HRC1 (High natural Rubber Content 1) ( Figure 3 A).

[0077] Insertion fragment (640 bp) in the rubber grass c112 mutant

[0078] AGTGTTTGTTATCATCTTCTGTCTACTGCGCT ATG AGCTTACTCGACCAATTCTGGGACGACACCGTCGCCGGGCCTCGGCCGGAGAAAGGCCTCGGAAAGCTGCGAAAACAGTCTACTTTTAGTTCCCGGTCATCGGATTCCGGCAAGGAATCGCCGCCGGTCACAAATACGGCAGTAGAGGATCCCACGATGAGAGTAACACGAAGCATAATGATC GTAAAACCAGAAAGAACTCTGAGCGACACTCCGCCGTCATCTCCCGCCGGATCTACACCTCCGGTATCTCCTTTCCCTGGAGGCAGATCAGAGGCATTTAGGTTTCGTCGGAAGTCTGCATCGGACGCTTTCGAGAAGGCAAGCGGGATTGGAACCAGGAGCCCTCGTGCTCCTTACGACCTG TGAAATCTGATCTATAAACGTCAGGGAAGAAGGCAGATCACTAGGGTTTAGCTAGCATGCTATAAATGTGTTCGGGTATTATGTCTTTTTGAGAGAGAGAGAGAGTTTTCTCGTATGTGTATATAGTTGATGTAAATACGCGTGGAAAGTTGTGTTAGGTTTATGTTACACTCGTACTTTTTCTGGATGCTTGTAGGCTTGCTTTATATCATAAGATGCAATGCTTCGTTTTCTTGAAG(SEQ ID NO:3)

[0079] Coding region (372bp) of the HRC1 gene:

[0080] ATGAGCTTACTCGACCAATTCTGGGACGACACCGTCGCCGGGCCTCGGCCGGAGAAAGGCCTCGGAAAGCTGCGAAAACAGTCTACTTTTAGTTCCCGGTCATCGGATTCCGGCAAGGAATCGCCGCCGGTCACAAATACGGCAGTAGAGGATCCCACGATGAGAGTAACACGAAGCATAATGATCGTAAAACCAGAAAGAACTCTGAGCGACACTCCGCCGTCATCTCCCGCCGGATCTACACCTCCGGTATCTCCTTTCCCTGGAGGCAGATCAGAGGCATTTAGGTTTCGTCGGAAGTCTGCATCGGACGCTTTCGAGAAGGCAAGCGGGATTGGAACCAGGAGCCCTCGTGCTCCTTACGACCTGTGA(SEQ ID NO:1)

[0081] Amino acid sequence (123aa) of the protein encoded by the HRC1 gene

[0082] MSLLDQFWDDTVAGPRPEKGLGKLRKQSTFSSRSSDSGKESPPVTNTAVEDPTMRVTRSIMIVKPERTLSDTPPSSPAGSTPPVSPFPGGRSEAFRFRRKSASDAFEKASGIGTRSPRAPYDL(SEQ ID NO:2)

[0083] In order to find the insertion site of the gene fragment, the whole genome resequencing analysis of the c112 mutant was performed, and the primers c112-Site-1D-Sense / c112-Site-1D-Anti and c112-Site-2D-Sense / c112-Site-2D-Anti were designed for PCR amplification of the predicted insertion site. The amplification template was the leaf DNA of the wild type (WT) and c112 mutant of rubber grass. The nucleotide sequences of the primers are as follows:

[0084] c112-Site-1D-Sense: 5'-GAATGCGGATATTTAATCGAAGTG-3' (SEQ ID NO: 8);

[0085] c112-Site-1D-Anti: 5'-CGATAGAAAACAAAATATAGCGCGC-3' (SEQ ID NO:9).

[0086] c112-Site-2D-Sense: 5'-GTTGATGTAAATACGCGTGGAAAG-3' (SEQ ID NO: 10);

[0087] c112-Site-2D-Anti: 5'-TCTTATATGCTCAACACATGAGCG-3' (SEQ ID NO: 11).

[0088] The PCR system was: 2×GC Buffer 10μL, 2mM dNTPs 2μL, TaKaRa Taq (5U / μL) 0.2μL, forward primer (2μM) 1.5μL, reverse primer (2μM) 1.5μL, DNA template 2μL, ddH2O to 20μL. The PCR program was: 94℃3min; 94℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃10min.

[0089] The results showed that the c112-Site-1D primer pair had no amplification product in the wild type of rubber grass, but had a specific amplification product in the c112 mutant, indicating that the insertion site was amplified in the c112 mutant; c112-Site-2D had amplification products of the same size in the wild type and c112 mutant of rubber grass, indicating that this predicted insertion site was a false positive result. Analysis of the amplified sequence of the c112-Site-1D primer revealed that the insertion position of the fragment was located near 137706793 of chromosome 3 of rubber grass. The site was located in the intergenic region, with no gene within 4Kb upstream of the site, and the gene utg1288.20( Figure 3 A, 3B).

[0090] The root cDNAs of wild-type plants of Rubber Grass 1151 and c112 mutant plants were used as templates to generate the utg1288.20 gene ( http: / / bigd.big.ac.cn / gwh / , accession number: PRJCA000437, Gene ID: evm.model.utg1288.20) specific fluorescence quantitative PCR primers qc112-insertion-sense / qc112-insertion-anti, HRC1 gene specific fluorescence quantitative PCR primers qHRC1-Sense / qHRC1-Anti, and internal reference TkGAPDH gene ( http: / / bigd.big.ac.cn / gwh / , accession number: PRJCA000437, Gene ID: evm.model.utg9113.3) fluorescent quantitative PCR primers qTkGAPDH-Sense / qTkGAPDH-Anti, using Bio-Rad's SsoFast Supermix (Cat#1725201) was used to prepare the fluorescence quantitative PCR reaction system according to the instructions, and the fluorescence quantitative PCR was performed using the real-time fluorescence quantitative PCR instrument CFX96 from BIO-RAD. The nucleotide sequences of the primers are as follows:

[0091] qc112-insertion-sense: 5'-TAGCGGTCCACAAAGTCCAC-3' (SEQ ID NO: 12);

[0092] qc112-insertion-anti: 5'-CGGATTCAATTGAGCAGCCG-3' (SEQ ID NO: 13).

[0093] qHRC1-Sense: 5'-CTACACCTCCGGTATCTCCT-3' (SEQ ID NO: 14);

[0094] qHRC1-Anti: 5'-GAGGGCTCCTGGTTCCAATC-3' (SEQ ID NO: 15).

[0095] qTkGAPDH-Sense: 5'-AGTTGGTTTCGTGGTATGAC-3' (SEQ ID NO: 16);

[0096] qTkGAPDH-Anti: 5'-ACATGTCAGTGAACAGGTAGAC-3' (SEQ ID NO: 17).

[0097] The PCR reaction system was: 2×SsoFast mix 5μL; cDNA 2μL; forward and reverse primers (1μM) 1.5μL each. The PCR operation program was: 98℃, 30s; (98℃, 5s→60℃, 5s→information acquisition), 40 cycles; 60-95℃, 0.5℃ / 5s, information acquisition / 5s. After the program was completed, the data was analyzed using BIO-RAD CFX Manager software. The results showed that the transcription of the utg1288.20 gene in the c112 mutant was not affected ( Figure 3 C), the expression of HRC1 gene was significantly upregulated ( Figure 3 D), therefore the phenotype of the c112 mutant may be caused by overexpression of the HRC1 gene.

[0098] Example 3: Functional verification of HRC1 gene

[0099] To verify the function of the HRC1 gene, transgenic plants with 35S::HRC1 were obtained using transgenic technology and phenotype analysis was performed. 1. Construction of the overexpression vector 35S::HRC1

[0100] Gene amplification: Use the primer combination GW-HRC1-F and GW-HRC1-R, and use the root cDNA of the wild-type plant of Rubber Grass 1151 as a template to amplify the HRC1 gene fragment. The nucleotide sequence of the primers is as follows:

[0101] GW-HRC1-F:

[0102] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGCTTACTCGACCAATTCTG-3' (SEQ IDNO: 18);

[0103] GW-HRC1-R:

[0104] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCACAGGTCGTAAGGAGCAC-3' (SEQ ID NO: 19).

[0105] The PCR system was: 10×KOD Plus Buffer 5μL, 2mM dNTPs 5μL, 25mM MgSO4 2μL, KODPlus (1.0U / μL) 1μL, GW-HRC1-F (10μM) 0.75μL, GW-HRC1-R (10μM) 0.75μL, cDNA template 2μL, ddH2O to 50μL, mix, centrifuge. The PCR program was: 94℃3min; 98℃10s, 55℃30s, 68℃1min, 30 cycles; 68℃10min.

[0106] After the PCR reaction was completed, 1 μL of the reaction product was taken and tested for correct band size using 1% agarose gel electrophoresis. An agarose gel recovery kit (Biomed Biotech, catalog number DH101-01) was used to recover HRC1 gene fragments with correct band size according to the operating steps provided in the kit instructions.

[0107] BP reaction: The recovered HRC1 gene fragment was recombined into the pDONR221 intermediate vector by BP reaction to obtain pDONR221-HRC1. The BP reaction system and reaction conditions are as follows: 150 ng of HRC1 gene fragment, 150 ng of pDONR221 vector, 1 μL of BP Clonase II Enzyme Mix, 1×TE (pH=8.0) to 10 μL; incubate at 25°C for 10 h; add 1 μL of Proteinase K, incubate at 37°C for 10 min, obtain the recombinant product pDONR221-HRC1, and place on ice.

[0108] Transformation: Take the E. coli DH5α competent cells stored at -80℃ and melt them on ice. Add 5μL of the recombinant product pDONR221-HRC1, mix gently, and let it stand on ice for 30min. Heat shock at 42℃ for 45s, and let it stand on ice for 2min. Add 500μL of LB liquid culture medium without any antibiotics, and activate it at 37℃, 180rpm for 1h. Centrifuge the activated E. coli at 3,000rpm for 1min, remove most of the supernatant, and resuspend the bacteria in 100μL of supernatant. Spread the entire bacterial liquid on a solid LB plate containing 50μg / ml kanamycin, and culture it at 37℃ overnight.

[0109] Identification of positive colonies: Pick a single colony from the LB solid plate, inoculate it into 500 μL LB liquid medium containing 50 μg / ml kanamycin, activate it at 37°C, 200 rpm for 3 hours, and then perform bacterial liquid PCR to identify positive clones. The primers for bacterial liquid PCR are qHRC1-Sense (SEQ ID NO: 14) and M13R primers.

[0110] M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO: 20).

[0111] The PCR system was: 2×GC Buffer 10μL, 2mM dNTPs 2μL, TaKaRa Taq (5U / μL) 0.2μL, qHRC1-Sense (10μM) 0.3μL, M13R (10μM) 0.3μL, bacterial solution 2μL, ddH2O supplemented to 20μL. The PCR program was: 94℃3min; 94℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃10min. After the reaction, the band size of the PCR product was detected with 1% agarose gel, and the bacterial solution of the positive colony with the correct band size was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0112] Plasmid extraction: Take 200 μL of bacterial solution with correct sequencing results, inoculate it into 10 mL of LB liquid medium containing 50 μg / ml kanamycin, and culture it overnight at 37°C and 200 rpm. Use a plasmid small-scale rapid extraction kit (centrifugal column type) (Cat. No. DP102-01, Beijing Bomed Gene Technology Co., Ltd.) to extract the plasmid according to the kit instructions.

[0113] LR reaction: The HRC1 gene fragment was homologously recombined into the binary vector pB7WG2D (Gateway) using the LR reaction. The LR reaction system and reaction conditions were as follows: 150 ng of pDONR221-HRC1 plasmid, 150 ng of pB7WG2D plasmid, 1 μL of LR ClonaseII Enzyme Mix, supplemented to 10 μL with 1×TE (pH=8.0); incubated at 25°C for 10 h; added 1 μL of Proteinase K, incubated at 37°C for 10 min, and the overexpression vector 35S::HRC1 was obtained, which was placed on ice.

[0114] Transformation: Take the E. coli DH5α competent cells stored at -80℃ and thaw on ice. Add 5μL of the overexpression vector 35S::HRC1, mix gently, and let it stand on ice for 30min. Heat shock at 42℃ for 45s, and let it stand on ice for 2min. Add 500μL of LB liquid culture medium without any antibiotics, and activate at 37℃, 180rpm for 1h. Centrifuge the activated E. coli at 3,000rpm for 1min, remove most of the supernatant, and leave 100μL of the supernatant to resuspend the bacteria. Spread the entire bacterial liquid on a solid LB plate containing 50μg / ml spectinomycin, and culture at 37℃ overnight.

[0115] Identification of positive colonies: Pick a single colony from the LB solid plate, inoculate it into 500 μL LB liquid medium containing 50 μg / ml spectinomycin, activate it at 37°C, 200 rpm for 3 hours, and then perform bacterial liquid PCR to identify positive clones. The bacterial liquid PCR primers are qHRC1-Sense (SEQ ID NO: 14) and T35S-R1 (SEQ ID NO: 5).

[0116] The PCR system was: 2×GC Buffer 10μL, 2mM dNTPs 2μL, TaKaRa Taq (5U / μL) 0.2μL, qHRC1-Sense (10μM) 0.3μL, T35S-R1 (10μM) 0.3μL, bacterial solution 2μL, ddH2O supplemented to 20μL. The PCR program was: 94℃3min; 94℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃10min. After the reaction, the band size of the PCR product was detected with 1% agarose gel, and the bacterial solution of the positive colony with the correct band size was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0117] Plasmid extraction: Take 200 μL of bacterial solution with correct sequencing results, inoculate it into 10 mL of LB liquid medium containing 50 μg / ml spectinomycin, and culture it at 37°C, 200 rpm overnight. Use a plasmid small-volume rapid extraction kit (centrifugal column type) (Bomaide Biotechnology, product number DP102-01) to extract the plasmid according to the kit instructions.

[0118] The formula of LB liquid medium is: peptone 10g / L + yeast extract 5g / L + NaCl 10g / L, pH 7.0. LB solid medium is based on LB liquid medium and agar 15g / L is added.

[0119] 2. Obtaining Agrobacterium tumefaciens containing overexpression vector 35S::HRC1

[0120] The correctly sequenced overexpression vector 35S::HRC1 was transformed into Agrobacterium tumefaciens AGL1 by electroporation. The method is as follows: the competent cells of Agrobacterium tumefaciens AGL1 stored at -80°C were thawed in an ice bath. 150 ng of plasmid was added to each 50 μL competent cells, and then the competent cells were electroporated at 1.8 kV. After the electroporation, LB liquid medium without antibiotics was added and cultured at 28°C and 200 rpm for 3 hours. The cultured bacterial solution was centrifuged at 5000 rpm for 1 minute to collect the bacteria, most of the supernatant was discarded, and about 100 μL of the supernatant was retained. The bacterial block was resuspended by blowing gently, and then spread on a YEP solid culture medium plate containing 25 mg / L rifampicin and 50 mg / L spectinomycin. After dark culture at 28°C for 48 h, a single clone was picked and inoculated into a YEP liquid culture medium containing 25 mg / L rifampicin and 50 mg / L spectinomycin. After shaking culture at 28°C overnight, the bacterial solution was inoculated into a YEP liquid culture medium containing 25 mg / L rifampicin and 50 mg / L spectinomycin at a volume ratio of 1:100, and acetosyringone was added to a final concentration of 100 μM, and cultured to an OD of 0. 600 =0.6-0.8, collect the bacteria by centrifugation at room temperature, remove the supernatant, and fully suspend them with an equal volume of infection solution containing 100 μM acetosyringone to obtain the Agrobacterium tumefaciens infection solution for use.

[0121] The formula of YEP liquid medium is: peptone 10g / L + yeast extract 10g / L + NaCl 5g / L, pH 7.0. YEP solid medium is based on YEP liquid medium with 15g / L agar added.

[0122] The formula of the infection solution is: MS 4.4g / L+MES 0.5g / L+6-BA 0.5mg / L+NAA 0.01mg / L+sucrose 20g / L+glucose 10g / L, pH 5.2.

[0123] 3. Obtaining transgenic rubber grass T0 plants

[0124] The rubber grass receptor material used was the genome sequencing strain 1151 preserved in this laboratory. The roots of rubber grass tissue culture seedlings with good growth status were taken, cut into small segments of about 0.5 mm, and infected with the above-mentioned Agrobacterium tumefaciens infection solution for about 10-20 minutes, then the infected roots were scooped onto sterile filter paper to absorb the excess bacterial solution and dried, and co-cultured at 21-23°C in the dark for 2-3 days, and then the infected rubber grass roots were transferred to the resistance callus induction medium for screening and culture until green resistance buds appeared, and then transferred to the resistance seedling medium for screening and culture for 1-2 months, the culture temperature was 21°C, 16h light / 8h dark, and the light intensity was 60-80μmol s -1 m -2, and obtained rooted resistant rubber grass transgenic plants.

[0125] The formula of the resistant callus induction medium is: 400 mg / L Timentin + Basta 10 mg / L + MS 4.4 g / L + MES 0.5 g / L + 6-BA 0.5 mg / L + NAA 0.01 mg / L + sucrose 20 g / L + plant gel 3.6 g / L, pH 5.8.

[0126] The formula of the resistant seedling culture medium is: 400 mg / L Timentin + Basta 10 mg / L + MS 2.2 g / L + MES 0.5 g / L + sucrose 10 g / L + plant gel 3.6 g / L, pH 5.8.

[0127] 4. Screening of positive transgenic plants

[0128] The rooted resistant rubber grass transgenic plants were transplanted into a matrix with a volume ratio of 1:1 between nutrient soil and vermiculite. The culture temperature was 21°C, with 16 h light / 8 h dark and a light intensity of 60-80 μmol s -1 m -2 When new leaves grow out, take leaves and extract genomic DNA using the CTAB method as a template, amplify using primers qHRC1-Sense (SEQ ID NO: 14) and T35S-R1 (SEQ ID NO: 5), and screen positive transgenic plants.

[0129] The PCR system was: 2×GC Buffer 10μL, 2mM dNTPs 2μL, TaKaRa Taq (5U / μL) 0.2μL, qHRC1-Sense (10μM) 0.3μL, T35S-R1 (10μM) 0.3μL, DNA 2μL, and ddH2O was added to 20μL.

[0130] The PCR program was: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min. After the reaction, the amplified product was detected with 1% agarose gel, and the positive transgenic plants were identified with a band size of 372 bp.

[0131] Through PCR identification, a total of 23 positive overexpression transgenic T0 plants were obtained, and 3 positive transgenic plants (numbered 6#, 7#, and 15#) were randomly selected for in-depth analysis.

[0132] 5. Determination of HRC1 gene expression and rubber content

[0133] According to the method in Example 1, the three positive transgenic plants (numbered 6#, 7#, and 15#) were sliced ​​and broken into pieces to observe the root of rubber grass. The results showed that the number of latex cells in the transgenic lines was not much different from that in the wild-type plants ( Figure 4 A).

[0134] According to the method in Example 2, the root RNA of the transgenic line was extracted and reverse transcribed to obtain cDNA, and qRT-PCR detection was performed using the HRC1 gene-specific fluorescent quantitative primers qHRC1-Sense (SEQ ID NO: 14) and qHRC1-Anti (SEQ ID NO: 15), and the internal reference TkGAPDH gene fluorescent quantitative PCR primers qTkGAPDH-Sense (SEQ ID NO: 16) and qTkGAPDH-Anti (SEQ ID NO: 17). Bio-Rad's SsoFast Supermix (Cat#1725201) was used to prepare the fluorescence quantitative PCR reaction system according to the product instructions, and the real-time fluorescence quantitative PCR instrument (BIO-RAD, CFX96) was used for fluorescence quantitative PCR. The PCR reaction system was: 2×SsoFast mix 5μL; cDNA 2μL; forward and reverse primers (1μM) 1.5μL each. The PCR operation program was: 98℃, 30s; (98℃, 5s→60℃, 5s→information collection), 40 cycles; 60-95℃, 0.5℃ / 5s, information collection / 5s. After the program was completed, the data was analyzed using BIO-RAD CFX Manager software. The results showed that the expression levels of the HRC1 gene in the three strains were significantly increased ( Figure 4 B).

[0135] Furthermore, the rubber in the roots of the transgenic lines was extracted and its content was detected according to the method in Example 1. The results showed that the content of natural rubber in the three lines was significantly increased compared with that in the wild-type plants ( Figure 4 C).

[0136] The above results prove that overexpression of HRC1 gene can increase the rubber content of rubber grass.

[0137] Example 4: Analysis of the structure and expression pattern of the HRC1 gene

[0138] In order to explore the protein characteristics of HRC1, the NCBI database was used to predict the protein conserved domain. The results showed that the protein encoded by the HRC1 gene has an auxin repressed conserved domain and belongs to the auxin repressed super family ( Figure 5 A).

[0139] To explore the location of HRC1 gene function, latex and different organs such as roots, leaves, stem base, pedicels, flowers, and seeds of rubber grass were collected, and RNA was extracted from latex using a latex RNA extraction kit (Qiagen, Cat#74804), and RNA from different organs was extracted using an RNA rapid extraction kit (Beijing Huayueyang, Cat#0416-50). IIIFirst-Strand Synthesis System Kit (Invitrogen, Cat#18080051) was used for reverse transcription to obtain cDNA. Using cDNA as a template, fluorescent quantitative PCR was performed according to the method in Example 2 using the fluorescent quantitative primers qHRC1-Sense (SEQ ID NO: 14) and qHRC1-Anti (SEQ ID NO: 15) specific to the HRC1 gene, and the fluorescent quantitative PCR primers qTkGAPDH-Sense (SEQ ID NO: 16) and qTkGAPDH-Anti (SEQ ID NO: 17) of the internal reference TkGAPDH gene. SsoFast Supermix (Cat#1725201) was used to prepare the fluorescence quantitative PCR reaction system according to the instructions. The PCR reaction system was: 2×SsoFast mix 5μL; cDNA 2μL; forward and reverse primers (1μM) 1.5μL each. The PCR operation program was: 98℃, 30s; (98℃, 5s→60℃, 5s→information collection), 40 cycles; 60-95℃, 0.5℃ / 5s, information collection / 5s. After the program was completed, the data was analyzed using BIO-RAD CFX Manager software. The results showed that the HRC1 gene was expressed in different tissues and organs of rubber grass, and the expression level in latex was higher ( Figure 5 B).

Claims

1. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1, or the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

3.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the amino acid sequence shown in SEQ ID NO:

2.

3. An expression cassette, vector or host bacteria comprising the nucleic acid molecule according to claim 1 or 2.

4. Use of the nucleic acid molecule according to claim 1 or 2 in cultivating rubber grass plants with high rubber content.

5. Use of the nucleic acid molecule according to claim 1 or 2 in producing natural rubber from rubber grass plants.

6. A method for obtaining rubber grass plants with high rubber content, characterized in that: include: The expression of the coding gene of the protein in rubber grass is enhanced to obtain rubber grass plants with high rubber content; the amino acid sequence of the protein is shown in SEQ ID NO:

2.

7. The method according to claim 6, characterized in that The nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO:

1.

8. The method according to claim 6, characterized in that By introducing an overexpression vector of a nucleic acid molecule having a nucleotide sequence such as that shown in SEQ ID NO: 1 into a rubber grass receptor material, a rubber grass plant with a high rubber content is obtained.

9. The method according to claim 8, characterized in that The overexpression vector is introduced into the rubber grass receptor material through Agrobacterium-mediated.

10. A method for producing natural rubber, characterized in that: include: A transgenic rubber grass plant with a high rubber content is obtained by adopting the method described in any one of claims 6 to 9, and the transgenic rubber grass plant is cultivated to extract natural rubber from the roots.

11. A protein, whose amino acid sequence is shown in SEQ ID NO: 2.

Images