CN115927367A - High-gum-content gene HRC1 of rubbergrass and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to a high-gum-content gene HRC1 of turfgrass and application thereof. Providing a nucleic acid molecule comprising any one of the following nucleotide sequences (a 1) to (a 5): (a 1) a nucleotide sequence shown as SEQ ID NO: 1; (a 2) a nucleotide sequence shown as SEQ ID NO. 3; (a3) A nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOs 1 and 3; (a4) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2; (a5) A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2; wherein the nucleic acid molecule is capable of conferring a high rubber content to a turfgrass plant comprising the nucleic acid molecule. The invention provides a new gene resource for the cultivation of a new variety of the high-gum-content hevea brasiliensis and provides a new idea for the research of the regulation and control of the synthetic approach of the natural rubber.

Description

High-gum-content gene HRC1 of rubbergrass and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a high-gum-content gene HRC1 of rubber grass and application thereof.

Background

Natural rubber is a natural high molecular compound with cis-1, 4-polyisoprene as the main component, and there are about more than 2500 different plants in the world that can produce polymers similar to natural rubber, but the one really having commercial application value is Hevea brasiliensis Muell. When the surface of the rubber tree is cut, the mammary duct cells in the bark are cut off, latex flows out from the mammary duct cells, and the commercial natural rubber is prepared through processes such as later collection and processing. The natural rubber has high elasticity, plasticity, flexibility resistance, good electrical insulation property and the like at normal temperature, has wide application, and cannot be separated from the natural rubber in medical sanitation, transportation, industry, agriculture, national defense and daily life of people. Because rubber trees can only grow in tropical and subtropical regions, the produced natural rubber is in shortage day by day due to the influences of inappropriate climatic conditions, serious insect pests, low automation degree and the like.

The Hevea brasiliensis (TKS) is a perennial herb of Taraxacum of Compositae, is originally produced in Kazakhstan and Xinjiang in China, has a perennial root rubber content of more than 20% of dry weight, has the same rubber quality as that of Brazilian rubber tree, and can be directly used for producing rubber products. The rubber grass likes cold climate, has wide application range and is a rubber-producing plant with great development prospect. At present, the rubber grass is mostly a wild resource, the agronomic characters of the rubber grass are improved through genetic engineering, the rubber content is increased, and the rapid breeding of the high-rubber germplasm of the rubber grass can be realized.

Biosynthesis of natural rubber is a typical plant isoprenoid secondary metabolic pathway, and its synthetic precursors are mainly synthesized by Mevalonate (MVA) pathway in cytoplasm. Firstly, three molecules of acetyl CoA are catalyzed by acetyl CoA acyltransferase (AACT) and HMG-CoA synthetase (HMGS) to complete two-step condensation reaction to generate 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA), and then MVA is generated under the action of HMG-CoA reductase (HMGR); MVA is phosphorylated by Mevalonate Kinase (MK) and phosphomevalonate kinase (MPK), and decarboxylation is catalyzed by mevalonate decarboxylase pyrophosphate (MDC) to isopentenyl pyrophosphate (IPP). IPP is catalyzed by isomerase (isopropenyl pyrophosphate isocerase, 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), and IPP units are added successively on the basis of this to obtain other isopentenyl compounds: farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The MEP (2-C-methyl-D-erythrol-4-phosphate) pathway in the plastid also forms IPP and DMAPP, crossing the MVA pathway to some extent, and thus the MEP pathway may also be a source of rubber biosynthetic precursors. Natural rubber synthesis starts with 1 molecule of allyl pyrophosphate (e.g., DMAPP, GPP, FPP, or GGPP) in a trans-configuration as a starting material, and starts with biosynthesis of rubber molecules by regenerating 1 molecule of allyl pyrophosphate end group from the cleavage product of the methylene group of IPP with allyl pyrophosphate diphosphate under catalysis of cis-prenyltransferase (CPT), and then thousands of IPPs are incorporated into a long chain of isopentenyl pyrophosphate under the action of Small Rubber Particle Protein (SRPP) and Rubber Elongation Factor (REF) to form rubber molecules of different sizes. The efficiency of natural rubber synthesis can be improved by regulating and controlling the expression of natural rubber synthesis pathway genes.

Disclosure of Invention

We found a mutant c112 with increased rubber content from the earlier constructed pool of SARE (Sense/antisense RNA expression) mutants. The mutant plants are early blossoming, the leaf number is increased, the dry weight of roots is not changed greatly, and the number of mammary ducts in the roots is not changed greatly. By analyzing the insertion site in the c112 mutant, a gene associated with dormancy/auxin repression was found and isolated. The gene is from a hevea brasiliensis sequencing line 1151, is expressed in organs such as roots, leaves, flowers and seeds of the hevea brasiliensis, and has the highest expression level in latex. Since overexpression of the gene in Hevea brasiliensis can increase the natural Rubber Content in roots, the gene was named HRC1 (High natural Rubber Content 1) gene.

Based on the above results, the present invention provides a nucleic acid molecule comprising any one of the following nucleotide sequences (a 1) to (a 5):

(a1) 1, a nucleotide sequence shown in SEQ ID NO;

(a2) 3, a nucleotide sequence shown in SEQ ID NO;

(a3) A nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOs 1 and 3;

(a4) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2;

(a5) A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2;

wherein the nucleic acid molecule is capable of conferring a high rubber content to a turfgrass plant comprising the nucleic acid molecule.

Expression cassettes, vectors or host bacteria comprising the above-described nucleic acid molecules are also within the scope of the present invention.

The vector may be a cloning vector comprising the Geum japonicum high gum content gene HRC1 and other elements required for plasmid replication. The vector may also be an expression vector comprising the Geranium strictipes high gum content gene HRC1 and other elements enabling successful expression of the protein. In some embodiments, the expression vector is a pB7WG2D vector into which a pelelia tabacum high rubber content gene HRC1 is inserted.

The host bacterium can be a host bacterium containing a cloning vector, such as E.coli DH5 alpha, and the high rubber content gene HRC1 of the turfgrass is replicated by culturing the bacterium under proper conditions; or a host bacterium containing an expression vector, such as agrobacterium tumefaciens AGL1, and the expression vector is transferred into a rubber grass receptor material by an agrobacterium tumefaciens mediated genetic transformation technology to obtain a transgenic rubber grass plant.

The application of the nucleic acid molecule in culturing the rubber grass plants with high rubber content also belongs to the protection scope of the invention.

The application of the nucleic acid molecule in the production of natural rubber also belongs to the protection scope of the invention.

The invention also provides a method for obtaining a rubber grass plant with high rubber content, which is characterized by comprising the following steps: enhancing the expression of the coding gene of the protein in the kochia to obtain a kochia plant with high rubber content; the protein is (B1) or (B2) as follows:

(B1) Protein with amino acid sequence shown as SEQ ID NO. 2;

(B2) 2, the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

The coding gene of the protein is the nucleic acid molecule.

In some embodiments of the invention, a high rubber content hevea brasiliensis plant is obtained by introducing into the hevea brasiliensis receptor material an overexpression vector comprising the above-described nucleic acid molecule.

In some embodiments of the invention, the overexpression vector is mediated into the kochia rubber-grass recipient material by agrobacterium.

The turfgrass receptor material may be a turfgrass 1151 line.

The present invention also provides a method for producing natural rubber, comprising: any one of the above methods is used to obtain a transgenic hevea brasiliensis plant with high rubber content, the transgenic hevea brasiliensis plant is cultured and the natural rubber in the roots is extracted.

The invention also provides a protein which is (B1) or (B2) as follows:

(B1) Protein with amino acid sequence shown as SEQ ID NO. 2;

(B2) 2, and the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2.

The HRC1 gene separated by the invention belongs to a Dormancy/Auxin inhibitory protein gene family, has an Auxin repressed conserved domain, encodes a complete Dormancy/Auxin associated protein (DRM/ARP) with the size of 13.3KD, and has an amino acid sequence shown as SEQ ID NO: 2. Experimental results show that the HRC1 gene is overexpressed in the ruby grass 1151 strain, the content of natural rubber in the roots of the ruby grass can be increased, the expression level of a part of key enzyme genes of a natural rubber synthesis pathway in the roots of the rubber content increasing mutant c112 is increased, and the HRC1 gene is involved in expression regulation of the part of key enzyme genes in the natural rubber synthesis pathway. Therefore, the HRC1 gene can be used for genetic improvement of increasing the rubber content in the rubber grass roots, provides a new gene resource for the cultivation of a new variety of high-rubber-content rubber grass, and provides a new idea for researching the regulation and control of a natural rubber synthetic approach.

Drawings

FIG. 1 shows the results of phenotypic testing of the C112 rubber plant mutant. In the figure: a is a comparison graph of whole plants grown for 4 months by Wild Type (WT) and c112 mutant of the rubber grass, and the length of a ruler is 5 cm; b is the dry root wiredrawing phenotype of 3-month-old Wild Type (WT) and c112 mutant, with the length of the scale being 1 mm; c is the root section staining phenotype of 3-month old Wild Type (WT) and C112 mutant of Hevea brasiliensis, the length of the scale is 500 microns; d is the statistical result of the number of leaves of 2-month-old Wild Type (WT) and c112 mutant; e is the statistical result of the weight (g) of the single dry roots of the 3-month-old Wild Type (WT) and the c112 mutant of the kochia scoparia; f is the statistics of the lactiferous duct cell index (%) in the Wild Type (WT) and c112 mutant of the rubber grass growing in the soil for 3 months; g is the detection result of the natural rubber content in the 3-month-old Wild Type (WT) and the c112 mutant of the rubber grass. The values for D-F represent mean ± s.d., n =12 in D, n =20 in E, n ≧ 9 in F, the P values are as shown, and the significance analysis was performed using two-tailed student's t-test.

FIG. 2 shows rubberFourier infrared spectrum of the rubber content detection of the roots of the wild type and c112 mutant plants of the glue grass. In the figure: a is a Fourier infrared spectrogram for detecting the content of Wild Type (WT) rubber of the rubber; b is the local enlargement of a rectangular dotted line box in the A; c is a Fourier infrared spectrogram for detecting the rubber content of the mutant C112; d is the local enlargement of a rectangular dotted line box in the C; 699cm in the figure ^-1 The nearby peak position indicates the absorption peak of external standard narrow distribution polystyrene, 835cm ^-1 The nearby peak positions indicate natural rubber absorption peaks, and the hatched portions indicate integrated areas.

FIG. 3 shows the results of the insertion site analysis of the Hevea brasiliensis c112 mutant. In the figure: a is a mutant form of the c112 mutant, wherein HRC1 represents a CDS region, the two ends of the CDS region are UTR regions, dark arrows represent vector amplification primers, and light arrows represent vector sequencing primers; b is the PCR detection result of the insertion Site of the c112 mutant, wherein the left lane is the amplification product of the primer c112-Site-1D-Sense/c112-Site-1D-Anti, and the right lane is the amplification product of the primer c112-Site-2D-Sense/c 112-Site-2D-Anti; c is qRT-PCR detection result of gene utg1288.20 expression level in Wild Type (WT) and C112 mutant of the rubber grass; d is the qRT-PCR detection result of HRC1 expression level in the Wild Type (WT) and the c112 mutant of the rubber grass. Values in C and D represent mean ± s.d. (n = 3), P values are shown, and significance analysis was performed using a two-tailed student t-test.

FIG. 4 shows the results of phenotypic analysis of 35S HRC1 transgenic rubber grass. In the figure: a is the Wild Type (WT) and 35S of 3 months old kochia scoparia, the root-mammary duct section of HRC1 transgenic kochia scoparia, wherein the length of the scale is 500 microns; b is the Wild Type (WT) of the rubber grass growing for 3 months and 35S, the result of detecting the HRC1 gene transcription level of HRC1 transgenic rubber grass; c is Wild Type (WT) and 35S of 3-month-grown Hevea brasiliensis, and the result of detecting the natural rubber content in HRC1 transgenic Hevea brasiliensis. Values in B and C represent mean ± s.d. (n = 3), P values are shown, and significance analysis was performed using a two-tailed student t-test. 6#, 7# and 15# represent 35S-three lines of HRC1 transgenic Hevea brasiliensis.

FIG. 5 shows the structure and expression pattern of HRC1 gene. In the figure: a is the amino acid sequence alignment condition of HRC1 gene and homologous gene AT1G56220 in Arabidopsis, and the indication position of black bottom line is a conservative structure domain predicted by NCBI; and B is the relative expression of the HRC1 gene in different organs of the rubber grass detected by quantitative PCR, and the values in the graph are the mean value +/-s.d. (n = 3).

Detailed Description

The present invention is further described with reference to the following examples and accompanying drawings, it being understood that the following examples are illustrative and explanatory only and are not intended to limit the scope of the present invention.

The experimental methods used in the following examples, unless otherwise specified, are conventional in the art, and reference may be made to the relevant experimental manuals or manufacturer's instructions; the reagents and materials used, unless otherwise specified, are commercially available or may be prepared by conventional methods.

Plant material used in the following examples:

plants and seeds of the line Taraxacum kok-saghyz Rodin (TKS) 1151 were stored and bred in this laboratory. The kokura 1151 strain is disclosed in 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 proteins new energies in inter-rubber biosynthesis, nature Sci Rev 5,78-87, publicly available from institute of genetics and developmental biology, china academy of sciences.

The strains used in the following examples:

escherichia coli strain DH 5. Alpha. Was stored in the laboratory and was commercially available. Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain AGL1 is a strain stored in the laboratory, commercially available from Biotech, inc., of the Beijing Huayue ocean, under the product designation NRR01250.

The carriers used in the examples below:

the intermediate vector pDONR221, a DONR vector of Gateway system, was stored in the laboratory and commercially available from Invitrogen corporation under the accession number 12536017. The binary vector pB7WG2D (Gateway) was maintained in the laboratory and was purchased from the Biovector NTCC plasmid vector cell Gene Collection.

Reagents and consumables used in the following examples:

MS culture medium was purchased from Beijing Kiwiyi scientific Co., ltd, cat #: m0222.0050.MES, 6-BA, NAA, plant gel purchased from Sigma. Sucrose was purchased from national drug group chemical agents limited, cat #: 10021418. oil Red O, available from Sigma-Aldrich, oil Red O solution, cat # O1391. Polystyrene standards (NIM-RM 2068) were purchased from the chinese metrological academy of sciences. Natural rubber standards, available from Sigma-Aldrich, cat #431257-100G. The RNA rapid extraction kit is purchased from Beijing Huayuyo Biotech limited, cat 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), available from Beijing Bailingke Biotech, inc. TaKaRa Taq ^TM Purchased from Beijing Liuhe Jingmai Quiko, inc., having a product number of: and R001A. SsoFast

Supermix, available from Bio-Rad, cat #1725201. Agarose gel recovery kit (cat # DH 101-01) and plasmid small quantity rapid extraction kit (centrifugal column type) (cat # DP 102-01) were purchased from Beijing Bomaide Gene technology, inc. BP CLONASE II ENZYME MIX (cat # 11789100), LR CLONASE II ENZYME MIX (cat # 11791020) are Invitrogen corporation products. Latex extraction RNA kits were purchased from Qiagen, cat #: 74804.

example 1 acquisition and phenotypic characterization of rubber-enriched rubber grass mutants

1. Obtaining of the rubber grass mutant c112

In the earlier stage of the laboratory, a rubberized grass SARE (Sense/antisense RNA expression) mutant library is constructed by taking a rubberized grass (Taraxacuum kok-saghyz Rodin, TKS) 1151 strain as a material, and 8117 mutant plants are obtained. SARE is called sense/antisense RNA expression system, the system mainly overexpresses full-length cDNA and truncated cDNA, the fragment can be inserted into the carrier in a forward direction or a reverse direction, and the overexpression can not only cause the phenomena of gene function acquisition and cosuppression (cosuppression) but also cause antisense inhibition, thereby generating a large amount of mutant phenotypes. For the construction of the rubber grass SARE mutant library, reference is mainly made to published documents Mou, Z.L., wang, X.Q., fu, Z.M., dai, Y., han, C., ouyang, J., bao, F., hu, Y.X., and Li, J.Y. (2002) Silening of phosphoethanomine N-methyl transferase responses in temporal-sensitive mass reliability and salt sensitivity in Arabidopsis plant 14,2031-2043. The construction method of the mutant library is that the mutant library is constructed according to the modification of different plant binary expression vectors: taking roots, leaves, flowers and seeds of the kohlrabi, respectively extracting total RNA, separating mRNA, constructing a cDNA library, adopting pB7WG2D (Gateway) as a terminal plant binary expression vector, electrically transducing the cDNA library plasmid into AGL1 competent cells of the agrobacterium tumefaciens, transforming the kohlrabi, and screening by a herbicide to obtain a mutant plant.

Because the Hevea brasiliensis is incompatible by self and cannot obtain homozygous progeny through seeds, the tissue culture is used for carrying out clone propagation on the Hevea brasiliensis. Taking leaves of wild type and mutant plants of a strong growing rubberella 1151 strain, disinfecting the leaves by using 12% (v/v) queen bee, inoculating the leaves into a callus induction culture medium, inoculating the leaves into a strong seedling culture medium after a wound has a bud, carrying out subculture for 2-3 times, differentiating the bud into a plantlet, transplanting the plantlet into a greenhouse after a sterile seedling takes root, wherein a transplanting matrix is nutrient soil: vermiculite =1 (v/v). The greenhouse culture conditions are 21 deg.C, long day (16 h light/8 h dark), and light intensity of 80-120 μ E.m ^-2 ·s ^-2 。

The formula of the callus induction culture medium is as follows: MS 4.4g/L, MES 0.5g/L, 6-BA 0.5mg/L, NAA 0.01mg/L, sucrose 20g/L, plant gel 3.6g/L, and pH5.8.

The formula of the strong seedling culture medium comprises: MS 2.2g/L, MES 0.5g/L, sucrose 10g/L, plant gel 3.6g/L, pH5.8.

By observing the phenotype of the mutant plants, detecting the rubber content of the mutant plants and comparing with the wild type plants, a mutant with increased rubber content is found, and the number of the mutant is c112. The rubber grass mutant c112 bloomed earlier and more leaves than the wild type (FIG. 1A, 1D).

2. Phenotypic characterization of the rubber grass mutant c112

(1) Root slicing and breaking method wire drawing observation of kochia scoparia

Roots of the wild type and mutant c112 plants of the kochia scoparia were planted in a greenhouse for 3 months, and small sections of about 1.5cm in length were fixed in 80% (v/v) ethanol for 24 hours, then sectioned by a shaking microtome with a thickness of 100 μm, stained with oil red O for 1min, then rinsed once with 45% (v/v) glacial acetic acid, washed 2 times with distilled water, and sealed with 60% (v/v) glycerol, observed with a stereoscope (OLYMPUS SZX 16), and photographed. Milk tube area statistics were performed with ImageJ software, milk tube cell index = milk tube cell area/root section area x 100%. Cleaning the roots of the rubber grass, placing the roots of the rubber grass in a 50 ℃ oven to dry the roots to constant weight, and judging the density degree of rubber threads in the roots by a breaking method.

The results showed that the c112 mutant showed more stringiness in the dry roots (fig. 1B), less weight change in the dry roots (fig. 1E), and more but not significant differences in the milk ducts in the root sections (fig. 1c, 1f) compared to the wild type.

(2) Detection of natural rubber content of rubber grass root

Extraction of natural rubber: roots of wild type rubbergrass and mutant c112 plants are respectively planted in a greenhouse for 3 months, the roots are washed, placed in an oven at 50 ℃ and dried to constant weight, then frozen and ground into powder, 100mg of the powder is weighed and placed in a 2.0mL centrifuge tube, 1mL of toluene is added to completely suspend the powder, then the powder is rotated for 1min, the powder is extracted at 50 ℃,750rpm for 2h,10,000rpm is used for centrifuging at room temperature for 10min, and the supernatant is taken, and the operation is repeated for 3 times. Concentrating the supernatant, diluting to 500 μ L, adding 1mL of methanol, standing at 4 deg.C for 30min, centrifuging to remove supernatant, adding 1mL of distilled water, washing at room temperature for 10min, centrifuging to remove supernatant, adding 1mL of acetone, washing at room temperature for 10min, centrifuging to remove supernatant, and storing the dried rubber in a refrigerator at-20 deg.C.

Detection of the content of the natural rubber:

natural rubber standards (Sigma-Aldrich, cat # 431257-100G) were prepared in toluene at various concentrations (0.5,1,2,4,6,8 mg/mL) and each concentration was measuredThe standard solution (2) is prepared by mixing 150 μ L of the standard solution with 15 μ L of narrow-distribution polystyrene external standard with concentration of 10mg/mL, sucking 70 μ L by a pipette, uniformly coating the mixture on potassium bromide wafers which are pressed by a tablet press in advance, baking the potassium bromide wafers in an oven, and detecting the potassium bromide wafers by a far infrared spectrometer (BRUKER, TENSOR 27), wherein the wafers are scanned 32 times in the detection process. The obtained infrared spectrogram is subjected to simple atmosphere compensation and baseline correction, and then 835cm is calculated ^-1 (absorption peaks of Natural rubber) and 699cm ^-1 Peak area ratio of (narrow distribution polystyrene absorption peak). Drawing a standard curve by taking the peak area ratio and the concentration of the natural rubber as coordinates to obtain a standard curve equation X = (Y-0.0698)/0.2596 ² =0.9961, wherein X represents the natural rubber concentration and Y represents 835cm ^-1 And 699cm ^-1 Peak area ratio of (a).

Dissolving root rubber of the extracted wild type and mutant c112 plants in 1mL of toluene, respectively, collecting 150 μ L of the solution, adding 15 μ L of narrow-distribution polystyrene with concentration of 10mg/mL as external standard, mixing, sucking 70 μ L with pipette, performing infrared spectrum detection according to the above method, and calculating 835cm ^-1 (absorption peaks of Natural rubber) and 699cm ^-1 Peak area ratio of (narrow distribution polystyrene absorption peak). Substituting the peak area ratio of the obtained natural rubber absorption peak of the Hevea brasiliensis and the narrow-distribution polystyrene absorption peak into the standard curve X = (Y-0.0698)/0.2596, and calculating the concentration of the natural rubber. The relative content of natural rubber = concentration of natural rubber (mg/mL) × volume of toluene for extraction (mL)/mass of powder of root for extraction of rubber (mg) × 100%.

As shown in FIG. 1G, the rubber content of the c112 mutant was almost 2 times that of the wild-type rubber.

Example 2 analysis of insertion site of the rubber grass mutant c112

1. Extraction and PCR amplification of Hedychium elaterium genome DNA

Collecting young leaves of wild type Hedychium spicatum and c112 mutant, grinding into powder with liquid nitrogen, extracting DNA with 2% CTAB extractive solution, incubating at 65 deg.C in water bath for about 1 hr, extracting with equal volume of chloroform for about 10min, centrifuging at 4 deg.C and 8,000rpm for 10min, sucking supernatant into new centrifuge tube, adding into the centrifuge tube, and mixingMixing 80% isopropanol, shaking, standing at room temperature for 10min, centrifuging at 4 deg.C and 12,000rpm for 5min, rinsing the precipitate with 70% ethanol, air drying at room temperature, and dissolving in sterilized ddH ₂ And O, obtaining leaf DNA for PCR identification and re-sequencing.

2. Extraction and reverse transcription of rubber grass root RNA

RNA extraction: roots of the wild type plants and c112 mutant plants of the Hevea brasiliensis 1151 strain which grow for 3 months are taken, washed, ground by liquid nitrogen and extracted by a Huayuanyang RNA rapid extraction kit (Cat # 0416-50), and the specific method refers to the kit specification. The kit is provided with DNase, so that DNA pollution in total RNA can be removed. Total RNA was dissolved in elution buffer EB in the kit. The OD values at 230nm, 260nm and 280nm were measured by an ultraviolet spectrophotometer to detect and quantify the RNA quality.

Reverse transcription: use of

III First-Strand Synthesis System reverse transcription kit (Invitrogen, cat # 18080051) by Oligo (dT) according to the product instructions ₂₀ For reverse transcription of the primers, the system and conditions were as follows: oligo (dT) ₂₀ mu.L (50. Mu.M), 1. Mu.L of 10mM dNTPs, 2. Mu.g of RNA, complement ddH ₂ O to total volume of 10 μ L, mixing, centrifuging, heating at 65 deg.C for 5min, and standing on ice for 5min; to this system was added 10 × RT buffer 2 μ L,25mM MgCl ₂ mu.L, 0.1M DTT 2. Mu.L, RNaseOUT (40U/. Mu.L) 1. Mu.L, superScript III RT (200U/. Mu.L) 1. Mu.L, mixed well, centrifuged, heated at 50 ℃ for 50min, at 85 ℃ for 5min, placed on ice for 5min, added with 1. Mu.L RNaseH, incubated at 37 ℃ for 20min, and heated at 65 ℃ for 20min. Obtaining root cDNA of wild type plants and c112 mutant plants, using ddH ₂ Diluting with 10 times of O, and storing in a refrigerator at-20 deg.C.

3. Identification of inserts in the C112 mutant of Hevea brasiliensis

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

P35S-F1：5’-CCAACCACGTCTTCAAAGC-3’(SEQ ID NO:4)；

T35S-R1：5’-TCGCATGCCTGCAGGTCACT-3’(SEQ ID NO:5)。

the PCR system is as follows: 2 XKOD FX Buffer 25. Mu.L, 2mM dNTPs 10. Mu.L, KOD FX (1.0U/. Mu.L) 1. Mu.L, P35S-F1 (10. Mu.M) 0.75. Mu.L, T35S-R1 (10. Mu.M) 0.75. Mu.L, DNA template 2. Mu.L, ddH ₂ Make up to 50 μ L, mix well and centrifuge. The PCR procedure was: 3min at 94 ℃; 10s at 98 ℃,30s at 55 ℃, 1.5min at 68 ℃ and 30 cycles; 10min at 68 ℃.

After the reaction, the PCR product was sent to Beijing Rui Boxing scientific Biotech Co., ltd and the nucleotide sequence of the insert was obtained by sequencing with the vector sequence sequencing primer P35S-F2/T35S-R2. The nucleotide sequences of the primers are as follows:

P35S-F2：5’-TCATTTGGAGAGGACTCCGGT-3’(SEQ ID NO:6)；

T35S-R2：5’-GAGAGAGATAGATTTGTAGAGAG-3’(SEQ ID NO:7)。

PCR sequencing results show that a fragment with the length of 640bp is inserted into the c112 mutant in the forward direction, the nucleotide sequence of the fragment is shown as SEQ ID NO. 3, and the fragment comprises a 5'UTR region with the length of 32bp, a 372bp coding region and a 3' UTR region with the length of 236 bp. The coding region of this fragment was subjected to blastx sequence alignment at the NCBI website, and as a result, it was shown that the coding region encodes a complete Dormancy/auxin associated protein (DRM/ARP) of 13.3KD, and this gene was named HRC1 (High natural Rubber Content 1) (fig. 3A).

Insertion in the Hevea brasiliensis c112 mutant (640 bp)

AGTGTTTGTTATCATCTTCTGTCTACTGCGCTATGAGCTTACTCGACCAATTCTGGGACGACACCGTCGCCGGGCCTCGGCCGGAGAAAGGCCTCGGAAAGCTGCGAAAACAGTCTACTTTTAGTTCCCGGTCATCGGATTCCGGCAAGGAATCGCCGCCGGTCACAAATACGGCAGTAGAGGATCCCACGATGAGAGTAACACGAAGCATAATGATCGTAAAACCAGAAAGAACTCTGAGCGACACTCCGCCGTCATCTCCCGCCGGATCTACACCTCCGGTATCTCCTTTCCCTGGAGGCAGATCAGAGGCATTTAGGTTTCGTCGGAAGTCTGCATCGGACGCTTTCGAGAAGGCAAGCGGGATTGGAACCAGGAGCCCTCGTGCTCCTTACGACCTGTGAAATCTGATCTATAAACGTCAGGGAAGAAGGCAGATCACTAGGGTTTAGCTAGCATGCTATAAATGTGTTCGGGTATTATGTCTTTTTGAGAGAGAGAGAGAGTTTTCTCGTATGTGTATATAGTTGATGTAAATACGCGTGGAAAGTTGTGTTAGGTTTATGTTACACTCGTACTTTTTCTGGATGCTTGTAGGCTTGCTTTATATCATAAGATGCAATGCTTCGTTTTCTTGAAG(SEQ ID NO:3)

Coding region of HRC1 gene (372 bp):

ATGAGCTTACTCGACCAATTCTGGGACGACACCGTCGCCGGGCCTCGGCCGGAGAAAGGCCTCGGAAAGCTGCGAAAACAGTCTACTTTTAGTTCCCGGTCATCGGATTCCGGCAAGGAATCGCCGCCGGTCACAAATACGGCAGTAGAGGATCCCACGATGAGAGTAACACGAAGCATAATGATCGTAAAACCAGAAAGAACTCTGAGCGACACTCCGCCGTCATCTCCCGCCGGATCTACACCTCCGGTATCTCCTTTCCCTGGAGGCAGATCAGAGGCATTTAGGTTTCGTCGGAAGTCTGCATCGGACGCTTTCGAGAAGGCAAGCGGGATTGGAACCAGGAGCCCTCGTGCTCCTTACGACCTGTGA(SEQ ID NO:1)

amino acid sequence of protein encoded by HRC1 Gene (123 aa)

MSLLDQFWDDTVAGPRPEKGLGKLRKQSTFSSRSSDSGKESPPVTNTAVEDPTMRVTRSIMIVKPERTLSDTPPSSPAGSTPPVSPFPGGRSEAFRFRRKSASDAFEKASGIGTRSPRAPYDL(SEQ ID NO:2)

In order to search for the gene fragment insertion Site, the c112 mutant is subjected to whole genome re-sequencing analysis, primers c112-Site-1D-Sense/c112-Site-1D-Anti and c112-Site-2D-Sense/c112-Site-2D-Anti are designed for the predicted insertion Site, PCR amplification is carried out, and amplification templates are leaf DNA of the Wild Type (WT) of the kochia and the c112 mutant. The nucleotide sequences of the primers are as follows:

c112-Site-1D-Sense：5’-GAATGCGGATATTTAATCGAAGTG-3’(SEQ ID NO:8)；

c112-Site-1D-Anti：5’-CGATAGAAAACAAAATATAGCGCGC-3’(SEQ ID NO:9)。

c112-Site-2D-Sense：5’-GTTGATGTAAATACGCGTGGAAAG-3’(SEQ ID NO:10)；

c112-Site-2D-Anti：5’-TCTTATATGCTCAACACATGAGCG-3’(SEQ ID NO:11)。

the PCR system is as follows: 2 XGC Buffer 10. Mu.L, 2mM dNTPs 2. Mu.L, taKaRa Taq (5U/. Mu.L) 0.2. Mu.L, forward primer (2. Mu.M) 1.5. Mu.L, reverse primer (2. Mu.M) 1.5. Mu.L, DNA template 2. Mu.L, ddH ₂ Make up to 20. Mu.L of O. The PCR procedure was: 3min at 94 ℃; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 1min; 10min at 72 ℃.

The result shows that the c112-Site-1D primer pair has no amplification product in the wild type of the kochia, and has a specific amplification product in the c112 mutant, which indicates that the insertion Site is amplified in the c112 mutant; the c112-Site-2D has the same size of amplification product in the wild type and the c112 mutant of the rubber grass, indicating that the predicted insertion Site is a false positive result. The amplified sequence of the c112-Site-1D primer was analyzed, and it was found that the insertion Site of the fragment was located near 137706793 of chromosome 3 of Geranium strictum, the Site was located in the intergenic region, no gene was present within 4Kb upstream of the Site, and the gene utg1288.20 was present near 4Kb downstream (FIG. 3A, 3B).

Respectively taking root cDNAs of a wild type plant and a c112 mutant plant of a Hevea brasiliensis 1151 strain as templates, and utilizing utg1288.20 gene(s) ((r))http://bigd.big.ac.cn/gwh/And the login number is as follows: PRJCA000437, gene ID: model, utg1288.20) specific fluorescent quantitative PCR primer qc112-insertion-Sense/qc112-insertion-Anti, HRC1 gene specific fluorescent quantitative PCR primer qHRC1-Sense/qHRC1-Anti, and internal reference TkGAPDH gene (TkGAPDH gene) ((qHRC 1-Anti)http://bigd.big.ac.cn/gwh/And the login number: PRJCA000437, gene ID: model. Utg9113.3) using Ssofast of Bio-Rad company as a fluorescent quantitative PCR primer qTkGAPDH-Sense/qTkGAPDH-Anti

Supermix (Cat # 1725201) was used to prepare a fluorescent quantitative PCR reaction system according to the instructions, and fluorescent quantitative PCR was performed using a real-time fluorescent quantitative PCR instrument CFX96 from BIO-RAD. The nucleotide sequences of the primers are as follows:

qc112-insertion-sense：5’-TAGCGGTCCACAAAGTCCAC-3’(SEQ ID NO:12)；

qc112-insertion-anti：5’-CGGATTCAATTGAGCAGCCG-3’(SEQ ID NO:13)。

qHRC1-Sense：5’-CTACACCTCCGGTATCTCCT-3’(SEQ ID NO:14)；

qHRC1-Anti：5’-GAGGGCTCCTGGTTCCAATC-3’(SEQ ID NO:15)。

qTkGAPDH-Sense：5’-AGTTGGTTTCGTGGTATGAC-3’(SEQ ID NO:16)；

qTkGAPDH-Anti：5’-ACATGTCAGTGAACAGGTAGAC-3’(SEQ ID NO:17)。

the PCR reaction system is as follows: 2 × SsoFast mix 5 μ L;2 mu L of cDNA; forward and reverse primers (1. Mu.M) were 1.5. Mu.L each. The PCR run program was: 30s at 98 ℃; (98 ℃,5s → 60 ℃,5s → information acquisition), 40 cycles; 60-95 ℃,0.5 ℃/5s, information acquisition/5 s. And after the program is operated, analyzing the data by BIO-RAD CFX Manager software. The results showed that the transcription of utg1288.20 gene in the C112 mutant was not affected (fig. 3C), the expression level of HRC1 gene was significantly up-regulated (fig. 3D), and thus the phenotype of the C112 mutant was probably due to overexpression of HRC1 gene.

Example 3 functional verification of HRC1 Gene

In order to verify the function of the HRC1 gene, a transgenic technology is utilized to obtain 35S-HRC 1 transgenic plants and perform phenotype analysis. 1. Construction of overexpression vector 35S HRC1

Gene amplification: and (3) performing PCR by using a primer combination of GW-HRC1-F and GW-HRC1-R and taking root cDNA of a wild plant of a rubberella 1151 strain as a template to amplify the HRC1 gene segment. The nucleotide sequences of the primers are as follows:

GW-HRC1-F：

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGCTTACTCGACCAATTCTG-3’(SEQ ID NO:18)；

GW-HRC1-R：

5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCACAGGTCGTAAGGAGCAC-3’(SEQID NO:19)。

the PCR system is as follows: 10 XKOD Plus Buffer 5. Mu.L, 2mM dNTPs 5. Mu.L, 25mM MgSO ₄ 2 μ L,1 μ L of KOD Plus (1.0U/. Mu.L), 0.75 μ L of GW-HRC1-F (10 μ M), 0.75 μ L of GW-HRC1-R (10 μ M), 2 μ L of cDNA template, ddH ₂ Make up to 50 μ L, mix well and centrifuge. The PCR procedure was: 3min at 94 ℃; 10s at 98 ℃,30s at 55 ℃, 1min at 68 ℃ and 30 cycles; 10min at 68 ℃.

After the PCR reaction, 1. Mu.L of the reaction product was collected and subjected to electrophoresis on 1% agarose gel to determine whether the band size was correct. The HRC1 gene fragment with the correct band size was recovered using agarose gel recovery kit (Bomaide organism, cat # DH 101-01) following the protocol provided in the kit instructions.

BP reaction: and recombining the recovered HRC1 gene fragment to a pDONR221 intermediate vector by using BP reaction to obtain pDONR221-HRC1. The BP reaction system and reaction conditions were as follows: HRC1 gene fragment 150ng, pDONRR 221 vector 150ng, BP clone II Enzyme Mix 1. Mu.L, 1 XTE (pH = 8.0) to 10. Mu.L; incubating for 10h at 25 ℃; adding 1 mu L of protease K, incubating for 10min at 37 ℃ to obtain a recombinant product pDONR221-HRC1, and placing on ice.

And (3) transformation: coli DH 5. Alpha. Competent cells, stored at-80 ℃ were thawed on ice. Adding 5 μ L of recombinant product pDONR221-HRC1, mixing gently, and standing on ice for 30min. Heat shock at 42 deg.C for 45s, and standing on ice for 2min. 500. Mu.L of LB liquid medium without any antibiotic was added and activated at 37 ℃ and 180rpm for 1h. The activated E.coli was centrifuged at 3,000rpm for 1min to remove most of the supernatant, leaving 100. Mu.L of the supernatant as a resuspended cell, and the whole of the cell suspension was spread on LB solid plate containing 50. Mu.g/ml kanamycin and cultured overnight at 37 ℃.

And (3) positive colony identification: single colonies were picked from LB solid plates, inoculated into 500. Mu.L of LB liquid medium containing 50. Mu.g/ml kanamycin, activated at 37 ℃ for 3 hours at 200rpm, and then subjected to PCR of the bacterial solution to identify positive clones. PCR primers for the bacterial liquid are qHRC1-Sense (SEQ ID NO: 14) and M13R primers.

M13R：5’-CAGGAAACAGCTATGACC-3’(SEQ ID NO:20)。

The PCR system is as follows: 2 XGC Buffer 10. Mu.L, 2mM dNTPs 2. Mu.L, taKaRa Taq (5U/. Mu.L) 0.2. Mu.L, qHRC1-Sense (10. Mu.M) 0.3. Mu.L, M13R (10. Mu.M) 0.3. Mu.L, bacterial suspension 2. Mu.L, ddH ₂ Make up to 20. Mu.L of O. The PCR procedure was: 3min at 94 ℃; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 1min; 10min at 72 ℃. After the reaction, 1% agarose gel was used to detect whether the band size of the PCR product was correct, and the bacterial solution of the positive colony with the correct band size was sent to Beijing Rui Boxing Corp Biotech Co.

Plasmid extraction: 200. Mu.L of the bacterial suspension with the correct sequencing result was inoculated into 10mL of LB liquid medium containing 50. Mu.g/mL kanamycin and cultured overnight at 37 ℃ and 200 rpm. A small-scale plasmid rapid extraction kit (centrifugal column type) (product number DP102-01, beijing Bomaide Gene technology Co., ltd.) was used to extract plasmids according to the kit instructions.

LR reaction: the HRC1 gene fragment was homologously recombined onto binary vector pB7WG2D (Gateway) using LR reaction. The LR reaction system and reaction conditions were as follows: pDONR221-HRC1 plasmid 150ng, pB7WG2D plasmid 150ng, LR clone II Enzyme Mix 1. Mu.L, 1 XTE (pH = 8.0) to 10. Mu.L; incubating for 10h at 25 ℃; adding protease K: mu.L, incubate 10min at 37 ℃ to obtain over-expression vector 35S, HRC1, and place on ice.

And (3) transformation: coli DH 5. Alpha. Competent cells, stored at-80 ℃ were thawed on ice. 5 μ L of overexpression vector 35S (HRC 1) was added, mixed gently and left on ice for 30min. Heat shock at 42 deg.C for 45s, and standing on ice for 2min. 500. Mu.L of LB liquid medium without any antibiotic was added and activated at 37 ℃ and 180rpm for 1h. The activated E.coli was centrifuged at 3,000rpm for 1min to remove most of the supernatant, leaving 100. Mu.L of the supernatant to resuspend the cells, and the whole of the cell suspension was spread on LB solid plate containing 50. Mu.g/ml spectinomycin and cultured overnight at 37 ℃.

And (3) positive colony identification: single colonies were picked from LB solid plates, inoculated into 500. Mu.L of LB liquid medium containing 50. Mu.g/ml spectinomycin, activated at 37 ℃ for 3 hours at 200rpm, and then subjected to PCR of bacterial liquid to identify positive clones. The PCR primers of the bacterial liquid are qHRC 1-sensor (SEQ ID NO: 14) and T35S-R1 (SEQ ID NO: 5).

The PCR system is as follows: 2 XGC Buffer 10. Mu.L, 2mM dNTPs 2. Mu.L, taKaRa Taq (5U/. Mu.L) 0.2. Mu.L, qHRC1-Sense (10. Mu.M) 0.3. Mu.L, T35S-R1 (10. Mu.M) 0.3. Mu.L, bacterial suspension 2. Mu.L, ddH ₂ Make up to 20. Mu.L of O. The PCR procedure was: 3min at 94 ℃; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 1min; 10min at 72 ℃. After the reaction, 1% agarose gel was used to detect whether the band size of the PCR product was correct, and the bacterial solution of the positive colony with the correct band size was sent to Beijing Rui Boxing Corp Biotech Co.

Plasmid extraction: 200. Mu.L of the bacterial suspension with the correct sequencing result was inoculated into 10mL of LB liquid medium containing 50. Mu.g/mL spectinomycin and cultured overnight at 37 ℃ and 200 rpm. A small-scale plasmid rapid extraction kit (centrifugal column type) (Bomaide organism, cat # DP 102-01) was used to extract plasmids according to the kit instructions.

The LB liquid culture medium has the formula: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl, and pH7.0. The LB solid medium was supplemented with agar 15g/L based on the LB liquid medium.

2. Obtainment of Agrobacterium tumefaciens containing overexpression vector 35S HRC1

HRC1 is transformed into Agrobacterium tumefaciens AGL1 by an electric shock method. The method comprises the following steps: agrobacterium tumefaciens AGL1 competent cells stored at-80 ℃ were thawed in an ice bath. 150ng of plasmid was added per 50. Mu.L of competent cells, followed by electric shock at 1.8kV. After completion of the electric shock, LB liquid medium without antibiotics was added and cultured at 28 ℃ and 200rpm for 3 hours. Centrifuging the cultured bacteria liquid at 5000rpm for 1 minute to collect bacteria, discarding most supernatant, leaving about 100 mu L of supernatant, gently blowing and beating, re-suspending bacteria blocks, spreading on a YEP solid culture medium plate containing 25mg/L rifampicin and 50mg/L spectinomycin, after dark culture at 28 ℃ for 48 hours, selecting monoclone to inoculate in YEP liquid culture medium containing 25mg/L rifampicin and 50mg/L spectinomycin, after overnight shaking culture at 28 ℃, inoculating the bacteria liquid in YEP liquid culture medium containing 25mg/L rifampicin and 50mg/L spectinomycin according to the volume ratio of 1 ₆₀₀ And =0.6-0.8, centrifugally collecting thalli at room temperature, removing supernatant, and fully suspending by using an equal volume of infection liquid containing 100 mu M acetosyringone to obtain an agrobacterium tumefaciens infection bacterial liquid for later use.

The YEP liquid culture medium comprises the following components in percentage by weight: 10g/L of peptone, 10g/L of yeast extract and 5g/L of NaCl, and pH7.0.YEP solid Medium agar was added at 15g/L based on YEP liquid medium.

The formula of the staining solution is as follows: MS 4.4g/L + MES 0.5g/L +6-BA 0.5mg/L + NAA 0.01mg/L + sucrose 20g/L + glucose 10g/L, pH5.2.

3. Transgenic Hevea brasiliensis T ₀ Obtaining plants

The turfgrass recipient material used was the laboratory-maintained genome sequencing line 1151. Cutting the tissue culture seedling root of RUBENCAO with good growth state into small segments of about 0.5mm, infecting with the Agrobacterium tumefaciens infection bacterial liquid for about 10-20min, and collecting the infected rootTaking out to sterile filter paper, removing excess bacteria liquid, air drying, co-culturing at 21-23 deg.C in dark for 2-3 days, transferring infected herba Gei Elegantis root to resistant callus induction culture medium for screening culture until green resistant bud appears, transferring to resistant strong seedling culture medium for screening culture for 1-2 months at 21 deg.C under 16h illumination/8 h dark with illumination intensity of 60-80 μmol s ^-1 m ^-2 And obtaining the rooting resistant transgenic rubber grass plant.

The formula of the resistant callus induction culture medium is as follows: 400mg/L timentin, 10mg/L Basta, 4.4g/L MS, 0.5g/L MES, 0.5 mg/L6-BA, 0.01mg/L NAA, 20g/L sucrose, 3.6g/L plant gel and pH5.8.

The formula of the resistant strong seedling culture medium comprises: 400mg/L timentin, 10mg/L Basta, 2.2g/L MS, 0.5g/L MES, 10g/L sucrose and 3.6g/L plant gel, and the pH value is 5.8.

4. Screening for Positive transgenic plants

Transplanting the rooted resistant rubber grass transgenic plant to nutrient soil: in a medium with the volume ratio of 1 to 1 of vermiculite, the culture temperature is 21 ℃, the illumination is 16 h/8 h dark, and the illumination intensity is 60-80 mu mol s ^-1 m ^-2 . When new leaves grow out, taking the leaves, extracting genome DNA by a CTAB method as a template, amplifying by using a primer qHRC1-Sense (SEQ ID NO: 14) and T35S-R1 (SEQ ID NO: 5), and screening positive transgenic plants.

The PCR system is as follows: 2 XGC Buffer 10. Mu.L, 2mM dNTPs 2. Mu.L, taKaRa Taq (5U/. Mu.L) 0.2. Mu.L, qHRC1-Sense (10. Mu.M) 0.3. Mu.L, T35S-R1 (10. Mu.M) 0.3. Mu.L, DNA 2. Mu.L, ddH ₂ O make up to 20. Mu.L.

The PCR procedure was: 3min at 94 ℃; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 1min; 10min at 72 ℃. After the reaction is finished, detecting the amplified product by using 1% agarose gel, and identifying the transgenic plant with the band size of 372bp as a positive transgenic plant.

Through PCR identification, 23 positive over-expression transgenic T parts are obtained ₀ And (4) generating plants, and randomly selecting 3 positive transgenic plants (numbered 6#, 7#, and 15 #) for deep analysis.

5. Measurement of HRC1 Gene expression level and rubber content

The 3 positive transgenic plants (nos. 6#, 7#, and 15 #) were subjected to root section of Hevea brasiliensis and wire drawing by the snap method according to the method described in example 1, and the number of breast duct cells in the transgenic line was not much different from that in the wild type (FIG. 4A).

The root RNA of the transgenic line was extracted and reverse-transcribed to obtain cDNA according to the method described in example 2, 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 TkGAPDH gene-specific fluorescent quantitative PCR primers qTkGAPDH-Sense (SEQ ID NO: 16) and qTkGAPDH-Anti (SEQ ID NO: 17). SsoFast from Bio-Rad was used

Supermix (Cat # 1725201) prepared the fluorescent quantitative PCR reaction system according to the product instructions, and performed fluorescent quantitative PCR using a real-time fluorescent quantitative PCR apparatus (BIO-RAD, CFX 96). The PCR reaction system is as follows: 2 × SsoFast mix 5 μ L;2 mu L of cDNA; forward and reverse primers (1. Mu.M) were 1.5. Mu.L each. The PCR run program was: 30s at 98 ℃; (98 ℃,5s → 60 ℃,5s → information acquisition), 40 cycles; 60-95 ℃,0.5 ℃/5s, and information acquisition/5 s. And after the program is operated, analyzing the data by BIO-RAD CFX Manager software. The results showed that the HRC1 gene expression level was significantly increased in all 3 lines (FIG. 4B).

Further, the rubber in the root of the transgenic line was extracted and the content thereof was measured according to the method in example 1. The results show that the natural rubber content in all 3 lines is significantly increased compared to the wild type plants (FIG. 4C).

The results prove that the rubber content of the hevea brasiliensis can be improved by over-expressing the HRC1 gene.

Example 4 analysis of the structure and expression Pattern of HRC1 Gene

In order to explore protein characteristics of HRC1, protein conserved domain prediction was performed using NCBI database, and the results showed that the protein encoded by HRC1 gene has a Auxin suppressed (Auxin suppressed) conserved domain, belonging to the Auxin suppressed super family (fig. 5A).

To explore HRC1Taking latex of Hevea brasiliensis and different organs such as roots, leaves, stem bases, flower stalks, flowers and seeds, extracting RNA of the latex by using a latex extraction RNA kit (Qiagen, cat # 74804), extracting RNA of different organs by using an RNA rapid extraction kit (Cat # 0416-50) and then extracting RNA of different organs by using a Beijing Huayue

III First-Strand Synthesis System kit (Invitrogen, cat # 18080051) was reverse transcribed to obtain cDNA. The quantitative fluorescence PCR was carried out in the same manner as in example 2 using cDNA as a template, and using fluorescence quantitative primers qHRC1-Sense (SEQ ID NO: 14) and qHRC1-Anti (SEQ ID NO: 15) specific to HRC1 gene, and fluorescence quantitative PCR primers qTkGAPDH-Sense (SEQ ID NO: 16) and qTkGAPDH-Anti (SEQ ID NO: 17) of the internal reference TkGAPDH gene. Ssofast @ was used from Bio-Rad>

Supermix (Cat # 1725201) was used to prepare fluorescent quantitative PCR reaction system according to the instructions. The PCR reaction system is as follows: 2 × SsoFast mix 5 μ L;2 mu L of cDNA; forward and reverse primers (1. Mu.M) were 1.5. Mu.L each. The PCR run program was: 30s at 98 ℃; (98 ℃,5s → 60 ℃,5s → information acquisition), 40 cycles; 60-95 ℃,0.5 ℃/5s, and information acquisition/5 s. And after the program is operated, analyzing the data by BIO-RAD CFX Manager software. The results showed that the HRC1 gene was expressed in different tissues and organs of Hevea brasiliensis and was expressed in higher amounts in the latex (FIG. 5B). />

Claims (10)

1. A nucleic acid molecule comprising any one of the following nucleotide sequences (a 1) to (a 5):

(a1) 1, SEQ ID NO;

(a2) 3, the nucleotide sequence shown in SEQ ID NO;

(a3) A nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOs 1 and 3;

(a4) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2;

(a5) A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2;

wherein the nucleic acid molecule is capable of conferring a high rubber content on a turfgrass plant comprising the nucleic acid molecule.

2. An expression cassette, vector or host bacterium comprising the nucleic acid molecule of claim 1.

3. Use of the nucleic acid molecule of claim 1 for growing a high rubber content hevea brasiliensis plant.

4. Use of the nucleic acid molecule of claim 1 in the production of natural rubber.

5. A method of obtaining a high rubber content tarragon plant comprising: enhancing the expression of the coding gene of the protein in the kochia to obtain a kochia plant with high rubber content; the protein is (B1) or (B2) as follows:

(B1) Protein with amino acid sequence shown as SEQ ID NO. 2;

(B2) 2, the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

6. The method of claim 5, wherein the gene encoding the protein is the nucleic acid molecule of claim 1.

7. The method of claim 5, wherein a high rubber content hevea plant is obtained by introducing into the hevea brasiliensis receptor material an overexpression vector comprising the nucleic acid molecule of claim 1.

8. The method of claim 7, wherein the overexpression vector is mediated into the koenen receptor material by agrobacterium.

9. A method of producing natural rubber, comprising: a transgenic Hevea plant with high rubber content obtained by the method of any one of claims 5 to 8, said transgenic Hevea plant being cultured and the natural rubber in the roots being extracted.

10. A protein which is (B1) or (B2) below:

(B1) A protein with an amino acid sequence shown as SEQ ID NO. 2;

(B2) 2, the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

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