High-yield gene hry of rubber grass natural rubber and application thereof

Abstract

The application belongs to the technical field of biology and relates to a high-yield gene HRY of natural rubber of rubber grass and application thereof. A nucleic acid molecule is provided, which comprises any one of the following nucleotide sequences: a nucleotide sequence shown in SEQ ID NO:1 or 3; a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1 or 3; a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO:2 or 4; 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 or 4; wherein the nucleic acid molecule can impart higher natural rubber content to rubber grass containing the nucleic acid molecule. The nucleic acid molecule can be used for genetic improvement of rubber grass to improve the yield of natural rubber of rubber grass and provides a new genetic resource for cultivation of a new variety of high-yield rubber grass.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a high-yield gene HRY of natural rubber of Taraxacum kok-saghyz and application thereof. BACKGROUND

[0002] Natural rubber (NR) is a natural high molecular compound with cis-1, 4-polyisoprene as the main component. Natural rubber has high elasticity, plasticity, flex resistance, good electrical insulation and other properties at room temperature, and has a wide range of applications in medical and health care, transportation, industry, agriculture, national defense, and people's daily life. The global annual consumption of natural rubber exceeds 12 million tons, with a market value of about 20 billion US dollars. Due to the excellent performance of natural rubber, many application fields cannot be replaced by synthetic rubber (SR). There are more than 2500 plants in the world that can produce polymers similar to natural rubber, but only Hevea brasiliensis Muell. Arg. has commercial application value. When the surface of rubber tree is cut, the latex cells in the bark are cut off, and the latex flows out from the latex cells, which is collected and processed to obtain the commercially available natural rubber. Due to the fact that rubber trees can only grow in tropical and subtropical regions, affected by the unsuitable climate, serious pests and diseases, and low degree of automation, the production of natural rubber is becoming increasingly scarce, and it is urgent to develop other rubber crops as alternative sources.

[0003] Taraxacum kok-saghyz Rodin (TKS) is a perennial herb of the family Asteraceae, which is originally from Kazakhstan and Xinjiang, China. The rubber content in the perennial roots of TKS can be as high as more than 20% of the dry weight, and the quality of the rubber is equivalent to that of the rubber produced by Hevea brasiliensis, which can be directly used to produce rubber products such as tires. TKS prefers cool and cool climate and has a wide range of adaptability, and is a very promising rubber-producing plant. At present, TKS is mostly a wild resource. In order to make it truly become an economic crop with production and application value, it is necessary to improve its agronomic traits and increase the rubber yield through genetic engineering, so as to realize the rapid breeding of high-rubber TKS germplasm.

[0004] The root system of rubber grass is the main organ for the synthesis and storage of natural rubber, and it has a significant impact on natural rubber yield. Root biomass, the proportion of latex in the root system (i.e., latex duct cell density), and the proportion of natural rubber in the latex (i.e., natural rubber synthesis efficiency) all significantly influence the natural rubber yield of rubber grass. However, a relatively small root biomass greatly limits the natural rubber yield of rubber grass. Therefore, increasing the root biomass of rubber grass is one of the key ways to improve its rubber yield. The morphological structure of the root system is an important component of the ideal plant architecture and a key factor determining its yield. Understanding the molecular regulatory network is crucial for cultivating an ideal plant architecture. Therefore, in-depth research on the structure and growth and development regulatory network of rubber grass roots is a necessary prerequisite for modifying the root morphology and structure of rubber grass, increasing root biomass, and ultimately improving natural rubber yield. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to increase the natural rubber content in rubber grass.

[0006] To address the aforementioned technical problems, the present invention provides a nucleic acid molecule comprising any one of the following nucleotide sequences (a1)-(a4):

[0007] (a1) The nucleotide sequence shown in SEQ ID NO:1 or 3;

[0008] (a2) A nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1 or 3;

[0009] (a3) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 or 4;

[0010] (a4) A nucleotide sequence that encodes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2 or 4;

[0011] The nucleic acid molecules described therein can impart a higher natural rubber content to rubber grass containing the nucleic acid molecules.

[0012] Expression cassettes, vectors, or host bacteria containing the nucleic acid molecules are also within the scope of protection of this invention.

[0013] The expression cassette consists of a promoter capable of initiating the expression of the nucleic acid molecule, the nucleic acid molecule, and a transcription terminator.

[0014] The vector can be a cloning vector, containing the aforementioned nucleic acid molecules and other elements required for plasmid replication. The vector can also be an expression vector, containing the aforementioned nucleic acid molecules and other elements enabling successful protein expression. The expression vector can be any suitable plant expression vector, such as the pFGC5941 vector.

[0015] The host bacterium can be a host bacterium containing the above-mentioned cloning vector, such as E. coli DH5α. The nucleic acid molecules are replicated by culturing the bacteria under appropriate conditions. Alternatively, the host bacterium can be a host bacterium containing the above-mentioned expression vector, such as Agrobacterium tumefaciens AGL1. The expression vector is transferred into rubber grass recipient material using Agrobacterium tumefaciens-mediated genetic transformation technology to obtain transgenic rubber grass plants.

[0016] The application of the nucleic acid molecules in cultivating rubber grass plants with high rubber content is also within the scope of protection of this invention.

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

[0018] The present invention also provides a method for increasing the natural rubber content in rubber grass, comprising increasing the content or activity of HRY protein in rubber grass; wherein the HRY protein is as follows (B1) or (B2):

[0019] (B1) Proteins with amino acid sequences as shown in SEQ ID NO:2 or 4;

[0020] (B2) Proteins with the same function 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 or 4.

[0021] In the above method, the content or activity of HRY protein in rubber grass is increased by overexpressing the nucleic acid molecule in rubber grass.

[0022] The present invention also provides a method for obtaining rubber grass with increased natural rubber content, comprising: introducing an overexpression vector for overexpressing the nucleic acid molecule into rubber grass recipient material, and culturing to obtain transgenic rubber grass plants with increased natural rubber content.

[0023] In the above method, the overexpression vector is introduced into the rubber grass receptor material via Agrobacterium-mediated transformation.

[0024] The rubber grass recipient material can be any suitable rubber grass (Taraxacum kok-saghyz Rodin, TKS) variety or strain, such as strain 1151.

[0025] The present invention also provides a method for producing natural rubber, comprising: obtaining transgenic rubber grass plants with increased natural rubber content using the above method, cultivating the transgenic rubber grass plants and extracting natural rubber from the roots.

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

[0027] (B1) Proteins with amino acid sequences as shown in SEQ ID NO:2 or 4;

[0028] (B2) Proteins with the same function 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 or 4.

[0029] This invention isolated the natural rubber yield control gene HRY (High natural Rubber Yield) from rubber grass. Two HRY genes with 99% homology exist in rubber grass, named HRY1 and HRY2, with their coding sequences shown in SEQ ID NO:1 and 3, and the amino acid sequences of the HRY proteins they encode shown in SEQ ID NO:2 and 4. Experiments showed that overexpression of HRY1 and HRY2 in rubber grass significantly increased the diameter of the taproot, the number of lateral roots and fibrous roots, and the dry root weight per rubber grass plant. Figure 3 It significantly increases the natural rubber content in the roots of rubber grass. Figure 4 Therefore, HRY1 and HRY2 can be used for genetic improvement to increase the natural rubber yield of rubber grass. This invention provides new genetic resources for the breeding of new high-yielding rubber grass varieties. Attached Figure Description

[0030] Figure 1 The results of phenotypic analysis of the rubber grass rc-1 mutant are shown. A represents the underground part phenotype of wild-type (WT) and rc-1 mutant plants after 3 months of growth, with a scale bar of 5 cm. B represents the taproot diameter of wild-type (WT) and rc-1 mutant plants after 3 months of growth; values ​​in the figure are mean ± SD (n = 11). Significance analysis was performed using Student's t-test, with ns indicating no significant difference. C represents the dry root weight per plant of wild-type (WT) and rc-1 mutant plants after 3 months of growth; values ​​in the figure are mean ± SD (n = 11). Significance analysis was performed using Student's t-test. The t-test was used, with ns indicating no significant difference; D shows the cross-sectional analysis of roots of wild-type (WT) and rc-1 mutant rubber grass plants, with a scale bar length of 500 μm; E shows the statistical results of the latex duct cell index of roots of wild-type (WT) and rc-1 mutant rubber grass plants after 3 months of growth, with values ​​in the figure representing mean ± SD (n = 6), and the significance analysis was performed using Student's t-test, with ns indicating no significant difference; F shows the detection results of the relative content of natural rubber in wild-type (WT) and rc-1 mutant rubber grass plants, with values ​​in the figure representing mean ± SD (n = 3), and the significance analysis was performed using Student's t-test, with * indicating P < 0.05.

[0031] Figure 2 The results of the insertion site analysis of the rubber grass rc-1 mutant are shown. A is a schematic diagram of the inserted gene sequence in the rubber grass rc-1 mutant; the yellow box represents the inserted CDS fragment, the green box represents the inserted 3'UTR fragment, the gray box represents the vector fragment, and the blue arrow represents the primer for insertion site identification. B shows the PCR detection results of the rubber grass rc-1 mutant insertion site; the upper figure shows the amplification results of the insertion site primers, and the lower figure shows the amplification results of the CPTL1 primers (as a positive control). The first lane from the left shows the DNA amplification of wild-type (WT) rubber grass, and the second lane shows the DNA amplification of the rc-1 mutant. C shows the transcriptional level of the inserted fragment in the rubber grass rc-1 mutant detected by qRT-PCR. The vertical axis represents the relative expression level of the inserted fragment, and the values ​​are mean ± SD (n = 3). Significance analysis was performed using Student's... t-test, *** indicates P<0.001; D represents the amino acid sequence alignment results of HRY1 and HRY2 proteins; E represents the expression pattern analysis results of HRY1 and HRY2 in various tissues of rubber grass, and a heatmap using standardized RPKM values ​​is used to display the expression patterns of HRY1 and HRY2 in various tissues of rubber grass.

[0032] Figure 3The root phenotypes of HRY1 and HRY2 overexpressing lines of rubber grass are shown. A represents the phenotypes of wild-type (WT), HRY1 overexpressing line (35S::HRY1), and HRY2 overexpressing line (35S::HRY2) after 3 months of growth, with a scale bar of 5 cm. B represents the transcriptional levels of the HRY gene in the HRY1 overexpressing line (35S::HRY1) and HRY2 overexpressing line (35S::HRY2) of rubber grass, as detected by qRT-PCR. The vertical axis represents the relative expression levels of the HRY gene (HRY1 and HRY2), with values ​​representing mean ± SD (n = 3). Significance analysis was performed using Student's method. The t-test was used, with *** indicating P < 0.001; C represents the taproot diameter of wild-type rubber grass (WT), HRY1 overexpression line (35S::HRY1), and HRY2 overexpression line (35S::HRY2) after 3 months of growth. The values ​​in the figure are mean ± SD (n = 25). The significance analysis was performed using Student's t-test, with ns indicating no significant difference and *** indicating P < 0.001; D represents the dry root weight of each plant of wild-type rubber grass (WT), HRY1 overexpression line (35S::HRY1), and HRY2 overexpression line (35S::HRY2) after 3 months of growth. The values ​​in the figure are mean ± SD (n = 25). The significance analysis was performed using Student's t-test, with * indicating P < 0.05; ** indicating P < 0.01; *** indicating P < 0.001.

[0033] Figure 4This paper presents the root section results, relative natural rubber content, and yield analysis of HRY1-overexpressing and HRY2-overexpressing rubber grass. Section A shows the cross-sectional analysis of roots from wild-type (WT), HRY1-overexpressing (35S::HRY1), and HRY2-overexpressing (35S::HRY2) rubber grass after 3 months of growth, with a scale bar of 500 μm. Section B shows the statistical analysis of latex duct cell indices from roots of wild-type (WT), HRY1-overexpressing (35S::HRY1), and HRY2-overexpressing (35S::HRY2) rubber grass after 3 months of growth. Values ​​in the figures are mean ± SD (n = 9). Significance analysis was performed using Student's method. The t-test was performed, with ns indicating no significant difference. C represents the analysis results of the relative natural rubber content in the roots of wild-type (WT), HRY1 overexpression line (35S::HRY1), and HRY2 overexpression line (35S::HRY2) after 3 months of growth. The values ​​in the figure are mean ± SD (n=3). The significance analysis was performed using Student's t-test, with * indicating P<0.05; ** indicating P<0.01; *** indicating P<0.001. D represents the analysis results of the natural rubber yield per plant of wild-type (WT), HRY1 overexpression line (35S::HRY1), and HRY2 overexpression line (35S::HRY2) after 3 months of growth. The values ​​in the figure are mean ± SD (n=25). The significance analysis was performed using Student's t-test, with *** indicating P<0.001. Detailed Implementation

[0034] The present invention will be described in detail below with reference to the embodiments. It should be understood that the following embodiments are only for explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0035] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and can be found in relevant laboratory manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] The rubber grass (Taraxacum kok-saghyz Rodin, TKS) used in the following examples is strain 1151, described in the article "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." This strain is preserved and propagated in our laboratory and is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. It is used solely for verifying this invention.

[0037] The Escherichia coli DH5α and Agrobacterium tumefaciens AGL1 strains used in the following examples are commercially available strains.

[0038] The pFGC5941 vector used in the following examples is an Agrobacterium binary expression vector, described in the article "Lei, X.; Tan, B.; Liu, Z.; Wu, J.; Lv, J.; Gao, C. (2021). ThCOL2 Improves the Salt Stress Tolerance of Tamarix hispida. Front Plant Sci 12, 653791." This vector is preserved in our laboratory and is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. It is used solely for verifying this invention.

[0039] Some of the reagents used in the following examples:

[0040] MS medium was purchased from Beijing Qiweiyicheng Technology Co., Ltd., catalog number M0222.0050. MES, 6-BA, NAA, and plant gel were all purchased from Sigma-Aldrich. Sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd. Oil Red O solution was purchased from Sigma-Aldrich, Cat#O1391. The RNA rapid extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd., catalog number 0416-50. A reverse transcription kit was also provided. III. The First-Strand Synthesis System is an Invitrogen product, catalog number 18080051. KOD FX is a Toyobo product, catalog number KFX-101. KOD Plus is a Toyobo product, catalog number KOD-201. The basic PCR enzyme is TaKaRa Taq. TM (R001A) and the Seamless Cloning Kit In-Fusion Snap Assembly Master Mix (638949) are Takara products. Agarose gel extraction kit (DH101-01) and plasmid mini-rapid extraction kit (DP102-01) were purchased from Beijing Bomed Gene Technology Co., Ltd. Natural rubber standards were purchased from Sigma-Aldrich, Cat#431257-100G. Polystyrene standards (NIM-RM2068) were purchased from the National Institute of Metrology, China.

[0041] Example 1: Obtaining and phenotypic identification of the rubber grass rc-1 mutant

[0042] In our previous work, we constructed a SARE (Sense / Antisense RNA Expression) mutant library of *Taraxacum kok-saghyz Rodin* (TKS) 1151 as material, obtaining 8117 mutant plants. The construction method of the SARE mutant library followed the method described in "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 male sterility and salt hyperpersensitivity in Arabidopsis. Plant Cell 14, 2031-2043." Because *Taraxacum kok-saghyz* is self-incompatible, hybridization was used to obtain mutant seeds. The obtained seeds were planted and the roots were screened, revealing a progeny mutant with a significantly altered root phenotype, named rc-1.

[0043] Because rubber grass is self-incompatible and cannot produce homozygous offspring through seeds, asexual propagation of rubber grass is achieved through tissue culture. Leaves from robust wild-type rubber grass plants and RC-1 mutant plants were sterilized with 12% (v / v) bleach and inoculated into callus induction medium. Once buds emerged from the wounds, they were transferred to a seedling growth medium. After 2-3 subcultures, the buds differentiated into seedlings. After the aseptic seedlings rooted, they were transplanted to a greenhouse. The cultivation substrate was a 1:1 (v / v) mixture of potting soil and vermiculite. Greenhouse cultivation conditions included a temperature of 21℃, long day (16h light / 8h darkness), and a light intensity of 80-120 μmol·m⁻². -2 ·s -1 .

[0044] The formula for callus induction medium is: 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. The formula for seedling strengthening medium is: MS 2.2 g / L + MES 0.5 g / L + sucrose 10 g / L + plant gel 3.6 g / L, pH 5.8.

[0045] (1) Observation of rubber grass roots

[0046] After transplanting tissue-cultured sterile seedlings of wild-type and rc-1 mutant rubber grass into a greenhouse for 3 months, the roots were dug out of the soil, washed, photographed with a Canon camera (PowerShot G16), and the diameter of the main root was measured using calipers. Then, they were placed in a 50℃ oven to dry to constant weight, and the weight of the dry roots of each plant was counted.

[0047] (2) Observation of rubber grass root slices

[0048] After transplanting tissue-cultured aseptic seedlings of wild-type and rc-1 mutant rubber grass into a greenhouse for 3 months, the roots were dug up from the soil, washed, and fresh roots with a diameter of about 2-3 mm were cut into segments about 1.5 cm long using a scalpel. These segments were fixed in 80% (v / v) ethanol for 24 h and then sectioned using a Leica VT1200S microtome to a thickness of 100 μm. The sections were stained with Oil Red O for 1 min, rinsed once with 45% (v / v) glacial acetic acid, and then washed twice with distilled water. The sections were mounted with 60% (v / v) glycerol and observed and photographed under a stereomicroscope (OLYMPUS SZX16). The area of ​​latex duct cells and the area of ​​root sections were statistically analyzed using ImageJ software, and the latex duct cell index was calculated. The latex duct cell index was calculated as: latex duct cell area / root section area × 100%.

[0049] (3) Extraction of rubber from rubber grass roots

[0050] After transplanting tissue-cultured sterile seedlings of wild-type and rc-1 mutant rubber grass into a greenhouse for 3 months, the roots were dug up from the soil, washed, and dried in a 50℃ oven until constant weight. They were then freeze-ground into powder. 100 mg of powder was placed in a 2.0 mL centrifuge tube, suspended completely with 1 mL of toluene, and vortexed thoroughly for 1 min. Extraction was performed at 50℃ and 750 rpm for 2 h, followed by centrifugation at 10,000 rpm for 10 min at room temperature. The supernatant was collected, and the precipitate was extracted twice more using the same method. The supernatants were combined, concentrated, and brought to a final volume of 500 μL. 1 mL of methanol was added, and the mixture was incubated at 4℃ for 30 min. The supernatant was discarded by centrifugation. 1 mL of distilled water was added, and the mixture was washed at room temperature for 10 min. The supernatant was discarded by centrifugation. 1 mL of acetone was added, and the mixture was washed at room temperature for 10 min. The supernatant was discarded by centrifugation, yielding the rubber roots. After drying, the roots were stored at -20℃.

[0051] (4) Detection of rubber content in rubber grass roots

[0052] The natural rubber content in the roots of wild-type and rc-1 mutant rubber grass was detected using Fourier Transform Infrared (FT-IR) spectroscopy, as described in the article "Rolere, S., Liengprayoon, S., Vaysse, L., Sainte-Beuve, J., and Bonfils, F. (2015). Investigating natural rubber composition with Fourier Transform Infrared (FT-IR) spectroscopy: a rapid and non-destructive method to determine both protein and lipid contents simultaneously. Polym. Test. 43, 83-93."

[0053] Plotting the standard curve: First, prepare solutions of natural rubber standards (Sigma-Aldrich, Cat#431257-100G) at different concentrations (0.5, 1, 2, 4, 6, 8 mg / mL) using toluene. Take 150 μL of each concentration of standard solution and mix it with 15 μL of a 10 mg / mL polystyrene standard solution (National Institute of Metrology, NIM-RM2068). Then, pipette 70 μL of the mixture and spread it evenly onto potassium bromide wafers pre-pressed using a tablet press. Dry the wafers in an oven and then detect them using a far-infrared spectrometer (BRUKER, TENSOR 27). The wafers are scanned 32 times during the detection process. After simple atmosphere compensation and baseline correction, the obtained infrared spectra are calculated to be at 835 cm⁻¹. -1 (Natural rubber absorption peak) and 699cm-1 The peak area ratio of the polystyrene absorption peak was used as the coordinate system. A standard curve was plotted using the peak area ratio and natural rubber concentration as coordinates, yielding the standard curve equation X = (Y - 0.0698) / 0.2596, R... 2 = 0.9961, where X represents the concentration of natural rubber (mg / mL) and Y represents 835cm. -1 and 699cm -1 The peak area ratio.

[0054] The root rubber extracted from the wild-type and rc-1 mutant of *Hedysarum heterotropoides* in (3) above was detected by the following method: The root rubber was dissolved in 1 mL of toluene. 150 μL of the rubber solution was then added to 15 μL of 10 mg / mL polystyrene standard (National Institute of Metrology, NIM-RM2068, China) as an external standard. After mixing, 70 μL of the mixture was pipetted and evenly spread onto a potassium bromide wafer pre-pressed by a tablet press. The wafer was then dried in an oven and detected using a far-infrared spectrometer (BRUKER, TENSOR 27). The wafer was scanned 32 times during the detection process. After simple atmosphere compensation and baseline correction, the obtained infrared spectrum was calculated to be 835 cm⁻¹. -1 (Natural rubber absorption peak) and 699cm -1 The peak area ratio of (polystyrene absorption peak). (835 cm⁻¹) -1 and 699cm -1 Substituting the peak area ratio into the standard curve X = (Y - 0.0698) / 0.2596, the concentration of natural rubber was calculated. Then, the relative content of natural rubber in the wild-type and rc-1 mutant of rubber grass was calculated. The relative content of natural rubber (%) = concentration of natural rubber (mg / mL) × volume of toluene used to dissolve the rubber (mL) / mass of root powder used for rubber extraction (mg) × 100%.

[0055] The results showed that, compared with wild-type rubber grass plants, the rc-1 mutant plants had significantly more lateral roots and fibrous roots. Figure 1 A) The relative content of natural rubber increased significantly. Figure 1 F), while the diameter of the main root ( Figure 1 B) Weight of dry roots per plant ( Figure 1 C) and ductal cell index ( Figure 1 E) No significant changes.

[0056] Example 2: Insertion site analysis in the rubber grass rc-1 mutant

[0057] 1. Extraction and PCR amplification of DNA from rubber grass

[0058] Young leaves of wild-type and rc-1 mutant rubber grass were ground into powder in liquid nitrogen, and 2% CTAB extraction solution was added and mixed thoroughly. The mixture was then incubated in a 65℃ water bath for 1 hour, followed by extraction with an equal volume of chloroform for about 10 minutes. The mixture was then centrifuged at 8,000 rpm for 10 minutes at 4℃. The supernatant was transferred to a new centrifuge tube, and 80% volume of isopropanol was added. The mixture was gently shaken and allowed to stand at room temperature for 10 minutes. The mixture was then centrifuged at 12,000 rpm for 5 minutes at 4℃. The resulting precipitate was washed with 70% ethanol and allowed to air dry at room temperature. The precipitate was then dissolved in an appropriate volume of sterile ddH2O for PCR identification and resequencing.

[0059] 2. Extraction and reverse transcription of RNA from rubber grass roots

[0060] Wild-type and rc-1 mutant tissue-cultured sterile seedlings of rubber grass (1151 strain) were transplanted to a greenhouse and cultured for 3 months. Roots were harvested, washed, and ground into powder in liquid nitrogen. Total RNA was extracted using the Huayueyang RNA Rapid Extraction Kit (Cat#0416-50) according to the kit's instructions. This kit includes DNase to remove DNA from the total RNA. The total RNA was dissolved in elution buffer EB provided in the kit. The OD values ​​of the RNA solution at 230 nm, 260 nm, and 280 nm were measured using a UV spectrophotometer for RNA quality detection and quantification.

[0061] Using a reverse transcription kit III. First-Strand Synthesis System (Invitrogen, Cat#18080051): Total RNA was reverse transcribed according to the kit instructions. The reverse transcription system and conditions are as follows:

[0062] Oligo(dT) 20 (50 μM): 1 μL, 10 mM dNTPs: 1 μL, RNA: 2 μg, add ddH2O to a total volume of 10 μL, mix well, centrifuge, heat at 65 °C for 5 min, and place on ice for 5 min; add 10×RT buffer: 2 μL, 25 mM MgCl2: 4 μL, 0.1 M DTT: 2 μL, RNaseOUT (40 U / μL): 1 μL, SuperScript III RT (200 U / μL): 1 μL to the above system, mix well, centrifuge, heat at 50 °C for 50 min, heat at 85 °C for 5 min, place on ice for 5 min, add 1 μL RNaseH, incubate at 37 °C for 20 min, and heat at 65 °C for 20 min. Obtain root cDNA from wild-type and rc-1 mutant rubber grass, dilute 10-fold with ddH2O and store at -20 °C.

[0063] 3. Identification of the inserted fragment in the rubber grass rc-1 mutant

[0064] Using leaf DNA from the rc-1 mutant as a template, PCR was performed using vector sequence amplification primers P35S-F1 / T35S-R1 and DNA polymerase KOD FX (Toyobo, catalog number KFX-101) to amplify the inserted gene fragment in the rc-1 mutant. The nucleotide sequences of the primers are as follows:

[0065] Upstream primer P35S-F1: 5'-CCAACCACGTCTTCAAAGC-3';

[0066] Downstream primer T35S-R1: 5'-TCGCATGCCTGCAGGTCACT-3'.

[0067] The PCR system was as follows: 2×KOD FX Buffer: 25 μL, 2 mM dNTPs: 10 μL, KOD FX (1.0 U / μL): 1 μL, P35S-F1 (10 μM): 0.75 μL, T35S-R1 (10 μM): 0.75 μL, template DNA: 2 μL, and ddH2O was added to a final volume of 50 μL. The mixture was then centrifuged.

[0068] The PCR program was as follows: 94℃ for 3 min; 98℃ for 10 s, 55℃ for 30 s, 68℃ for 1.5 min, 30 cycles; 68℃ for 10 min.

[0069] After the reaction, the PCR product was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The sequencing primers were P35S-F2 / T35S-R2, and the nucleotide sequence is as follows:

[0070] Upstream primer P35S-F2: 5'-TCATTTGGAGAGGACTCCGGT-3';

[0071] Downstream primer T35S-R2: 5'-GAGAGAGATAGATTTGTAGAGAG-3'.

[0072] Sequencing results showed that a 1186 bp fragment was inserted forward into the rc-1 mutant, with its nucleotide sequence shown in SEQ ID NO:5, containing a 965 bp coding region and a 221 bp non-coding region. The gene corresponding to this coding region was named HRY (High Naturnal Rubber Yield). Alignment of the nucleotide sequence of this coding region with the reference genome of the Rubbergrass 1151 strain (https: / / ngdc.cncb.ac.cn / gwh / Assembly / 1 / show) revealed two genes with 99% homology, named HRY1 and HRY2, respectively. The coding region of HRY2 is completely identical to the corresponding segment of HRY1. Figure 2 A) The coding sequence of the HRY1 gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2. The coding sequence of the HRY2 gene is shown in SEQ ID NO:3, and its encoded amino acid sequence is shown in SEQ ID NO:4.

[0073] To gain a more detailed understanding of the expression patterns of HRY1 and HRY2 in rubber grass, we analyzed RNA-seq data from 12 tissues, including rubber grass flowers, latex, and mature leaves, which were previously measured by our research group. Figure 2 E shows the relative expression levels of HRY1 and HRY2 in these 12 tissues, indicating that HRY1 and HRY2 are broadly expressed genes, expressed in all tissues.

[0074] To locate gene insertion sites, whole-genome resequencing analysis was performed on the rc-1 mutant. Leaf DNA from both wild-type and rc-1 mutant rubber plants was used as templates for PCR amplification of the predicted insertion sites using the primer combination rc-1-Site-F / rc-1-Site-R. The TkCPTL1 gene was amplified using the CPTL1-F / CPTL1-R primer combination as a control. The nucleotide sequence of the TkCPTL1 gene is shown in SEQ ID NO:6.

[0075] The primers used to amplify the insert fragment are:

[0076] Upstream primer rc-1-Site-F: 5'-CGTTCCATAAATTCCCCTCGGTA-3';

[0077] Downstream primer rc-1-Site-R: 5'-GGCATTCTAGTGTCTCTAGAAGGT-3'.

[0078] The primers used to amplify the TkCPTL1 gene are:

[0079] Upstream primer CPTL1-F: 5'-ATGGATCTGTTAGACGGACCCCAG-3';

[0080] Downstream primer CPTL1-R: 5'-TTATGAACCTGGTACATGTCAAAAC-3'.

[0081] The PCR system consisted of: 2×GC Buffer: 10 μL, 2 mM dNTPs: 2 μL, TaKaRa Taq (5 U / μL): 0.2 μL, Primer-F (2 μM): 1.5 μL, Primer-R (2 μM): 1.5 μL, template DNA: 2 μL, and ddH2O to a final volume of 20 μL.

[0082] The PCR program was as follows: 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 10 min.

[0083] Resequencing results showed that the rc-1 mutant insertion fragment was located near chromosome 22152945 on chromosome A4 of *Hedysarum heterotropoides*, with no genes within 5 kb upstream and downstream of the site. Figure 2 A). PCR results showed that the rc-1-Site-F / rc-1-Site-R primer combination did not amplify in wild-type rubber grass, but specifically amplified in the rc-1 mutant, indicating that the insertion site was amplified in the rc-1 mutant; the CPTL1-F / CPTL1-R primer combination amplified the same size gene fragment in both wild-type and rc-1 mutant rubber grass, indicating that the amplification system was functioning correctly. Figure 2 B).

[0084] Using root cDNA from wild-type and rc-1 mutant rubber grass as templates, respectively, quantitative real-time PCR was performed using PCR primers qHRY-F1 / qHRY-R1 for the inserted fragment and qTkGAPDH-F / qTkGAPDH-R for the internal reference gene TkGAPDH (http: / / bigd.big.ac.cn / gwh / , accession number PRJCA000437, Gene ID: evm.model.utg9113.3). Bio-Rad's SsoFast... The Supermix (Cat#1725201) was used to prepare the real-time PCR reaction system according to the instructions, and the reaction was performed using a BIO-RAD CFX96 real-time PCR instrument.

[0085] The primers used for qRT-PCR detection of insert expression levels are:

[0086] Upstream primer qHRY-F1: 5'-TGTTTTCCGCGACTTTTCCG-3';

[0087] Downstream primer qHRY-R1: 5'-GATCGATTGGTTGATTTGAAGC-3'.

[0088] The primers used for qRT-PCR detection of TkGAPDH expression levels are:

[0089] Upstream primer qTkGAPDH-F: 5'-AGTTGGTTTCGTGGTATGAC-3';

[0090] Downstream primer qTkGAPDH-R: 5'-ACATGTCAGTGAACAGGTAGAC-3'.

[0091] The PCR reaction system consisted of: 5 μL of 2×SsoFast mix; 2 μL of cDNA; and 1.5 μL each of forward and reverse primers (1 μM). The PCR program was as follows: 98℃, 30 s; (98℃, 5 s → 60℃, 5 s → data acquisition), 40 cycles; 60-95℃, 0.5℃ / 5 s, data acquisition / 5 s. After the program was completed, the data were analyzed using BIO-RAD CFX Manager software. The results showed that the expression level of the inserted fragment HRY was significantly upregulated in the rc-1 mutant. Figure 2 C).

[0092] Example 3: Functional verification of HRY1 and HRY2 genes

[0093] To verify the functions of the HRY1 and HRY2 genes, transgenic rubber grass overexpressing HRY1 (35S::HRY1) and HRY2 overexpressing transgenic rubber grass (35S::HRY2) were obtained using transgenic technology and phenotypic analysis was performed.

[0094] 1. Construction of HRY1 and HRY2 overexpression vectors

[0095] Using root cDNA from wild-type Rubbergrass 1151 plants as a template, PCR was performed using primers PF-HRY-F / PF-HRY-R and DNA polymerase KOD Plus (Toyobo, catalog number KOD-201) to amplify the full-length coding sequences of HRY1 and HRY2. The nucleotide sequences of the primers are as follows:

[0096] Upstream primer PF-HRY-F:

[0097] 5'-CATTTACAATTACCATGGATGAATAACAACGGACGTGGAG-3';

[0098] Downstream primer PF-HRY-R:

[0099] 5'-CTAGACTCACCTAGGATCCTCAACTACAATAAATAGATTGA-3'.

[0100] The PCR system was as follows: 10×KOD Plus Buffer: 5 μL, 2 mM dNTPs: 5 μL, 25 mM MgSO4: 2 μL, KOD Plus (1.0 U / μL): 1 μL, PF-HRY-F (10 μM): 0.75 μL, PF-HRY-R (10 μM): 0.75 μL, cDNA: 2 μL, and ddH2O was added to a final volume of 50 μL. The mixture was then centrifuged.

[0101] The PCR program was as follows: 94℃ for 3 min; 98℃ for 10 s, 55℃ for 30 s, 68℃ for 1 min, 30 cycles; 68℃ for 10 min.

[0102] After the reaction, take 1 μL of the reaction product and check the band size using 1% agarose gel electrophoresis. Use an agarose gel recovery kit (Beijing Bomed Gene Technology Co., Ltd., catalog number DH101-01) to recover the HRY1 and HRY2 gene fragments with the correct band size according to the instructions.

[0103] The recovered HRY1 and HRY2 gene fragments were recombined into the pFGC5941 vector, which had been double-digested with restriction endonucleases BamH1 and Nco1, using a recombination reaction. The recombination reaction mixture consisted of 150 ng of the HRY gene fragment, 150 ng of the double-digested pFGC5941 vector, 2 μL of 5×In-Fusion Snap Assembly Master Mix (Takara, catalog number 638949), and ddH2O to a final volume of 10 μL. The reaction was carried out at 50 °C for 15 min, and the recombinant product was then placed on ice.

[0104] Transformation: Take E. coli DH5α competent cells stored at -80℃ and thaw them on ice. Add 5 μL of the above recombinant product, mix gently, and incubate on ice for 30 min. Heat shock at 42℃ for 45 s, and incubate on ice for 2 min. Add 500 μL of LB liquid medium without any antibiotics and activate at 37℃ and 180 rpm for 1 h. Centrifuge the activated E. coli at 3,000 rpm for 1 min, remove most of the supernatant, and resuspend the cells in 100 μL of supernatant. Spread the entire bacterial suspension onto LB agar plates containing 50 μg / ml kanamycin and incubate overnight at 37℃.

[0105] Positive colony identification: Single colonies were picked from LB agar plates and inoculated into 500 μL of LB liquid medium containing 50 μg / ml kanamycin. The medium was activated at 37°C and 200 rpm for 3 h, followed by culture PCR to identify positive clones. Culture PCR used the forward primer qHRY-F for HRY gene quantification and the primer PF-R for the pFGC5941 vector sequence.

[0106] Upstream primer qHRY-F: 5'-GCAACAACAACAACAACAGTGG-3';

[0107] Downstream primer PF-R: 5'-CGTGCACAACAGAATTGAAAGC-3'.

[0108] The PCR system was as follows: 2×GC Buffer: 10 μL, 2 mM dNTPs: 2 μL, TaKaRa Taq (5 U / μL): 0.2 μL, qHRY-F (10 μM): 0.3 μL, PF-R (10 μM): 0.3 μL, bacterial culture: 2 μL, and ddH2O to a final volume of 20 μL.

[0109] The PCR program was as follows: 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 10 min.

[0110] After the reaction, the band size of the PCR products was checked using a 1% agarose gel electrophoresis to verify its accuracy. The bacterial cultures of positive colonies with correct band size were then sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Sequencing yielded positive colonies carrying only the HRY1 recombinant plasmid (pFGC5941-HRY1) and positive colonies carrying only the HRY2 recombinant plasmid (pFGC5941-HRY2).

[0111] Plasmid extraction: Take 200 μL of bacterial culture with correct sequencing results and inoculate it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 200 rpm. Extract plasmids using a rapid plasmid extraction kit (centrifuge column type, Beijing Bomeide Gene Technology Co., Ltd., catalog number DP102-01) according to the instructions to obtain recombinant plasmids pFGC5941-HRY1 and pFGC5941-HRY2.

[0112] 2. Obtaining Agrobacterium tumefaciens containing recombinant expression vector

[0113] The obtained recombinant plasmids pFGC5941-HRY1 and pFGC5941-HRY2 were transformed into Agrobacterium tumefaciens AGL1 cells via electroporation to obtain recombinant Agrobacterium AGL1-pFGC5941-HRY1 and AGL1-pFGC5941-HRY2, respectively. The method was as follows: Agrobacterium AGL1 competent cells stored at -80℃ were thawed in an ice bath. 150 ng of plasmid was added to every 50 μL of competent cells, followed by electroporation at 1.8 kV. After electroporation, antibiotic-free LB broth was added, and the cells were incubated at 28℃ and 200 rpm for 3 hours. The cultured bacterial suspension was centrifuged at 5000 rpm for 1 minute to collect the bacterial cells. Most of the supernatant was discarded, leaving approximately 100 μL. The supernatant was gently resuspended by pipetting and spread onto YEP solid medium plates containing 25 mg / L rifampicin and 50 mg / L kanamycin. After incubation in the dark at 28°C for 48 h, single colonies were picked and inoculated into YEP liquid medium containing 25 mg / L rifampicin and 50 mg / L kanamycin. The plates were then incubated overnight at 28°C with shaking. The bacterial suspension was then inoculated at a 1:100 volume ratio into YEP liquid medium containing 25 mg / L rifampicin and 50 mg / L kanamycin, and acetylsyleugenol was added to a final concentration of 100 μM. The plates were then incubated until OD500 reached 100 μM. 600nm =0.6-0.8, collect bacterial cells by centrifugation at room temperature, remove supernatant, and fully suspend the bacterial cells in an equal volume of infection solution containing 100 μM acetylsyl syringone to obtain AGL1-pFGC5941-HRY1 infection solution and AGL1-pFGC5941-HRY2 infection solution for later use.

[0114] The YEP liquid medium formula is: peptone 10g / L + yeast extract 10g / L + NaCl 5g / L, pH 7.0. YEP solid medium is the YEP liquid medium with the addition of 15g / L agar. The infection solution formula 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.

[0115] 3. Obtaining T0 transgenic rubber grass plants

[0116] The rubber grass used was strain 1151, whose genome was sequenced and preserved in our laboratory. Tissue culture seedlings of healthy rubber grass were taken and cut into segments approximately 0.5 mm in length. These segments were divided into two parts and infected with AGL1-pFGC5941-HRY1 and AGL1-pFGC5941-HRY2 bacterial inoculum solutions, respectively, for approximately 10-20 minutes. The infected root segments were then transferred to sterile filter paper to absorb excess bacterial solution and air-dried. After co-culturing at 21-23℃ in the dark for 2-3 days, the infected rubber grass root segments were transferred to a resistance callus induction medium for selection culture until green resistant buds appeared. Then, they were transferred to a resistance seedling strengthening medium for selection culture for 1-2 months at 21℃, with a 16h light / 8h dark cycle and a light intensity of 80-120 μmol·m⁻¹. -2 ·s -1 Rooted resistant rubber grass seedlings were obtained, namely T0 generation HRY1 overexpression transgenic rubber grass (35S::HRY1) and HRY2 overexpression transgenic rubber grass (35S::HRY2).

[0117] The formulation of the resistance 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. The formulation of the resistance seedling strengthening 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. Timentin is an Agrobacterium-mediated growth regulator. Basta is glufosinate-ammonium.

[0118] 4. Identification of transgenic rubber grass overexpressing HRY1 and HRY2

[0119] Rooted resistant rubber grass seedlings were transplanted into a greenhouse for cultivation. The cultivation substrate was a 1:1 (v / v) mixture of nutrient soil and vermiculite. The cultivation conditions were: temperature 21℃, long day (16h light / 8h dark), and light intensity of 80-120 μmol·m⁻². -2 ·s -1 After three months of greenhouse cultivation, the plants were dug up from the soil, washed, and dried. A portion of the root tissue was taken and rapidly frozen in liquid nitrogen at -80°C. Total RNA was extracted from the roots of T0 generation HRY1-overexpressing transgenic rubber grass (35S::HRY1) and HRY2-overexpressing transgenic rubber grass (35S::HRY2) according to the kit instructions using the Huayueyang RNA Rapid Extraction Kit (Cat#0416-50). Then, reverse transcription was performed using a kit... III. First-Strand Synthesis System (Invitrogen, Cat#18080051): The extracted total RNA was reverse transcribed according to the kit instructions to obtain root cDNA.

[0120] Using root cDNA from wild-type rubber grass, T0 generation 35S::HRY1, and T0 generation 35S::HRY2 as templates, quantitative real-time PCR was performed using PCR primers for the HRY gene and PCR primers for the internal reference gene TkGAPDH (http: / / bigd.big.ac.cn / gwh / , accession number PRJCA000437, Gene ID: evm.model.utg9113.3). Bio-Rad's SsoFast... The Supermix (Cat#1725201) was used to prepare the real-time PCR reaction system according to the instructions, and the reaction was performed using a BIO-RAD CFX96 real-time PCR instrument.

[0121] The primers used for qRT-PCR detection of HRY expression levels are:

[0122] Upstream primer qHRY-F: 5'-GCAACAACAACAACAACAGTGG-3';

[0123] Downstream primer qHRY-R: 5'-CTGCTGGTGGGAGTCTTAATTG-3'.

[0124] The primers used for qRT-PCR detection of TkGAPDH expression levels are:

[0125] Upstream primer qTkGAPDH-F: 5'-AGTTGGTTTCGTGGTATGAC-3';

[0126] Downstream primer qTkGAPDH-R: 5'-ACATGTCAGTGAACAGGTAGAC-3'.

[0127] The PCR reaction mixture consisted of: 5 μL of 2×SsoFast mix; 2 μL of cDNA; and 1.5 μL each of forward and reverse primers (1 μM). The PCR program was as follows: 98℃, 30 s; (98℃, 5 s → 60℃, 5 s → data acquisition), 40 cycles; 60-95℃, 0.5℃ / 5 s, data acquisition / 5 s. After the program completed, the data were analyzed using BIO-RAD CFX Manager software.

[0128] The results showed that the relative expression levels of HRY1 and HRY2 in T0 generation 35S::HRY1 and 35S::HRY2 transgenic rubber grass were significantly higher than those in the wild type. Figure 3 B). The overexpression lines were propagated according to the method in Example 1. The propagated lines were observed, and the results showed that the root morphology of the HRY1 overexpression lines (#1 and #2) and the HRY2 overexpression lines (#3 and #4) was significantly larger than that of the wild type. Figure 3 A). The taproot diameters of the HRY1 and HRY2 overexpression lines were measured and statistically analyzed according to the method in Example 1. After drying, the dried roots were weighed and statistically analyzed. The results showed that, compared with the wild type, the taproot diameters of the HRY1 overexpression lines (#1 and #2) and the HRY2 overexpression lines (#3 and #4) were significantly increased. Figure 3 C), and the weight of the dry roots of each plant also increased significantly. Figure 3 D).

[0129] 5. Analysis of root sections and natural rubber yield of HRY gene-overexpressing rubber grass lines

[0130] Following the method described in Example 1, root sections of wild-type rubber grass, HRY1-overexpressing lines, and HRY2-overexpressing lines were observed. It was found that, compared to the wild type, there were no significant changes in the latex cells of the roots of the HRY1-overexpressing lines (#1 and #2) and the HRY2-overexpressing lines (#3 and #4). Figure 4 A) There was no significant difference in the ductal cell index. Figure 4 B). Rubber was extracted and detected from the roots of wild-type, HRY1-overexpressing, and HRY2-overexpressing rubber grass according to the method described in Example 1. The results showed that the relative content of natural rubber in the roots of the HRY1-overexpressing lines (#1 and #2) and the HRY2-overexpressing lines (#3 and #4) was significantly increased compared to the wild type. Figure 4 C). The natural rubber yield per plant was calculated based on the dry root weight test results (natural rubber yield per plant = relative natural rubber content in roots × dry root weight). The results showed that the natural rubber yield of the HRY1 overexpressing lines (#1 and #2) and the HRY2 overexpressing lines (#3 and #4) was significantly higher than that of the wild type. Figure 4 D).

[0131] The present invention has been described in detail above with reference to specific embodiments. Any modifications or improvements made without departing from the core of the present invention are within the scope of the present invention.

Claims

1. A nucleic acid molecule that encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2 or 4.

2. The nucleic acid molecule according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1 or 3.

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

4. The application of the nucleic acid molecule as described in claim 1 or 2 in cultivating rubber grass plants with increased rubber content.

5. A method for increasing the natural rubber content in rubber grass, comprising: Overexpression of the nucleic acid molecule as described in claim 1 or 2 in rubber grass.

6. A method for obtaining rubber grass with increased natural rubber content, comprising: An overexpression vector for overexpressing the nucleic acid molecule described in claim 1 or 2 was introduced into the rubber grass recipient material, and transgenic rubber grass plants with increased natural rubber content were obtained by culturing.

7. The method according to claim 6, characterized in that, The overexpression vector was introduced into the rubber grass receptor material via Agrobacterium-mediated transformation.

8. A method for producing natural rubber, comprising: A transgenic rubber grass plant with increased natural rubber content is obtained by using the method of claim 6 or 7, the transgenic rubber grass plant is cultured and natural rubber is extracted from its roots.

9. A protein having an amino acid sequence as shown in SEQ ID NO: 2 or 4.

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