Gene DEL related to natural rubber content in rubber grass and its application

Abstract

The present invention belongs to the field of biotechnology, and relates to genes DEL and applications thereof related to natural rubber content in rubber grass. A nucleic acid molecule is provided, which comprises any nucleotide sequence of the following (a1)-(a4): (a1) nucleotide sequence shown in SEQ ID NO:1,3 or 5; (a2) nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1,3 or 5; (a3) ​​nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2,4 or 6; (a4) nucleotide sequence encoding the amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2,4 or 6; wherein the nucleic acid molecule can give the rubber grass comprising the nucleic acid molecule more latex duct cell numbers and higher natural rubber content. The present invention provides new gene resources for increasing the natural rubber yield of rubber grass, helps to accelerate the domestication and improvement of rubber grass, and cultivates rubber grass varieties with high yield of natural rubber.

Description

Technical Field

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

[0002] Natural rubber (NR) is a natural polymer compound synthesized in plants with cis-1,4-polyisoprene as the main component. The global annual consumption exceeds 12 million tons, with a value of 20 billion US dollars. Due to its excellent performance, it cannot be replaced by synthetic rubber (SR) in many application fields. At present, the Brazilian rubber tree (Hevea brasiliensis Muell.Arg.) is the main commercial source of natural rubber, but due to the threat of pests and diseases, the long rubber production cycle, and the fact that it can only be planted in tropical areas, it is urgent to develop other rubber crops for the production of natural rubber.

[0003] Rubber grass (Taraxacum kok-saghyz Rodin, TKS) is a wild plant native to Xinjiang and Kazakhstan, my country. Its roots can synthesize high-quality natural rubber and have strong environmental adaptability, so it can be planted in most parts of the world. The latex cells in rubber grass are the site for the synthesis and storage of natural rubber. Laticifer cells are a highly specialized cell type that are elongated single cells or connected multi-cells that can form an intricate network structure and are distributed throughout the plant. The root biomass of rubber grass, the proportion of latex in the root (i.e., latex cell density), and the proportion of natural rubber in latex (i.e., the synthesis of natural rubber) have an important impact on the natural rubber yield of rubber grass.

[0004] In order to make wild rubber grass truly an economic crop with production and application value, its natural rubber content needs to be further increased. Summary of the invention

[0005] The present invention separates a gene related to natural rubber content from rubber grass, named DEL (Dense Laticifer Cells) gene, the nucleotide sequence of its coding region is shown in SEQ ID NO: 1, 3 or 5, and the amino acid sequence of the protein (DEL protein) encoded by it is shown in SEQ ID NO: 2, 4 or 6. Overexpression of the gene in rubber grass can increase the number of latex cells and the natural rubber content in the root of rubber grass.

[0006] The technical problem to be solved by the present invention is to increase the natural rubber content of rubber grass.

[0007] In order to solve the above technical problems, the present invention provides a nucleic acid molecule comprising any one of the following nucleotide sequences (a1) to (a4):

[0008] (a1) the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5;

[0009] (a2) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5;

[0010] (a3) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4 or 6;

[0011] (a4) 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, 4 or 6;

[0012] The nucleic acid molecule can give the rubber grass containing the nucleic acid molecule more latex duct cells and higher natural rubber content.

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

[0014] The vector may be a cloning vector, comprising the above nucleic acid molecule and other elements required for plasmid replication. The vector may also be an expression vector, comprising the above nucleic acid molecule and other elements that enable successful protein expression. In some embodiments, the expression vector is a pB7WG2D vector into which the above nucleic acid molecule is inserted.

[0015] The host bacteria may be a host bacteria containing the above cloning vector, such as E. coli DH5α, and the above nucleic acid molecules may be replicated by culturing the bacteria under appropriate conditions. The host bacteria may also be a host bacteria containing the above expression vector, such as Agrobacterium tumefaciens AGL1, and the expression vector may be transferred into the rubber grass receptor material by the genetic transformation technology mediated by Agrobacterium tumefaciens to obtain a transgenic rubber grass plant.

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

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

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

[0019] (B1) a protein with an amino acid sequence as shown in SEQ ID NO: 2, 4 or 6;

[0020] (B2) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.

[0021] In the above method, the gene encoding the DEL protein is the above nucleic acid molecule.

[0022] In the above method, rubber grass plants with high rubber content are obtained by introducing an overexpression vector containing the above nucleic acid molecule into a rubber grass receptor material.

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

[0024] The present invention also provides a method for producing natural rubber, which is characterized by comprising: adopting the above method to obtain transgenic rubber grass plants with high rubber content, cultivating the transgenic rubber grass plants and extracting natural rubber from the roots.

[0025] The present invention also provides a protein, which is as follows (B1) or (B2):

[0026] (B1) a protein with an amino acid sequence as shown in SEQ ID NO: 2, 4 or 6;

[0027] (B2) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.

[0028] Experiments have shown that compared with wild-type rubber grass, rubber grass with overexpression of the DEL gene showed a phenotype of light green leaves, a larger angle between the leaves and the ground, leaves clustered together, and an increased number of leaves. The central column of the root was significantly enlarged, the number of laticifer cells and the number of laticifer cells in the same layer increased significantly, the laticifer cell index (the proportion of the area occupied by laticifer cells in the cross section of the root in the tissue section) was significantly increased, and the natural rubber content increased by 2-3 times ( Figure 5 ).

[0029] The invention provides a new gene resource for increasing the natural rubber yield of rubber grass, helps to accelerate the domestication and improvement of rubber grass, and is of great significance for the cultivation of rubber grass varieties with high natural rubber yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The plant phenotype, leaf number, root threading and natural rubber content of the rubber grass mutant b318 are shown. A is the phenotype of the aboveground part of the rubber grass wild type (WT) and b318 mutant plants; the scale length is 5 cm. B is the statistical results of the leaf number of the rubber grass wild type (WT) and b318 mutant at 0.5 months and 1 month; the values ​​represent the mean ± sd (n = 20), the P value is shown in the figure, and the significance analysis is performed using a two-tailed Student's t test. C is the dry root threading phenotype of the rubber grass wild type (WT) and b318 mutant; the scale length is 1 mm. D is the relative content of natural rubber (%) in the roots of the rubber grass wild type (WT) and b318 mutant at different growth stages (2, 3, and 4 months); the values ​​represent the mean ± sd (n = 3), the P value is shown in the figure, and the significance analysis is performed using a two-tailed Student's t test.

[0031] Figure 2 The phenotypic analysis of the roots and laticifer cells of the b318 mutant is shown. A is a transverse section analysis of the roots of the rubber grass wild type (WT) and the b318 mutant; the white dotted circle indicates the area of ​​the root column, and the scale length is 500 μm. B is a longitudinal section analysis of the roots of the rubber grass wild type (WT) and the b318 mutant; the scale length is 500 μm. C is a statistical analysis of the number of laticifer cell layers in the roots of the rubber grass wild type (WT) and the b318 mutant cultured for 4 months; the values ​​represent the mean ± sd (n = 15), the P value is shown in the figure, and the significance analysis is performed using a two-tailed Student's t-test. D is a statistical analysis of the laticifer cell index (%) of the roots of the rubber grass wild type (WT) and the b318 mutant cultured for 4 months; the values ​​represent the mean ± sd (n = 15), the P value is shown in the figure, and the significance analysis is performed using a two-tailed Student's t-test. E is the statistical analysis of the ratio of the root pith area to the root cross section (%) of the rubber grass wild type (WT) and the b318 mutant cultured for 4 months; the values ​​are mean ± sd (n = 15), P values ​​are shown in the figure, and the significance analysis was performed using a two-tailed Student's t-test.

[0032] Figure 3 The amino acid sequence alignment results of DEL1, DEL2 and DEL3 proteins.

[0033] Figure 4The genetic analysis of the b318 mutant trait, the mutant form and the expression level of the DEL1 gene. A shows the plant phenotypes (upper row) and root cross sections (lower row) of the wild type (WT) and b318 mutant of rubber grass, the hybrid offspring (b318♀) with the b318 mutant as the female parent and the wild type as the male parent; the scale length in the figure is 500 μm. B shows the expression level of the DEL1 gene in the wild type (WT) and b318 mutant plants of rubber grass; the values ​​represent the mean ± sd (n = 3), the P values ​​are shown in the figure, and the significance analysis is performed using a two-tailed Student's t test. C shows the expression level of the DEL1 gene in the wild type (WT) and the hybrid offspring (b318♀) with the b318 mutant as the female parent and the wild type as the male parent of rubber grass; the values ​​represent the mean ± sd (n = 3), the P values ​​are shown in the figure, and the significance analysis is performed using a two-tailed Student's t test.

[0034] Figure 5 The results of phenotypic analysis of transgenic rubber grass. A shows the phenotype of wild type (WT) and transgenic rubber grass (35S::DEL1, 35S::DEL2, 35S::DEL3) of rubber grass at 3 months of growth; the upper row shows the root cross section, showing the number of root latex cells and the number of latex cells, with a scale of 500 μm; the middle row shows the side view of the plant, with a scale of 4 cm; the lower row shows the top view of the plant, with a scale of 5 cm. B shows the expression levels of the three DEL genes in the roots of wild type (WT) and transgenic rubber grass (35S::DEL1, 35S::DEL2, 35S::DEL3) of rubber grass at 3 months of growth. The values ​​represent the mean ± sd (n = 3), the P values ​​are shown in the figure, and the significance analysis was performed using a two-tailed Student's t test. C is the statistics of latex cell index (%) of wild type (WT) and transgenic rubber grass (35S::DEL1, 35S::DEL2, 35S::DEL3) at 3 months of growth; the values ​​are mean ± sd (n = 9), P values ​​are shown in the figure, and the significance analysis is performed using a two-tailed Student's t test. D is the relative content of natural rubber in the roots of wild type (WT) and transgenic rubber grass (35S::DEL1, 35S::DEL2, 35S::DEL3) at 3 months of growth (%); the values ​​are mean ± sd, (n = 3), P values ​​are shown in the figure, and the significance analysis is performed using a two-tailed Student's t test. DETAILED DESCRIPTION

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

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

[0037] The rubber grass (Taraxacum kok-saghyz Rodin, TKS) used in the following examples is the known 1151 strain, which is preserved and propagated by this laboratory. The public can obtain this biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0038] The strains used in the following examples are:

[0039] Escherichia coli strain DH5α is preserved in our laboratory and can be purchased commercially. Agrobacterium tumefaciens strain AGL1 is a strain preserved in our laboratory and can be purchased from Beijing Huayueyang Biotechnology Co., Ltd. with the catalog number NRR01250.

[0040] The vectors used in the following examples are:

[0041] The pDONR221 vector is a DONR vector of the Gateway system, which is preserved in this laboratory and can be purchased from Invitrogen, with the catalog number 12536017. The binary vector pB7WG2D (Gateway) is preserved in this laboratory and can be purchased from the BioVector NTCC Plasmid Vector Bacteria Cell Gene Collection Center.

[0042] Reagents and consumables used in the following examples:

[0043] Oil Red O was purchased from Sigma-Aldrich, Oil Red O solution, Cat#O1391. Natural rubber standard was purchased from Sigma-Aldrich, Cat#431257-100G. Polystyrene standard (NIM-RM2068) was purchased from the Chinese Academy of Metrology. KOD FX (Toyobo, Cat#KFX-101) and KOD Plus (Toyobo, Cat#KOD-201) were purchased from Beijing Bailingke Biotechnology Co., Ltd. TaKaRa Taq TM , purchased from Beijing Liuhetong Economic and Trade Co., Ltd., item number: R001A. RNA rapid extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd., item number: 0416-50. IIIFirst-Strand Synthesis System reverse transcription kit, purchased from Invitrogen, Cat#18080051. SsoFast Supermix was purchased from Bio-Rad, Cat#1725201. Agarose gel recovery kit (catalog number: DH101-01) and plasmid small-scale rapid extraction kit (centrifugal column type) (catalog number: DP102-01) were purchased from Beijing Bomed Gene Technology Co., Ltd. BP CLONASE II ENZYME MIX (catalog number: 11789100) and LR CLONASE II ENZYME MIX (catalog number: 11791020) were products of Invitrogen. Latex RNA extraction kit was purchased from Qiagen, catalog number: 74804. MS medium was purchased from Beijing Qiwei Yicheng Technology Co., Ltd., catalog number: M0222.0050. MES, 6-BA, NAA, and plant gel were purchased from Sigma. Sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10021418.

[0044] Example 1. Discovery and cloning of DEL gene

[0045] 1. Obtaining the rubber grass mutant b318

[0046] The rubber grass SARE (sense / antisense RNA expression system) mutant library used was constructed by our research group. The screening and identification method of the mutants is as follows: the rubber grass SARE mutants are cultured in the greenhouse for about 1 month and numbered according to the presence or absence of phenotypes in the aboveground parts. The numbered mutant materials are continued to be cultured in the greenhouse for 3 months. At this time, the mutants are dug out from the soil, the floating soil on the roots is washed off, and the root morphological changes are observed. Take part of the roots, wash and dry to constant weight, and use the fracture method to estimate the rubber content. Then select mutant plants with obvious wire drawing changes, perform tissue sections on their roots, and count the number of latex cell layers, latex cell index, and the proportion of the root column area to the root cross section. The method of tissue sectioning is based on the literature (Sando, T., Hayashi, T., Takeda, T., Akiyama, Y., Nakazawa, Y., Fukusaki, E., and Kobayashi, A. (2009). Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Heveabrasiliensis using spectral confocal laser scanning microscopy. Planta 230, 215-225.), which is as follows: a small section of rubber grass root about 1.5 cm long was taken and fixed in 80% ethanol for at least 12 h. Cut a small section of about 0.5 cm in the middle, embed it with low melting point agarose, slice it with an oscillating microtome, slice it with a thickness of 100 μm, remove the agarose around the root slice, dye it with Oil Red O for 1 min, then rinse it once with 45% glacial acetic acid, wash it twice with distilled water, and then place it under a stereoscope (OLYMPUS SZX16) to observe and take pictures after sealing it with 60% (v / v) glycerol. Use ImageJ software to perform statistics on the area of ​​the latex, and the index of latex cells = the area of ​​latex cells / root slice area × 100%. Use ImageJ software to perform statistics on the area of ​​the middle column, and the ratio of the middle column area = the area of ​​the middle column / root slice area × 100%.

[0047] After the above screening process, a rubber grass mutant with a significantly changed plant shape and a significantly increased number of latex cells was found, named b318. Compared with the wild-type rubber grass, the leaves of the b318 mutant were light green, the angle between the leaves and the ground was larger, the leaves were clustered together, and the number of leaves increased ( Figure 1 AB), the rubber fibers in the roots increased significantly ( Figure 1C), the root stele was significantly enlarged, the number of laticifer cells increased significantly, the number of laticifer cells in the same layer increased significantly, and the laticifer cell index increased significantly ( Figure 2 ).

[0048] The roots of the wild type and b318 mutant of rubber grass of the same period were used for natural rubber extraction and quantification. The extraction method of natural rubber was based on the reference (Kreuzberger, M., Hahn, T., Zibek, S., Schiemann, J., and Thiele, K. (2016). Seasonal pattern of biomass and rubber and inulin of wild Russian dandelion (Taraxacum koksaghyz L. Rodin) under experimental field conditions. Eur. J. Agron. 80, 66-77.) and was appropriately modified. The specific operation is as follows: After the rubber grass is planted in the greenhouse for a certain period of time, the roots are taken, washed, placed in a 50°C oven to dry to constant weight, and then frozen and ground into powder. 100 mg of the powder is weighed and placed in a 2.0 mL centrifuge tube, 1 mL of toluene is added, and the powder is fully suspended and vortexed for 1 minute. Extract at 750 rpm for 2 h at 50 °C, centrifuge at 10,000 rpm at room temperature for 10 min, take the supernatant, repeat this process 3 times. Concentrate the supernatant and make it volume to 500 μL, add 1 mL of methanol, place at 4 °C for 30 min, centrifuge to remove the supernatant, add 1 mL of distilled water, wash at room temperature for 10 min, centrifuge to remove the supernatant, add 1 mL of acetone, wash at room temperature for 10 min, centrifuge to remove the supernatant, dry, and obtain natural rubber, which is stored in a refrigerator at -20 °C.

[0049] The natural rubber content in the roots of the wild type and b318 mutant of rubber grass was detected by Fourier transform infrared spectroscopy as described in the literature (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.). The specific operation is as follows:

[0050] Natural rubber standards (Sigma-Aldrich, Cat#431257-100G) of different concentrations (0.5, 1, 2, 4, 6, 8 mg / mL) were prepared with toluene. 150 μL of each concentration of standard solution was taken and mixed with 15 μL of 10 mg / mL polystyrene standard (China Institute of Metrology, NIM-RM2068). 70 μL of the mixture was taken with a pipette and evenly applied on a potassium bromide wafer that had been pressed with a tablet press in advance, and dried in an oven. Then, it was detected with a far-infrared spectrometer (BRUKER, TENSOR 27). During the detection process, the wafer was scanned 32 times. After simple atmosphere compensation and baseline correction, the infrared spectrum obtained was calculated at 835 cm -1 (natural rubber absorption peak) and 699cm -1 The peak area ratio of (polystyrene absorption peak). The standard curve was drawn with the peak area ratio and natural rubber concentration as coordinates, and the standard curve equation was obtained as X = (Y-0.0698) / 0.2596, R 2 = 0.9961, where X represents the concentration of natural rubber and Y represents 835 cm -1 and 699cm -1 The peak area ratio.

[0051] The extracted root rubber of the wild type and b318 mutant of rubber grass was dissolved in 1 mL of toluene, and 150 μL was taken. 15 μL of polystyrene standard with a concentration of 10 mg / mL (NIM-RM2068, Chinese Academy of Metrology) was added as an external standard. The mixture was mixed well, and 70 μL of the mixture was taken with a pipette. The infrared spectrum was detected according to the above method. The wavelength of 835 cm -1 (natural rubber absorption peak) and 699cm -1 The peak area ratio of the absorption peak of natural rubber and the absorption peak of polystyrene is substituted into the above standard curve X = (Y-0.0698) / 0.2596 to calculate the concentration of natural rubber. The relative content of natural rubber (%) = the concentration of natural rubber (mg / mL) × the volume of toluene used for extraction (mL) / the powder mass of the root used to extract rubber (mg) × 100%.

[0052] After testing, it was found that the natural rubber content in the b318 mutant at different growth stages was significantly higher than that in the wild type. The natural rubber content in the b318 mutant was 2-3 times that in the wild rubber grass at the same period ( Figure 1 D).

[0053] 2. Identification of inserted sequences in mutants

[0054] The total DNA was extracted from the young leaves of the mutant using the CTAB method as a template, and the cDNA fragment inserted in the b318 mutant was amplified by polymerase chain reaction (PCR) using vector primers Primer F1 and Primer R1. The PCR system (50 μL) was prepared according to the product instructions of PCR enzyme KODFX (TOYOBO, KFX-101). The PCR product was sequenced using vector primers Primer F2 and Primer R2 to obtain the information of the inserted sequence.

[0055] The sequences of the vector primers are as follows:

[0056] Primer F1:5'-TCGCATGCCTGCAGGTCACT-3'

[0057] Primer R1:5'-CCAACCACGTCTTCAAAGC-3'

[0058] Primer F2:5'-GAGAGAGATAGATTTGTAGAGAG-3'

[0059] Primer R2:5'-TCATTTGGAGAGGACTCCGGT-3'.

[0060] The sequencing results showed that the b318 mutant was caused by a 1,291bp cDNA fragment inserted into the genome of the wild-type rubber grass. By comparing it with the NCBI and rubber grass 1151 reference genomes, it was found that the fragment contained a 70bp 5'UTR region, a 1,101bp coding region and a 120bp 3'UTR region. The gene was named DEL1 (Denselaticifer cell 1, DEL1), the nucleotide sequence of its coding region is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes (DEL1 protein) is shown in SEQ ID NO:2. By comparing the protein sequences encoded by all genes in the rubber grass 1151 reference genome, it was found that there was no protein in the sequenced strain 1151 that was completely consistent with the DEL1 protein sequence, but there were two proteins with more than 98% amino acid similarity to the DEL1 protein ( Figure 3). When constructing the SARE mutant library, the cDNA used was a mixed sample of multiple varieties. For the convenience of distinction, the two genes aligned in the reference genome were named DEL2 (Dense laticifer cell 2, DEL2) and DEL3 (Dense laticifer cell 3, DEL3). The nucleotide sequence of the coding region of the DEL2 gene is shown in SEQ ID NO:3, and the amino acid sequence of the protein encoded by it (DEL2 protein) is shown in SEQ ID NO:4. The nucleotide sequence of the coding region of the DEL3 gene is shown in SEQ ID NO:5, and the amino acid sequence of the protein encoded by it (DEL3 protein) is shown in SEQ ID NO:6.

[0061] RNA extraction and reverse transcription: Take the roots of the wild type and mutant b318 of rubber grass grown for 3 months, wash them, grind them with liquid nitrogen, and use the RNA rapid extraction kit (Huayueyang, 0416-50) to extract RNA according to the product instructions. The kit is equipped with DNase to remove DNA contamination in the total RNA. Dissolve the total RNA in the elution buffer EB in the kit. Use a UV spectrophotometer to measure the OD values ​​at 230nm, 260nm, and 280nm to detect the quality and quantify the RNA. Use Oligo dT as a primer and use The extracted RNA was reverse transcribed using III First-Strand Synthesis System Reverse Transcription Kit (Invitrogen, 18080051) according to the product instructions to obtain cDNA.

[0062] Quantitative analysis of DEL1 gene expression: Using cDNA as template, using SsoFast Supermix (Bio-Rad, 1725201) was used to prepare the fluorescence quantitative PCR reaction system according to the product manual. The PCR reaction was performed using a real-time fluorescence quantitative PCR instrument (BIO-RAD, CFX96). The PCR reaction system was: 2×SsoFast mix 5μL; cDNA 2μL; 1.5μL of each upstream and downstream primer (1μM). The PCR reaction program was: 98℃, 30s; (98℃, 5s→60℃, 5s→information acquisition), 40 cycles; 60-95℃, 0.5℃ / 5s, information acquisition / 5s. After the program was completed, the data was analyzed using the BIO-RAD CFX Manager software.

[0063] The primers used for real-time fluorescence quantitative PCR are as follows: primers qDEL1-F and qDEL1-R for detecting the expression level of DEL1 (amplifying a 105 bp fragment located at the 3' end of the DEL1 coding region), and primers qGAPDH-F and qGAPDH-R for detecting the expression level of the GAPDH reference gene (http: / / bigd.big.ac.cn / gwh / , accession number: PRJCA000437, Gene ID: evm.model.utg9113.3) (amplifying a 244 bp fragment located at the 3' end of the DEL1 coding region).

[0064] Upstream primer qDEL1-F: 5′-TATGGTGGTCAAGGATATGGTTAC-3′;

[0065] Downstream primer qDEL1-R: 5'-TGATTGCTGATCTCCACCACC-3'.

[0066] Upstream primer qGAPDH-F: 5′-AGTTGGTTTCGTGGTATGAC-3′;

[0067] Downstream primer qGAPDH-R: 5'-ACATGTCAGTGAACAGGTAGAC-3'.

[0068] The relative expression level of DEL1 mRNA was determined by reference (Livak, KJ, and Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCt Method. Methods 25, 402-408.) First, calculate ΔCt = Ct of DEL1 minus Ct of GAPDH; ΔΔCt = ΔCt in b318 mutant minus ΔCt in wild type; use formula 2 -ΔΔCt The expression level of each sample in the experimental group relative to the control group was calculated.

[0069] RT-PCR detection revealed that the expression of DEL1 gene in the b318 mutant and its hybrid offspring was significantly upregulated ( Figure 4 ). The results indicate that the phenotype of the b318 mutant is stably inherited and that overexpression of the DEL1 gene may be involved in regulating the mutant phenotype of the b318 mutant.

[0070] Example 2. Functional verification of DEL gene

[0071] 1. Construction of overexpression vector

[0072] Using primers GW-DEL-F and GW-DEL-R, the genomic DNA of the b318 mutant was used as a template to amplify the DEL1 gene fragment with a length of 1101 bp. Using primers GW-DEL-F and GW-DEL-R, the latex cDNA of the wild-type 1151 strain of rubber grass was used as a template to amplify the DEL2 gene fragment and the DEL3 gene fragment with a length of 1110 bp and 1101 bp respectively.

[0073] Upstream primer GW-DEL-F:

[0074] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAGTCAGACGAAGGGAAGCT-3';

[0075] Downstream primer GW-DEL-R:

[0076] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCTACTGGTAACCAGTCTGCCCATTA-3'.

[0077] The PCR system was: 10×KOD Plus Buffer 5μL, 2mM dNTPs 5μL, 25mM MgSO4 2μL, KODPlus (1.0U / μL) 1μL, GW-DEL-F (10μM) 0.75μL, GW-DEL-R (10μM) 0.75μL, DNA template 2μL, and ddH2O made up to 50μL.

[0078] The PCR program was: 94°C for 3 min; 98°C for 10 s, 55°C for 30 s, 68°C for 1 min, 30 cycles; 68°C for 10 min.

[0079] After the PCR reaction was completed, 1 μL of the reaction product was taken and detected by 1% agarose gel electrophoresis. The gene fragments with the correct band size were recovered using an agarose gel recovery kit (Biomed Biotechnology, catalog number DH101-01) according to the operating steps provided in the kit manual.

[0080] The DEL1 / DEL2 / DEL3 gene fragments were homologously recombined into the intermediate vector pDONR221 using the BP reaction. The BP reaction system and reaction conditions were as follows: 150 ng of gene fragments, 150 ng of pDONR221 vector, 1 μL of BP Clonase II EnzymeMix, 1×TE (pH=8.0) to 10 μL; incubate at 25°C for 10 h; add 1 μL of Proteinase K, incubate at 37°C for 10 min, obtain the BP reaction product, and place on ice.

[0081] The BP reaction product was used to transform E. coli DH5α competent cells, and the transformation product was spread on LB solid plates containing 50 μg / ml kanamycin and cultured overnight at 37°C. A single colony was picked and inoculated into 500 μL LB liquid culture medium containing 50 μg / ml kanamycin, activated at 37°C, 200 rpm for 3 hours, and then the bacterial liquid PCR was performed using primers DEL-TEST-F and M13R to identify positive clones.

[0082] Primer DEL-TEST-F: 5′-TATGGAAACCCTGGTGGACTG-3′

[0083] Primer M13R: 5′-CAGGAAACAGCTATGACC-3′

[0084] The PCR system was: 2×GC Buffer 10μL, 2mM dNTPs 2μL, TaKaRa Taq (5U / μL) 0.2μL, DEL-TEST-F (10μM) 0.3μL, M13R (10μM) 0.3μL, bacterial solution 2μL, and ddH2O was added to 20μL.

[0085] The PCR program was: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min.

[0086] The positive clones identified by PCR were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The positive clones with correct sequencing were inoculated into 10 mL of LB liquid culture medium containing 50 μg / ml kanamycin, cultured overnight at 37°C and 200 rpm, and the plasmids were extracted using a plasmid small-scale rapid extraction kit (Beijing Bomed, DP102-01) according to the kit instructions. Recombinant plasmids pDONR221-DEL1, pDONR221-DEL2, and pDONR221-DEL3 were obtained.

[0087] The DEL1 / DEL2 / DEL3 gene fragments were homologously recombined into the binary vector pB7WG2D using the LR reaction. The LR reaction system and reaction conditions were as follows: 150 ng of pDONR221-DEL1 / pDONR221-DEL2 / pDONR221-DEL3 plasmids, 150 ng of pB7WG2D plasmids, 1 μL of LR Clonase II Enzyme Mix, supplemented to 10 μL with 1×TE (pH=8.0); incubated at 25°C for 10 h; added 1 μL of Proteinase K, incubated at 37°C for 10 min, and the LR reaction product was obtained and placed on ice.

[0088] The LR reaction product was used to transform E. coli DH5α competent cells, and the transformation product was spread on LB solid plates containing 50 μg / ml spectinomycin and cultured overnight at 37°C. A single colony was picked and inoculated into 500 μL LB liquid medium containing 50 μg / ml spectinomycin, activated at 37°C, 200 rpm for 3 hours, and then the primers DEL-TEST-F and T35S-R were used to perform bacterial liquid PCR to identify positive clones.

[0089] Primer T35S-R: 5′-TCGCATGCCTGCAGGTCACT-3′

[0090] The PCR system was: 2×GC Buffer 10μL, 2mM dNTPs 2μL, TaKaRa Taq (5U / μL) 0.2μL, DEL-TEST-F (10μM) 0.3μL, T35S-R (10μM) 0.3μL, bacterial solution 2μL, and ddH2O was added to 20μL.

[0091] The PCR program was: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min.

[0092] The positive clones identified by PCR were sent to Beijing Ruiboxing Biotechnology Co., Ltd. for sequencing. The positive clones with correct sequencing were inoculated into 10 mL of LB liquid culture medium containing 50 μg / ml spectinomycin, cultured overnight at 37°C and 200 rpm, and the plasmids were extracted using a plasmid small-scale rapid extraction kit (Beijing Bomed, DP102-01) according to the kit instructions. The overexpression vectors 35S::DEL1, 35S::DEL2 and 35S::DEL3 of the DEL1, DEL2 and DEL3 genes were obtained.

[0093] 2. Transformation of Agrobacterium tumefaciens

[0094] The overexpression vectors 35S::DEL1, 35S::DEL2, and 35S::DEL3 with correct sequencing were used to transform Agrobacterium tumefaciens AGL1, respectively, to obtain strains AGL1-35S::DEL1, AGL1-35S::DEL2, and AGL1-35S::DEL3. The specific method is as follows: the competent cells of Agrobacterium tumefaciens AGL1 stored at -80°C were melted in an ice bath. 150 ng of plasmid was added to each 50 μL competent cells, and then the competent cells were electroporated at an electroporation parameter of 1.8 kV. After the electroporation, LB liquid culture medium without antibiotics was added and cultured at 28°C and 200 rpm for 3 hours. The cultured bacterial solution was centrifuged at 5000 rpm for 1 minute to collect the bacteria, most of the supernatant was discarded, and about 100 μL of the supernatant was retained. The bacterial block was resuspended by blowing gently, and then spread on a YEP solid culture medium plate containing 25 mg / L rifampicin and 50 mg / L spectinomycin. After dark culture at 28°C for 48 h, a single clone was picked and inoculated into a YEP liquid culture medium containing 25 mg / L rifampicin and 50 mg / L spectinomycin. After shaking culture at 28°C overnight, the bacterial solution was inoculated into a YEP liquid culture medium containing 25 mg / L rifampicin and 50 mg / L spectinomycin at a volume ratio of 1:100, and acetosyringone was added to a final concentration of 100 μM, and cultured to an OD of 0. 600 =0.6-0.8, collect the bacteria by centrifugation at room temperature, remove the supernatant, and fully suspend them with an equal volume of infection solution containing 100 μM acetosyringone to obtain the infection solution.

[0095] The formula of the above YEP liquid medium is: peptone 10g / L, yeast extract 10g / L, NaCl 5g / L, and the pH is adjusted to 7.0. YEP solid medium is based on YEP liquid medium and agar 15g / L is added.

[0096] The formula of the above-mentioned infection solution is: MS 4.405g / L, MES 0.5g / L, 6-BA 0.5mg / L, NAA 0.01mg / L, sucrose 20g / L, glucose 10g / L, and the pH is adjusted to 5.2.

[0097] 3. Obtaining transgenic rubber grass

[0098] The rubber grass 1151 strain was used as the transformation receptor material, and the rubber grass tissue culture roots were infected with Agrobacterium to obtain transgenic plants. The strains used were AGL1-35S::DEL1, AGL1-35S::DEL2 and AGL1-35S::DEL3, and each strain was independently transformed into the rubber grass tissue culture roots. The specific transformation method is as follows: the rubber grass tissue culture roots with good growth status were cut into small segments of about 0.5 mm, and infected with the infection solution for about 10-20 minutes, and then the infected roots were scooped onto sterile filter paper to absorb the excess bacterial solution and dried, and co-cultured at 21-23°C in the dark for 2-3 days, and then the infected rubber grass roots were transferred to the resistance callus induction medium for screening and culture, until the green resistance buds appeared, and then transferred to the resistance seedling medium for screening and culture, and cultured for 1-2 months, the culture temperature was 21°C, 16h light / 8h dark, and the light intensity was 60-80μmol s -1 m -2 , and obtained transgenic rubber grass.

[0099] The formula of the above-mentioned resistant callus induction medium is: 400 mg / L Timentin, 10 mg / L Basta, 4.405 g / L MS, 0.5 g / L MES, 0.5 mg / L 6-BA, 0.01 mg / L NAA, 20 g / L sucrose, 3.6 g / L plant gel, and the pH is adjusted to 5.8.

[0100] The formula of the above-mentioned resistant seedling culture medium is: 400 mg / L Timentin, 10 mg / L Basta, 2.286 g / L MS, 0.5 g / L MES, 10 g / L sucrose, 3.6 g / L plant gel, and the pH is adjusted to 5.8.

[0101] 4. Analysis of the expression level of DEL gene in transgenic rubber grass

[0102] The wild type rubber grass was used as the control, and the transgenic rubber grass was subjected to RT-PCR to analyze the expression level of the DEL gene.

[0103] RNA extraction and reverse transcription: The transgenic rubber grass grown for 3 months was dug out of the soil, washed and dried, and some root tissue was taken and quickly frozen with liquid nitrogen. RNA from the roots of the T0 generation transgenic rubber grass was extracted using an RNA rapid extraction kit (Huayueyang, 0416-50) according to the product instructions. Oligo dT was used as a primer and The extracted RNA was reverse transcribed using III First-Strand Synthesis System Reverse Transcription Kit (Invitrogen, 18080051) according to the product instructions to obtain cDNA.

[0104] Quantitative analysis of DEL gene expression: Using cDNA as template, using SsoFast Supermix (Bio-Rad, 1725201) was used to prepare the fluorescence quantitative PCR reaction system according to the product manual. The PCR reaction was performed using a real-time fluorescence quantitative PCR instrument (BIO-RAD, CFX96). The PCR reaction system was: 2×SsoFast mix 5μL; cDNA 2μL; 1.5μL of each upstream and downstream primer (1μM). The PCR reaction program was: 98℃, 30s; (98℃, 5s→60℃, 5s→information acquisition), 40 cycles; 60-95℃, 0.5℃ / 5s, information acquisition / 5s. After the program was completed, the data was analyzed using the BIO-RAD CFX Manager software.

[0105] The primers used for real-time fluorescence quantitative PCR are as follows: primers qDELs-F and qDELs-R for detecting the expression level of DELs, which are primers designed for the regions with consistent sequences of the three genes DEL1, DEL2, and DEL3; primers qGAPDH-F and qGAPDH-R for detecting the expression level of the GAPDH internal reference gene.

[0106] Upstream primer qDELs-F: 5′-GCCGTGGTGGAGGTTATCAA-3′;

[0107] Downstream primer qDELs-R: 5′-CATAGCCGGAGTAAGCTGGG-3′.

[0108] The results showed that compared with the wild-type rubber grass, the mRNA levels of the DEL gene in the T0 generation 35S::DEL1 transgenic rubber grass, the T0 generation 35S::DEL2 transgenic rubber grass, and the T0 generation 35S::DEL3 transgenic rubber grass were significantly increased ( Figure 5 B).

[0109] 5. Analysis of the number of latex cells and natural rubber content in transgenic rubber grass

[0110] Wild-type rubber grass and T0 transgenic rubber grass were cultured under conventional conditions: greenhouse culture conditions were 21°C, long day (16 h light / 8 h dark), and light intensity of 80-120 μE·m -2 ·s -2 After the plants grew for one month, the phenotypes of the aboveground parts of the plants were observed, and it was found that the phenotypes of the 35S::DEL1 transgenic rubber grass, 35S::DEL2 transgenic rubber grass, and 35S::DEL3 transgenic rubber grass were consistent with the mutant material b318 in terms of leaf shape, leaf color, and plant type ( Figure 5). After the plants have grown for 3 months, the roots of the wild-type rubber grass and the transgenic rubber grass are sliced, the latex cell index (the area ratio of the latex cell in the root cross section in the tissue slice) is counted, and the natural rubber content of the roots of the wild-type rubber grass and the transgenic rubber grass is determined. The method of tissue slicing, the statistical method of the latex cell index, and the method of determining the natural rubber content are the same as in Example 1.

[0111] The results showed that compared with the wild-type rubber grass, the plant types of 35S::DEL1 transgenic rubber grass, 35S::DEL2 transgenic rubber grass and 35S::DEL3 transgenic rubber grass were significantly changed, including light green leaves, larger angles between leaves and the ground, clustered leaves and an increase in the number of leaves ( Figure 5 A, middle and lower rows); the pith of the root was significantly enlarged, the number of laticifer cells increased significantly, and the number of laticifer cells in the same layer increased significantly ( Figure 5 A, upper row), the milk duct cell index increased significantly ( Figure 5 C); the natural rubber content in the root increased significantly ( Figure 5 D). The above results prove that DEL1, DEL2, and DEL3 genes are related to the number of latex tubes and the content of natural rubber in rubber grass, indicating that DEL1, DEL2, and DEL3 proteins and their encoding genes can provide a theoretical basis and genetic resources for the breeding of rubber grass, which is of great significance for the genetic improvement of rubber grass and the cultivation of rubber grass varieties with high rubber yield.

Claims

1. A nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO: 1, 3 or 5.

2. A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.

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

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

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

6. A method for obtaining rubber grass plants with high rubber content, characterized in that: include: The expression of the gene encoding the DEL protein in rubber grass is enhanced to obtain rubber grass with a high rubber content; the amino acid sequence of the DEL protein is shown in SEQ ID NO: 2, 4 or 6.

7. The method according to claim 6, characterized in that The gene encoding the DEL protein is the nucleic acid molecule according to claim 1.

8. The method according to claim 6, characterized in that By introducing an overexpression vector comprising the nucleic acid molecule of claim 1 into a rubber grass receptor material, a rubber grass plant having a rubber content higher than that of the receptor material is obtained.

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

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

11. A protein, whose amino acid sequence is shown in SEQ ID NO: 2, 4 or 6.

Images