Rubber tree rubber particle synthesis related gene HbSEIPIN2 and application thereof

Abstract

The invention belongs to the technical field of plant engineering, and provides a rubber tree HbSEIPIN2 gene, the nucleotide sequence of which is as shown in SEQ ID NO: 1. The invention provides a rubber tree HbSEIPIN2 gene cloned from a rubber tree for the first time, the gene is positioned in an endoplasmic reticulum, the research shows that the gene encoded protein can interact with a plurality of rubber particle membrane proteins such as HbSRPP204, HbSRPP216, HbSRPP243, HbREF175 and HbREF296, and the gene is heterologous expressed in kok-saghyz, so that the relative molecular weight of latex and the particle size of rubber particles can be remarkably improved, and the rubber particle membrane membrane protein can be further improved. Therefore, the gene can be used for regulating and controlling the biosynthesis of rubber particles and is expected to be used for genetic improvement of yield and quality of rubber trees. The invention provides a new candidate gene for research in the aspects of regulation and control of rubber particle synthesis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a rubber tree rubber particle synthesis-related gene HbSEIPIN2 and an application thereof. Background Art

[0002] Currently, over 2,500 plant species worldwide are known to biosynthesize natural rubber, but the primary source of commercial natural rubber remains the rubber tree. Natural rubber in rubber trees is synthesized and distributed within the latex duct cells, primarily composed of cis-1,4-polyisoprene. Rubber particles are specialized organelles within the latex ducts that polymerize and store rubber hydrocarbons. Their core group is a hydrophobic rubber hydrocarbon, enclosed by a single phospholipid membrane. Proteins on the surface of the rubber particle membrane, including small rubber particle protein (SRRP), rubber elongation factor (REF), cis-isopentenyltransferase (CPT), and rubber transferase (HRT), form a complex that polymerizes isopentenyl pyrophosphate (IPP) into high-molecular-weight rubber hydrocarbons. It is generally believed that rubber particles originate in the endoplasmic reticulum, from which particles of varying sizes bud and are released into the latex duct cytoplasm. However, the molecular mechanisms underlying the development of rubber particles in rubber trees remain unclear. Summary of the Invention

[0003] In order to overcome the deficiencies in the prior art, the present invention provides a HbSEIPIN2 Genes and their applications HbSEIPIN2 The protein encoded by the gene can interact with multiple rubber particle membrane proteins. By heterologous expression in rubber grass, the relative molecular weight of latex and the particle size of rubber particles can be significantly increased. It can be used to regulate the synthesis of rubber particles and is expected to be used for genetic improvement of rubber tree yield and quality.

[0004] In order to achieve the above object, the present invention adopts the following technical measures: A first aspect of the present invention is to provide a rubber tree HbSEIPIN2 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0005] The second aspect of the present invention is to provide the rubber tree described in the first aspect HbSEIPIN2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0006] The third aspect of the present invention is to provide a rubber tree comprising the rubber tree of the first aspect of the present invention. HbSEIPIN2 Recombinant plasmid containing the gene coding region.

[0007] The recombinant plasmid original vector can be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc., and the present invention is not limited thereto. In a specific embodiment of the present invention, the original vector is pCAMBIA1300-EGFP, pGWC, etc., but it should be understood that other plasmids or viruses can also be used in the present invention.

[0008] Preferably, the subcellular localization vector of the recombinant plasmid is pCAMBIA1300-EGFP, rubber tree HbSEIPIN2 The gene coding region is connected to the BamH I restriction site of the vector; or the overexpression vector of the recombinant plasmid is the modified pCAMBIA1303-Refp-GFP-GUS, the intermediate vector is pGWC, and the rubber tree HbSEIPIN2 The gene coding region was connected to the Ahd I restriction site of the intermediate vector.

[0009] The fourth aspect of the present invention is to provide a rubber tree comprising the rubber tree of the first aspect of the present invention. HbSEIPIN2 Host bacteria or expression cassette for the gene coding region.

[0010] The fifth aspect of the present invention is to provide a rubber tree as the first aspect of the present invention belongs to HbSEIPIN2 Use of the gene, or the protein described in the second aspect of the present invention, or the recombinant plasmid described in the third aspect of the present invention, or the host bacteria or expression cassette described in the fourth aspect of the present invention in regulating the biosynthesis of rubber particles.

[0011] The sixth aspect of the present invention is to provide the rubber tree as described in the first aspect of the present invention. HbSEIPIN2 The use of the gene, or the protein described in the second aspect of the present invention, or the recombinant plasmid described in the third aspect of the present invention, or the host bacteria or expression cassette described in the fourth aspect of the present invention in interacting with HbSRPP204, and / or HbSRPP216, and / or HbSRPP243, and / or HbREF175, and / or HbREF296.

[0012] Furthermore, the bimolecular fluorescence original vector of the recombinant plasmid is pCAMBIA1300-nYFP or pCAMBIA1300-cYFP, rubber tree HbSEIPIN2 The gene coding region is connected to the Xho I and Xba I restriction enzyme sites of the vector; the yeast two-hybrid vector of the recombinant plasmid is pGADT7, rubber tree HbSEIPIN2 The gene coding region is connected to the BamHI and Xho I restriction enzyme sites of the vector; or the yeast two-hybrid original vector of the recombinant plasmid is pGBKT7, rubber tree HbSEIPIN2 The gene coding region was ligated to the BamH I and Pst I restriction sites of the vector.

[0013] The seventh aspect of the present invention is to provide a primer pair, wherein the primer pair is: ATGGAGCAATCTAATCAAACCG and TCATTTAGTGAGATTGTTTGG.

[0014] The eighth aspect of the present invention is to provide a rubber tree as described in the first aspect of the present invention. HbSEIPIN2 The full-length sequence amplification method of the gene was as follows: the total RNA of rubber tree latex was extracted and reverse transcribed into cDNA, and the forward full-length primer ATGGAGCAATCTAATCAAACCG and the reverse full-length primer TCATTTAGTGAGATTGTTTGG were used to amplify the rubber tree gene by RT-PCR. HbSEIPIN2 Gene.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for cloning rubber trees from rubber trees for the first time. HbSEIPIN2 This gene is located in the endoplasmic reticulum. Studies have shown that the protein encoded by this gene can interact with multiple rubber particle membrane proteins, including HbSRPP204, HbSRPP216, HbSRPP243, HbREF175, and HbREF296. Heterologous expression of the protein encoded by this gene in rubber grass can significantly increase the relative molecular weight of latex and the particle size of rubber particles. This indicates that this gene can be used to regulate rubber particle synthesis and is expected to be used for genetic improvement of rubber tree yield and quality. This invention provides a new candidate gene for research on regulating rubber particle synthesis and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For Example 2 HbSEIPIN2 Subcellular localization.

[0017] Figure 2 For Example 3 HbSEIPIN2 Bimolecular fluorescence interaction analysis of membrane proteins with rubber particles.

[0018] Figure 3 For Example 4 HbSEIPIN2 Yeast two-hybrid interaction analysis of membrane proteins of rubber particles.

[0019] Figure 4 For Example 5 HbSEIPIN2 Detection of heterologously expressed rubber grass latex; In the figure, A, HbSEIPIN2 Detection of relative molecular weight of heterologously expressed rubber grass latex; B, HbSEIPIN2 Rubber particle size detection of heterologous expression rubber grass latex; the data on the right is the weight average molecular weight and rubber particle size average calculated by the testing instrument software based on the data on the left, WT-1, WT-2, WT-3 and HbSEIPIN2-1, HbSEIPIN2-2, HbSEIPIN2-3 All data represent three biological replicates, the vertical bars are standard deviations, and different letters indicate differences among groups. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings and in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not specified in the examples, the methods are based on those described in the literature in this field or on the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0021] Example 1: Rubber Tree HbSEIPIN2 Gene cloning Rubber tree "Reyan 73397" latex was collected on ice. Total RNA was extracted according to the instructions of the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). cDNA was obtained according to the instructions of the Reverse Transcription Kit (Takara Biotechnology Co., Ltd.). The reverse transcribed cDNA was used as a template to amplify the rubber tree ATGGAGCAATCTAAT CAAACCG (SEQ ID NO: 3) and TCATTTAGTGAGATTGTTTGG (SEQ ID NO: 4). HbSEIPIN2 The gene, whose sequence is shown in SEQ ID NO: 1.

[0022] PCR amplification reaction system 20 μL: 2 x PCR buffer 10 μL, primers shown in SEQ ID NO: 3 (10 μmol·L -1 ) 0.8 μL, primers shown in SEQ ID NO: 4 (10 μmol·L -1 ) 0.8 μL, cDNA template 2 μL, ddH2O 6.4 μL.

[0023] PCR amplification program: pre-denaturation at 95°C for 6 min; denaturation at 96°C for 20 s, annealing at 58°C for 20 s, and extension at 72°C for 40 s, for a total of 5 cycles; annealing at 56°C for 20 s, and extension at 72°C for 40 s, for a total of 5 cycles; annealing at 54°C for 20 s, and extension at 72°C for 40 s, for a total of 30 cycles; extension at 72°C for 12 min.

[0024] Example 2: Rubber Tree HbSEIPIN2 Subcellular localization of gene-encoded proteins The correct sequence was amplified using primers AGTAGATCTGTACCATGGAGCAATCTAAT CAAACCG (SEQ ID NO: 5) and GCCCTTGCTCACCATTT TAGTGAGATTGTTTGG (SEQ ID NO: 6). HbSEIPIN2 Gene. PCR amplification reaction system 20 μL: 2 x PCR buffer 10 μL, primers shown in SEQ ID NO: 5 (10 μmol·L -1 ) 0.8 μL, primers shown in SEQ ID NO: 6 (10 μmol·L -1 ) 0.8 μL, cDNA template 1 μL, and ddH2O 7.4 μL. The PCR amplification protocol was as follows: 95°C pre-denaturation for 6 min; 96°C denaturation for 20 s, 58°C annealing for 20 s, and 72°C extension for 40 s, for a total of 5 cycles; 56°C annealing for 20 s, 72°C extension for 40 s, for a total of 5 cycles; 54°C annealing for 20 s, 72°C extension for 40 s, for a total of 30 cycles; and 72°C extension for 12 min. The target fragment was recovered and purified, and then ligated into the plant expression vector pCAMBIA1300-EGFP, digested with BamH I, according to the instructions of the Homologous Recombination Cloning Kit (Novozymes Biotech Co., Ltd.) and verified by sequencing. The correctly sequenced pCAMBIA1300-HbSEIPIN2-EGFP was transformed into Agrobacterium GV3101 (p19) competent cells and injected into tobacco leaves grown for about 4-6 weeks with GV3101 (p19) Agrobacterium carrying CFP-HDEL (endoplasmic reticulum marker protein) plasmid. The leaves were incubated in the dark for 12 hours and cultured under normal 16-hour light conditions for 36 hours. The epidermis of the back of the leaves was torn off and stained with Nile red (lipid marker) for 30 minutes. The leaves were observed using a laser confocal microscope. The results showed that the leaves transformed with GFP empty vector had green fluorescence in the nucleus and cytoplasm, while the leaves transformed with HbSERIPIN2 The leaves of the plant have strong green fluorescence mainly in the endoplasmic reticulum, indicating that HbSEIPIN2 Mainly located in the endoplasmic reticulum ( Figure 1 ).

[0025] Example 3: Rubber Tree HbSEIPIN2 Bimolecular fluorescence interaction analysis of genetically encoded proteins and rubber particle membrane proteins.

[0026] According to the open reading frame sequences of HbSRPP204 (NCBI accession number NM_001405320), HbSRPP216 (NCBI accession number KX498026), HbSRPP243 (NCBI accession number KR076817), HbREF175 (NCBI accession number XM_021797894), and HbREF296 (NCBI accession number KX498027) submitted to the NCBI public database, primers corresponding to the homologous recombination linker were designed (linker primer F: TTTGGAGAGGACACG CTCGAG , linker primer R: TGATGAACCACCACC TCTAGA) amplified latex cDNA. The target fragment was recovered and purified, then ligated into the plant expression vector pCAMBIA1300-cYFP, which had been double-digested with Xho I and Xba I, according to the instructions of the Homologous Recombination Cloning Kit (Novozymes Biotech Co., Ltd.), and verified by sequencing.

[0027] TTTGGAGAGGACACG CTCGAG ATGGAGCAATCTAATCAAACCG (SEQ ID NO: 7) and TGATGAACCACCACC TCTAGA TTTAGTGAGATTGTTTGG (SEQ ID NO: 8) primers were used to amplify and sequence the correct HbSEIPIN2 The target fragment was recovered and purified, and then ligated into the plant expression vector pCAMBIA1300-nYFP after double digestion with Xho I and Xba I according to the operating instructions of the homologous recombination cloning kit (Novozymes Biotech Co., Ltd.), and sequenced and verified. The correctly sequenced pCAMBIA1300-HbSEIPIN2-nYFP plasmid was extracted and transformed into Agrobacterium GV3101 (p19) competent cells, and combined with GV3101 (p19) Agrobacterium carrying pCAMBIA1300-HbSRPP204-cYFP, pCAMBIA1300-HbSRPP216-cYFP, pCAMBIA1300-HbSRPP243-cYFP, pCAMBIA1300-HbREF175-cYFP, and pCAMBIA1300-HbSREF296-cYFP plasmids to transform tobacco leaves that were about 4-6 weeks old. The leaves were incubated in the dark for 12 hours, cultured under normal 16-hour light conditions for 36 hours, and then the back epidermis of the leaves was torn off and observed using a laser confocal microscope. The results showed that the leaves with the empty combination of nYFP and cYFP had no yellow fluorescence, while HbSEIPIN2 and HbSRPP204 、 HbSRPP216 、 HbSRPP243 、 HbREF175 as well as HbREF296 The combined leaves showed different degrees of yellow fluorescence, which indicated that HbSEIPIN2 There is a real interaction between the gene-encoded protein and the rubber particle membrane protein ( Figure 2 ).

[0028] Example 4: Rubber Tree HbSEIPIN2 Yeast two-hybrid analysis of gene-encoded proteins and rubber particle membrane proteins.

[0029] According to the open reading frame sequence of the rubber particle membrane protein submitted to the NCBI public database (same as Example 3), primers were designed to respond to the homologous recombination linker (linker primer F: GCCGAATTCCCGG GGATCC , linker primer R: GTTATGCGGCCG CTGCAG ) was used to amplify latex cDNA. The target fragment was recovered and purified, then ligated into the yeast two-hybrid vector pGBKT7, which had been double-digested with BamHI and PstI, according to the instructions of the Homologous Recombination Cloning Kit (Novozymes Biotech Co., Ltd.), and verified by sequencing.

[0030] GTGGGCATCGATACG GGATCC ATGGAGCAATCTAATCAAACCG (SEQ ID NO: 9) and ACGATTCATCTGCAG CTCGAG TTTAGTGAGATTGTTTGG (SEQ ID NO: 10) primers were used to amplify and sequence the correct HbSEIPIN2 The target fragment was recovered and purified, and then ligated into the yeast two-hybrid vector pGADT7 after double digestion with BamH I and Xho I according to the instructions of the Homologous Recombination Cloning Kit (Novozymes Biotech Co., Ltd.) and verified by sequencing. The correctly sequenced pGADT7-HbSEIPIN2 plasmid was extracted and combined with the pGBKT7-HbSRPP204, pGBKT7-HbSRPP216, pGBKT7-HbSRPP243, pGBKT7-HbREF175, and pGBKT7-HbREF296 plasmids according to the instructions of the Yeast Two-hybrid Kit (Cool Lab Biotech Co., Ltd.) and transformed into Y2HGold competent cells. They were cultured in SD / -Leu / -Trp (double deficiency, DDO) and SD / -Adc / -His / -Leu / -Trp / X-α-gal (quadruple deficiency, QDO) media for 3-5 days and observed. The results showed that HbSEIPIN2 and empty vector combination did not grow on the four-deficient plate, while HbSEIPIN2 and HbSRPP204, HbSRPP216, HbSRPP243, HbREF175 and HbREF296 combination showed blue on the four-deficient plate and the growth was the same as that on the two-deficient plate, which indicated that HbSEIPIN2 There is a real interaction between the gene-encoded protein and the rubber particle membrane protein ( Figure 3 ).

[0031] Example 5: Rubber Tree HbSEIPIN2 Application in the synthesis of latex rubber particles.

[0032] The correct sequence was amplified using primers AGCAGGCTTTGACTTTATGGAGCAATCTAATCAAACCG (SEQ ID NO: 11) and GCTGGGTCTAGAGACTTTCAGTTTCCATTATCTAAAGA (SEQ ID NO: 12). HbSEIPIN2 The target fragment was recovered and purified, then ligated into the intermediate vector pGWC after single-enzyme Ahd I digestion according to the instructions of the Homologous Recombination Cloning Kit (Novozymes Biotech Co., Ltd.) and verified by sequencing. Following the instructions of the Gateway Recombination Cloning Kit (Thermo Fisher Scientific (China) Co., Ltd.), the correctly sequenced pGWC-HbSEIPIN2 was ligated into the modified plant expression vector pCAMBIA1303-Refp-GFP-GUS. The vector was then transformed into rubber grass using Agrobacterium AGL1 competent cells. Sequence ID NOs: 3 and 4 were used to detect transgenic plants, resulting in three transgenic plant lines.

[0033] According to the operating instructions of the gel permeation chromatography system (Waters Technology (Shanghai) Co., Ltd.) and the particle size analyzer (Houliba Instrument (Shanghai) Co., Ltd.), the rubber grass latex of the three transgenic plant lines was analyzed and found to have HbSEIPIN2 The rubber grass latex with heterologous gene expression showed a significant increase in the weight average molecular weight of the latex ( Figure 4 A), the rubber particle size increased significantly ( Figure 4 B), indicating HbSEIPIN2 It has an obvious positive regulatory effect on the synthesis of rubber particles.

[0034] While the specific embodiments of the present invention have been described in detail above, they are intended only as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

[0035] SEQ ID NO: 1

[0036] SEQ ID NO: 2 MEQSNQTDENDHDPSSQTDQKDGCFFDVFEDFPFHDSDQSASDSTLLEPLPEVSYRRNILRRRSRPTSDRGIYGTLSNDSNHESSCISSYITQNYDHKTEYKEKGQRYYRDLKENENNLYFTESNRWNRDRVDLVEVTSKVGEDDDEEMENDDLVGDSVDTAGELGDNSSPNLLECMARLVIKAIWFEVRLFFKFITFPILALYNSYMFVIDPFGVIRRGRGFLMKKLIGLCNRIIWYLSSLIIGWLKDRISIRKLLVRFAWRMFWSFYVCMILCILVIFSMMVSGSLVINLLEKPVQIREELSFDYTENSPVAFAPIMSCGGVGCDFKLCKEELGRRVVQPNDKLEVNVLLTLPESAYNKNLGLFQVRVDLLSPYGKTLASKRQLCMLKFRSEPVRFLLNFFKIVPLVTGYTSEIQTLKVKIRGFKEGDEPTSCLKVTVEQRAEFQAEGGVPEIYDASLVLETDLSILKRIIWCWKNTIFACISMMLFIIELLFTIICCRPAIIPRTRSRGVPALNNSTPNNLTK

Claims

1. Rubber Tree HbSEIPIN2 Gene, the rubber tree HbSEIPIN2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. the rubber tree according to claim 1 HbSEIPIN2 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. Contain the rubber tree described in claim 1 HbSEIPIN2 Recombinant plasmid containing the gene coding region.

4. Contain the rubber tree described in claim 1 HbSEIPIN2 Host bacteria or expression cassette for the gene coding region.

5. The method according to claim 1 HbSEIPIN2 Use of the gene or the protein according to claim 2 or the recombinant plasmid according to claim 3 or the host bacteria or expression cassette according to claim 4 in regulating the biosynthesis of rubber particles.

6. The use according to claim 5, characterized in that The subcellular localization vector of the recombinant plasmid is pCAMBIA1300-EGFP, HbSEIPIN2 The gene coding region is connected to the BamH I restriction site of the vector; or the overexpression vector of the recombinant plasmid is the modified pCAMBIA1303-Refp-GFP-GUS, the intermediate vector is pGWC, and the rubber tree HbSEIPIN2 The gene coding region was connected to the Ahd I restriction site of the intermediate vector.

7. The method according to claim 1 HbSEIPIN2 Use of the gene or the protein according to claim 2 or the recombinant plasmid according to claim 3 or the host bacteria or expression cassette according to claim 4 in interacting with HbSRPP204, and / or HbSRPP216, and / or HbSRPP243, and / or HbREF175, and / or HbREF296.

8. The use according to claim 7, characterized in that The bimolecular fluorescence original vector of the recombinant plasmid is pCAMBIA1300-nYFP or pCAMBIA1300-cYFP, HbSEIPIN2 The gene coding region is connected to the Xho I and Xba I restriction enzyme sites of the vector; the yeast two-hybrid vector of the recombinant plasmid is pGADT7, rubber tree HbSEIPIN2 The gene coding region is connected to the BamH I and Xho I restriction enzyme sites of the vector; or the yeast two-hybrid original vector of the recombinant plasmid is pGBKT7, rubber tree HbSEIPIN2 The gene coding region was ligated to the BamH I and Pst I restriction sites of the vector.

9. A primer pair, characterized in that The primer pair is: ATGGAGCAATCTAATCAAACCG and TCATTTAGTGAGATTGTTTGG.

10. A rubber tree as claimed in claim 1 HbSEIPIN2 A method for amplifying the full-length sequence of a gene, characterized in that: Total RNA was extracted from rubber tree latex and reverse transcribed into cDNA. The forward full-length primer ATGGAGCAATCTAATCAAACCG and the reverse full-length primer TCATTTAGTGAGATTGTTTGG were used to amplify the rubber tree latex by RT-PCR. HbSEIPIN2 Gene.

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