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Cymbidium sinense mannose binding protein

A technology for binding protein and mannose, applied in the fields of plant peptides, biochemical equipment and methods, applications, etc., can solve the problem of unpublished mannose binding protein gene and other problems, and achieve the effect of enriching mannose binding protein members

Inactive Publication Date: 2018-05-15
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No published reports on the gene encoding the mannose-binding protein

Method used

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  • Cymbidium sinense mannose binding protein
  • Cymbidium sinense mannose binding protein
  • Cymbidium sinense mannose binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0008] Example 1: Molan total RNA extraction, quality detection and reverse transcription

[0009] 1. Materials and methods

[0010] 1.1 Materials

[0011] 1.1.1 Plant material: The whole plant of Molan was collected from Mount Emei, Sichuan, China, and was used directly or quickly frozen and stored at -80°C.

[0012] 1.1.2 Enzymes and kits: RNA extraction kits were purchased from TaKaRabao Bioengineering Co., Ltd. (Dalian, China), MLV reverse transcriptase, TdT (nucleotide terminal transferase), RNase Inhibitor

[0013] 1.2 Method

[0014] 1.2.1 Molan total RNA extraction

[0015] Smash the whole plant of Molanca officinalis, use the RNA extraction kit (TaKaRabao Bioengineering Co., Ltd.) to extract the total RNA, and then prepare the following reaction solution in a microcentrifuge tube to remove the DNA in the total RNA:

[0016]

[0017] React the reaction solution in the above microcentrifuge tube with 37°C for 20-30min, add 50μl DEPC-H 2 O, add 100 μl of phenol, ...

Embodiment 2

[0033] Example 2: 3' RACE PCR reaction

[0034] 1. Materials and methods

[0035] 1.1 Materials

[0036] 1.1.1 Source of mannose-binding protein gene

[0037] In NCBI's gene database (https: / / www.ncbi.nlm.nih.gov / gene / ), search for the mannose-binding protein genes of plants in the Aramiaceae family, perform BLAST comparison, and obtain the conservation of the mannose-binding protein genes The sequence was used to design a degenerate primer, and the degenerate primer was: 5'-GACTGCAACCTCGTCCTC-3'.

[0038] 1.1.2 Medium

[0039] SOC liquid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 0.5g / L, 250mmol / L KCl solution 10ml, adjust pH to 7.0 with 5mmol / L NaOH, 2mol / L sterilized MgCl 2 Solution 5ml, 20ml of 1mol / L glucose solution sterilized by suction filtration.

[0040] LB solid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L, adjust pH to 7.0 with 5mol / L NaOH, after autoclaving, add 10g / L agar powder.

[0041] 1.1.3 Enzymes and kits

[0042] Taq DNA polymerase a...

Embodiment 3

[0063] Example 3: 5' RACE PCR reaction

[0064] 1. Materials and methods

[0065] 1.1 Materials

[0066] 1.1.1 The PCR amplification template is the PCR product in 1.2.2 of this embodiment.

[0067] 1.1.2 Enzymes and kits

[0068] RNase A (nucleotidase), TdT (nucleotide terminal transferase), and 5'RACE kits were purchased from TaKaRabao Bioengineering Co., Ltd. (Dalian, China)

[0069] 1.2 Method

[0070] 1.2.1 Primer design

[0071] According to the above 3'RACE PCR amplification reaction product sequence, design the specific primers required for the 5'RACE reaction, as follows:

[0072] GSP: 5'-GACCTCGCCTATTCATCAGT-3'

[0073] GSP1: 5'-TTCACTCACCTTGTTCTGCTC-3'

[0074] GSP2: 5'-AATGGGGTGGCTGTATATAAC-3'

[0075] 1.2.2 Homopolymer tailing of cDNA

[0076] TdT (nucleotide terminal transferase) was used to tail the total cDNA obtained in Example 1, and the amount of cDNA used in the TdT tailing reaction was determined according to the abundance of the target mRNA. Add...

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Abstract

The invention obtains a conserved sequence of a sinense mannose binding protein gene through sequence alignment of sinense mannose binding protein genes of orchidaceae plants. A degenerate primer is designed on the basis of the conserved sequence, a cymbidium sinense mannose binding protein gene is cloned by a 3'RACE and 5'RACE method, a nucleotide sequence of the gene is shown as SEQ ID NO:1 in asequence table, and a cymbidium sinense mannose binding protein amino acid sequence obtained by translation is shown as SEQ ID NO:2 in the sequence table. Mannose binding proteins of the orchidaceaeplants are enriched, and a material foundation is provided for subsequent research and application of the gene and the protein thereof in resistance transgenic agriculture. In addition, references canbe provided for exploring of other unreported mannose binding proteins in the orchidaceae plants.

Description

technical field [0001] The invention belongs to the field of mannose-binding protein, and in particular relates to the gene and protein sequence of the mannose-binding protein from molantium. Background technique [0002] Molan (Cymbidium sinense (Jackson ex Andr.) Willd.), also known as Yearling Orchid, belongs to Angiospermae, Monocotyledoneae, Microspermae, Orchidoideae , Jianlan subgenus (Jensoa) ground plants. Growing in moist but well-drained shady places beside forests, shrubs or valleys. [0003] Existing reports have shown that monocot mannose-binding mannose-binding protein (mannose-binding protein) exists in monocotyledonous orchids, such as Orchid chinensis, Cynthia chinensis, etc., and the monocotyledon mannose-binding protein is mannose The sugar-binding protein specifically combined with its derivatives has a specific and strong inhibitory effect on tumor cells and retroviruses, and has good resistance to diseases and insect pests of agricultural crops. The...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/10
CPCC07K14/415C12N15/1003C12N15/1096
Inventor 鲍锦库吴传芳梁丹凤龙鑫袁媛
Owner SICHUAN UNIV
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