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ABC transport protein for transporting lycorine and coding gene and application thereof

A transport protein and protein technology, applied in the fields of biotechnology and plant biology, can solve the problems of unseparated and cloned

Active Publication Date: 2021-05-14
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the transporter of Amaryllidaceae alkaloids has not been isolated and cloned

Method used

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  • ABC transport protein for transporting lycorine and coding gene and application thereof
  • ABC transport protein for transporting lycorine and coding gene and application thereof
  • ABC transport protein for transporting lycorine and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the cloning of ABC transporter gene LaABCB11 coding gene

[0035] The two primers synthesized respectively have the nucleic acid sequences of SEQ ID NO:3 and SEQ ID NO:4 in the sequence listing.

[0036] Using the cDNA obtained by reverse transcription of RNA extracted from Hudixiao as a template, PCR was performed using the above two primers SEQ ID NO:3 and SEQ ID NO:4. The DNA polymerase is Phanta from Nanjing Novizan Biotechnology Co., Ltd. TM SuperFidelity DNA polymerase. The amplification system was 50 μL, and the program was: 98°C for 2 min; 98°C for 10 s, 56°C for 10 s, 72°C for 2 min, a total of 32 cycles; 72°C for 10 min, then lowered to 10°C. PCR products were detected by agarose gel electrophoresis.

[0037] Under the irradiation of ultraviolet light, cut out the target DNA band. Then DNA was recovered from the agarose gel with a DNA purification kit (Nanjing Novizan FastPureGel DNA Extraction Mini Kit), which was the amplified DNA fragment ...

Embodiment 2

[0039] Example 2, Detection of the expression level of the gene encoding the ABC transporter gene LaABCB11 in Hudixiao

[0040] Two primers of the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:6 in the sequence listing were synthesized. Using the cDNA of each tissue of Hudixiao as a template, quantitative PCR amplification was performed using qTOWER 2.2 fluorescent quantitative PCR instrument. The expression of LaABCB11 gene in leaves, scapes and stamens of Hudixiao was relatively high through calculation analysis.

Embodiment 3

[0041] Embodiment 3, the construction of ABC transporter LaABCB11 recombinant expression vector

[0042] Two primers respectively having the nucleotide sequences of SEQ ID NO:7 and SEQ ID NO:8 in the sequence listing were synthesized. The 5'-ends of the synthesized primers SEQ ID NO:7 and SEQ ID NO:8 are provided with two restriction sites, BamHI and EcoRI, and their protected base sequences, respectively, and the cDNA of Hudixiao is used as a template for PCR amplification . The PCR amplification procedure is the same as that in Example 1. The PCR amplification product is detected by agarose gel electrophoresis, separated, gel-cut and recovered, and then digested by BamHI and EcoRI double enzymes, and connected by T4DNA of Takara Bioengineering (Dalian) Co., Ltd. (Takara) The pYES2 vector (Invitrogen) which was also digested with BamHI and EcoRI was ligated. The ligation product was transformed into Escherichia coli (E.coli) DH5α (purchased from Nanjing Qingke Biotechnology...

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Abstract

The invention relates to an ABC transport protein and a coding gene and application thereof. The invention discloses the ABC transport protein of lycoris aurea as an amaryllidaceae plant for the first time, and the ABC transport protein has the functions of being combined with lycorine and enabling the lycorine to be transported across cell membranes. The invention also discloses polynucleotide for coding the ABC transporter protein, a vector and a host cell for expressing the ABC transporter protein, and a method for realizing inward transport of lycorine across a biological membrane.

Description

technical field [0001] The technology relates to the fields of biotechnology and plant biology; more specifically, the invention relates to an ABC transporter derived from Amaryllidaceae plants, its coding gene and application. Background technique [0002] The transport of biomolecules across the cell membrane is one of the most important events in the physiological process of plants, and it is an important step in the exchange, transportation and accumulation of substances in plants. ABC transporter (ATP binding cassette transporter) transports substrates including polysaccharides, polypeptides, alkaloids, hormones and other metabolites. It uses the energy generated by the hydrolysis of ATP to achieve transmembrane transport of specific substrates. It plays an important role in plants. Physiological function. ABC transporters not only participate in the transport process of substrates but also have positive and close relationship with the production of substrates. For ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/81C12N1/19C12P17/18C12R1/865
CPCC07K14/415C12N15/81C12P17/18Y02A50/30
Inventor 王蓉汪仁李晓丹江玉梅刘彦彤周正雄张越徐晟
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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