CN-121495947-B - ChABCB1 gene for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis and application thereof

CN121495947BCN 121495947 BCN121495947 BCN 121495947BCN-121495947-B

Abstract

The invention discloses ChABCB genes for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis and application thereof, wherein the nucleotide sequence of the ChABCB genes is shown as SEQ ID NO. 1, the amino acid sequence of protein coded by the ChABCB genes is shown as SEQ ID NO. 2, and expression cassettes, vectors, engineering bacteria or transgenic cell lines containing the genes, the ChABCB genes, the protein coded by the ChABCB genes, the expression cassettes containing the ChABCB genes, the vectors, the engineering bacteria or transgenic cell lines are used for transferring cephalotaxine from outside to inside of cells.

Inventors

SUN HUAPENG
FU XIAONA
WANG RONGXIANG
CONG HANQING
BAI YIBO
QIAO FEI

Assignees

中国热带农业科学院热带作物品种资源研究所

Dates

Publication Date: 20260508
Application Date: 20260113

Claims (6)

1. The ChABCB gene for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis is characterized in that the nucleotide sequence of the ChABCB gene is shown as SEQ ID NO. 1.
2. The protein encoded by ChABCB gene according to claim 1, wherein the amino acid sequence of said protein is shown in SEQ ID NO. 2.
3. An expression cassette comprising the ChABCB gene of claim 1.
4. A vector comprising the ChABCB gene according to claim 1.
5. An engineered bacterium or transgenic cell line comprising the ChABCB gene of claim 1, the expression cassette of claim 3, the vector of claim 4.
6. Use of ChABCB gene of claim 1, chABCB gene encoded protein of claim 2, expression cassette of claim 3, vector of claim 4, engineering bacterium of claim 5 or transgenic cell line for extracellular to intracellular transport of cephalotaxine in yeast cells or tobacco.

Description

ChABCB1 gene for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis and application thereof Technical Field The invention belongs to the technical field of plant genetic engineering, and particularly relates to ChABCB gene for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis and application thereof, in particular to an ATP binding cassette transporter (ATP-binding cassette transporter, ABC transporter for short) ChABCB gene cloned in the process of cephalotaxine trans-cell membrane transportation in cephalotaxus hainanensis and encoded protein and application thereof. Background Cephalotaxus hainanensis (Cephalotaxus hainanensis) is a rare medicinal plant containing natural anticancer drugs cephalotaxine and homocephalotaxine. The cephalotaxine and homocephalotaxine are ester alkaloids with cephalotaxine as mother nucleus structure, and the medicinal products can be used for clinical treatment of non-lymphocytic leukemia and malignant tumors such as breast cancer, choriocarcinoma, cervical cancer, etc., and are few natural anticancer drugs with obvious curative effect and clear pharmacological mechanism. However, the production costs of these drugs remain high due to the scarcity and slow growth of cephalotaxus plants. Furthermore, cephalotaxus plants have been listed as secondary important protection endangered plants in countries, prohibiting access to raw materials by felling. Although cephalotaxine esters can be produced by artificial synthesis, commercial production is not realized due to the great difficulty and high cost of synthesis. Therefore, solving the problem of raw material sources of cephalotaxine ester alkaloids by using genetic engineering or cell engineering technology becomes a key point of current research, and aims to reduce production cost and improve clinical application value thereof. ATP-binding cassette (ABC) transporters are a class of membrane transporters that are widely found in organisms and function to transport a variety of substrate molecules. In plants, ABC transporters are involved in a number of important physiological processes including transport of plant hormones, accumulation of secondary metabolites, and excretion of foreign toxic substances. For example, ABC transporters in certain plants are found to be closely related to drug resistance, disease resistance, and transport of secondary metabolites. Cephalotaxus hainanensis is an important medicinal plant, and the roots, stems and leaves of cephalotaxus hainanensis contain anticancer active alkaloids. Cephalotaxine is a parent nucleus structure of anticancer compounds cephalotaxine and homocephalotaxine in cephalotaxine, and its synthesis and transport directly affect the content of cephalotaxine and homocephalotaxine. However, it is not clear about the transport of cephalotaxine in cephalotaxus hainanensis plants. Disclosure of Invention The invention aims to provide ChABCB gene for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis and coded protein thereof. The invention also aims to provide an expression cassette, a vector, engineering bacteria or a transgenic cell line containing ChABCB genes for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis. The final object of the present invention is to provide the use of ChABCB gene, the protein encoded by ChABCB gene, the expression cassette, vector, engineering bacteria or transgenic cell line containing ChABCB1, etc. in transferring cephalotaxine from outside to inside. The first aim of the invention can be achieved by the following technical scheme that a ChABCB gene for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis is adopted, and the nucleotide sequence of the ChABCB gene is shown as SEQ ID NO. 1. According to the invention, through analysis of a cephalotaxus hainanensis full-length transcriptome sequencing result, a pair of specific primers is designed, and PCR amplification is carried out on cephalotaxus hainanensis leaf sample cDNA, so that a CDS sequence of ChABCB gene of cephalotaxine is obtained, and the CDS sequence of ChABCB gene is specifically shown as a nucleotide sequence shown as SEQ ID NO. 1. The invention also provides the protein encoded by the ChABCB gene, the amino acid sequence of the protein is shown as SEQ ID NO. 2, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids. The second object of the present invention can be achieved by the following technical scheme that the expression cassette, the vector, the engineering bacteria or the transgenic cell line containing ChABCB genes for transferring cephalotaxine from outside to inside in cephalotaxus hainanensis is contained. As a preferred embodiment of the invention, the engineering bacteria provided by the invention are constructed by constructing ChABCB1 genes on a vector pDR196, transformin