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Recombinant vector pLD-TS for expressing taxadiene in chloroplast

A chloroplast expression and taxadiene technology, applied in the field of plant bioengineering, can solve the problems of complex process, difficult industrialization, complex culture process and difficult control, etc.

Active Publication Date: 2020-04-03
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complex process of chemical total synthesis, 25 to 27 steps of reaction are required, and because the yield of several steps of the reaction is particularly low, the cost of paclitaxel prepared by this method is expensive
However, the method of obtaining paclitaxel through plant cell or tissue culture is also difficult to realize industrialization due to factors such as too low yield, complicated and difficult to control the culture process, and difficulty in maintaining high yield.

Method used

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  • Recombinant vector pLD-TS for expressing taxadiene in chloroplast
  • Recombinant vector pLD-TS for expressing taxadiene in chloroplast
  • Recombinant vector pLD-TS for expressing taxadiene in chloroplast

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Experimental program
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Effect test

Embodiment 1

[0035] In this example, the construction process of the recombinant vector pLD-TS expressing taxadiene in chloroplast is briefly introduced as follows.

[0036] (1) Construction of chloroplast expression vector pLD-MCS

[0037] Referring to the prior art (Verma D et al. A protocol for expression of foreign genes inchloroplasts, Nat Protoc, 2008), the chloroplast expression vector pLD-MCS was designed. The structure of the chloroplast expression vector pLD-MCS is as follows figure 1 As shown, the vector is on the tobacco chloroplast genome trnI and trnA For left and right homologous sequences, use Prrn Promoter Regulatory Screening Markers aad A gene (spectinomycin resistance), using psB The promoter and 5'UTR of the gene regulate the expression of the target gene (that is, the TS gene in this application), use the terminator of TpsbA to terminate transcription, and design a multiple cloning site between the promoter PpsbA+5'UTR and the terminator TpsbA ( MCS).

[...

Embodiment 2

[0051] On the basis of the recombinant vector pLD-TS constructed in Example 1, the inventors further carried out chloroplast transformation and homogeneity screening. The related experimental process is briefly introduced as follows in this example.

[0052] (1) Chloroplast transformation

[0053] In the process of chloroplast transformation, use the PDS 1000 / He gene gun (Bio-Rad, USA) to operate. In the gene gun: the diameter of the gold powder particles is 0.6 μm, the bombardment distance is 9 cm, and 1100 psi can be used to split the membrane. The gold powder was coated with 0.2 μg of plasmid DNA (ie, the recombinant vector pLD-TS prepared in Example 1).

[0054] The specific operation process is:

[0055] Select the 3rd to 4th piece of healthy leaves with a diameter of about 3 cm from the aseptic seedlings of the cultivated tobacco safflower Dajinyuan as explants, cut them out and place them on the RMOP medium with the back facing up;

[0056] Tobacco leaves were bombard...

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Abstract

The invention belongs to the technical field of plant bioengineering, and particularly relates to a recombinant vector pLD-TS for expressing taxadiene in chloroplast. The preparation method of the vector comprises the following steps: obtaining a taxadiene synthase (TS) gene, constructing the vector, transforming, verifying and the like. According to the vector, in tobacco chloroplast, trnI and trnA on a tobacco chloroplast genome are left and right homologous sequences, a Prrn promoter is used for regulating and controlling a screening marker aadA gene, and meanwhile, a promoter of a psbA gene and 5'UTR are used for regulating and controlling expression of the taxadiene synthetase gene TS. The tobacco is used as a heterologous host, and a taxadiene synthetic route is reconstructed by means of genetic engineering. The preliminary experiment results show that the taxadiene is synthesized by high-efficiency expression heterologous synthesis in the tobacco chloroplast, and the content ofthe taxadiene in tobacco transformed plant leaves can reach up to 127.69 [mu]g / g, which presents has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of plant bioengineering, in particular to a recombinant vector expressing taxadiene in chloroplast. Background technique [0002] Paclitaxel (trade name Taxol) was developed by Wani et al. in the 1970s Taxus brevifolia Tetracyclic diterpenoids extracted from Nutt bark with unique anti-cancer effects. Since it was certified by the US FDA in 1992, paclitaxel has become the first-choice anticancer drug in hospital clinics due to its unique anti-tumor mechanism and significant tumor-inhibiting effect. At present, it is mainly used clinically for advanced breast cancer, advanced ovarian cancer, esophageal cancer, nasopharyngeal cancer, bladder cancer, lymphatic cancer, prostate cancer, malignant melanoma and head and neck tumors, upper gastrointestinal tract cancer, small cell and non-cancerous tumors. The treatment of small cell lung cancer also has a certain effect on lung cancer, colorectal cancer and bra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/82A01H5/00A01H6/82C12P5/00
CPCC12N15/66C12N15/8243C12P5/002
Inventor 张剑锋郑庆霞谢小东徐国云金静静王中武明珠罗朝鹏曹培健杨军
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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