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Oil-producing yeast expression vector utilizing Zeocin to screen aleurites montana SAD genes and construction method

A technology of expression vector and oleaginous yeast, which is applied in the field of genetic engineering to achieve the effect of simplifying the genetic transformation process

Inactive Publication Date: 2010-03-10
RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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Problems solved by technology

pCAMBIA2300 also belongs to this type of expression vector, but the resistance marker gene it carries - neomycin phosphotransferase gene (NPTII) has kanamycin resistance, while oleaginous yeast is not very sensitive to kanamycin , so it cannot be directly used as an expression vector for oleaginous yeast

Method used

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  • Oil-producing yeast expression vector utilizing Zeocin to screen aleurites montana SAD genes and construction method
  • Oil-producing yeast expression vector utilizing Zeocin to screen aleurites montana SAD genes and construction method
  • Oil-producing yeast expression vector utilizing Zeocin to screen aleurites montana SAD genes and construction method

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Embodiment Construction

[0025] The present invention is further described in detail through the following examples, but the content of the present invention is not limited thereto.

[0026] 1 Experimental materials

[0027] 1.1 Plant material

[0028] From August 25th to September 10th, 2006, the growing fruits of Tunga paulica were collected from the Arboretum of the Subtropical Forestry Research Institute of the Chinese Academy of Forestry, and the seeds were peeled off and quick-frozen in liquid nitrogen, and stored in a -80°C refrigerator for later use.

[0029] 1.2 Strains and plasmids

[0030] Escherichia coli E.coli DH5α and Agrobacterium tumefaciens EHA 105 were preserved in our laboratory. The plasmid pCAMBIA2300 was preserved in our laboratory, and the cloning vector pGEM-T Easy was purchased from Promega.

[0031] 1.3 Main biochemical reagents

[0032] RNA extraction kits were purchased from Tianze Gene Engineering Co., Ltd. (Mianyang, Sichuan); cDNA synthesis kits, IPTG, X-gal were pu...

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Abstract

The invention mainly relates to an oil-producing yeast expression vector which utilizes Zeocin to screen aleurites montana SAD genes, wherein, the vector takes pCAMBIA2300 as a basic framework, and the oil-producing yeast expression vector contains aleurites montana SAD genes, or contains Sh ble genes simultaneously. The construction method comprises the following steps: 1. acquiring a full-lengthsequence of a cDNA coding region of the aleurites montana SAD genes; 2. constructing an SAD sense expression vector; 3. cloning a resistant marker gene Sh ble of Zeocin; and 4. constructing the yeastexpression vector and converting agrobacterium tumfaciens. The invention has the beneficial effects that in the invention, the SAD genes separated from the total RNA of aleurites montana seeds are constructed on the yeast expression vector pCAMBIA2300, thus providing a gene transfer donor for improving the content of oleic acid in the oil-producing yeast; and in the invention, a primer is designed according to the expression vector pPICZ B of the pichia pastoris to separate the resistant gene Sh ble of Zeocin, and a new vector is constituted by connecting with the above recombinant vector, thus greatly simplifying the genetic transformation process of the oil-producing yeast and playing an important role in screening the oil-producing yeast of SAD transgenosis with Zeocin resistance.

Description

technical field [0001] The present invention belongs to the field of genetic engineering, and mainly relates to an oleaginous yeast expression vector and a construction method for screening the SAD gene of the Millennium Tung tree with Zeocin, and specifically relates to the recombination of the yeast expression vector. oleaginous yeast transformants. Background technique [0002] In response to the intensifying oil crisis, countries around the world are actively formulating and implementing new energy development strategies, hoping to find new oil substitutes through various possible channels, and biodiesel is one of them. At present, the raw materials for the production of biodiesel mainly depend on oil crops, woody oil plantations, animal fats and waste oils. Extracting oil from oil trees has a series of restrictive factors such as occupying arable land area, long cycle and high cost. The development of animal fat also faces a series of constraints. Therefore, it is of...

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Application Information

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IPC IPC(8): C12N15/81C12N15/65
Inventor 范正琪李纪元陈东亮范妙华田敏李辛雷
Owner RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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