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Method for constructing Dunaliella salina chloroplast transformation vector

A Dunaliella salina and construction method technology, applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of complex background, complicated operation steps, and low efficiency of exogenous gene expression, and achieve fast growth rate and convenient Homogenization, to achieve the effect of efficient expression

Inactive Publication Date: 2011-07-13
ZHENGZHOU UNIV
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Problems solved by technology

[0002] Although the traditional plant genetic engineering using the nucleus as the receptor of exogenous genes has matured and been widely used, with the deepening of research, people have gradually realized that there are a series of difficult problems in the genetic transformation of the nucleus genome: For example, due to the large nuclear genome and complex background, it is difficult to artificially control the integration site and integrated copy number of exogenous genes, resulting in low expression efficiency of exogenous genes and prone to gene silencing; the operation steps are too complicated when transferring multiple genes at the same time , the expressed prokaryotic gene must be modified, and the environmental safety is difficult to guarantee
The homogenization process is a process in which the resistance of transformants gradually increases. If the selection pressure is too small, the untransformed chloroplast genome cannot be removed; if the selection pressure is too high, it may be difficult to complete the homogenization process of the transformed chloroplast genome due to the rapid death of the host.

Method used

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  • Method for constructing Dunaliella salina chloroplast transformation vector
  • Method for constructing Dunaliella salina chloroplast transformation vector

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1. Cloning and vector construction of Dunaliella salina chloroplast light-independent protochlorophyllide reductase gene. The chlN gene is used as an example here, and the cloning and vector construction of homologous fragments of chlL and chlB genes are similar.

[0027] 1. Cultivation of Dunaliella salina

[0028] The medium formula is as follows: NaCl 58.5 g / L, MgCl 2 ·6H 2 O 15g / L, MgSO 4 ·7H 2 O 0.5 g / L, KCl 0.2 g / L, CaCl 2 0.151g / L, KNO 3 0.5 g / L, NaHCO 3 0.043 g / L, KH 2 PO 4 0.035 g / L, iron salt solution 10 mL / L, trace element solution 10 ml / L.

[0029] Iron salt solution formula: Na 2 EDTA·2H 2 O 209 mg / L, FeCl 3 ·H 2 O 244 mg / L. Trace element solution formula: H 3 BO 3 61 mg / L, (NH 4 ) 6 Mo 7 o 24 4H 2 O 38 mg / L, CuSO 4 ·5H 2 O 6 mg / L, CoC1 2 ·H 2 O 5.1 mg / L, ZnCl 2 4.1 mg / L, MnCl 2 ·H 2 O 4.1 mg / L, adjust the pH value to 7.5 with HCl, and sterilize at 0.11 MPa for 20 min.

[0030] The algae liquid was inoculated at a c...

Embodiment 2

[0047] Example 2. Cloning and vector construction of chloroplast promoter and terminator elements. Here, specific steps are illustrated by taking Chlamydomonas reinhardtii chloroplast atpA promoter and rbcL terminator elements as examples.

[0048] 1. Cultivation of Chlamydomonas reinhardtii

[0049] Chlamydomonas reinhardtii was grown in TAP (Tris-acetate-phosphate) liquid medium at 20-25°C with a light intensity of 3000 Lux and a light cycle of 12 h light / 12 h dark.

[0050] The formula of TAP liquid medium is as follows:

[0051] Tris base 2.42 g, Beijinercke buffer 5 ml, phosphate buffer 5 ml, trace element solution 1 ml, glacial acetic acid 1 ml, adjust the pH value to 6.8-7.3, add water to 1000 ml. 0.11 MPa sterilize for 20 min and set aside .

[0052] Beijinercke buffer recipe:

[0053] NH 4 Cl lO g ,MgSO 4 ·7H 2 O 4 g, CaCl 2 2H 2 0 2 g, add water to 1000 ml.

[0054]Phosphate Buffer Recipe:

[0055] K 2 HP0 4 ·3H 2 0 373 g, KH 2 P0 4 144 g, add water t...

Embodiment 3

[0074] Example 3. Cloning and Vector Construction of Double Selectable Marker Gene Sequence

[0075] 1. Chloramphenicol resistance selection marker gene chloramphenicol acetyltransferase (CAT) gene cloning

[0076] The chloramphenicol resistance selectable marker gene chloramphenicol acetyltransferase (CAT) gene was cloned on the plasmid vector pCAT3-Control (Promega, GenBank accession number: U57025.2), and the primers were designed as follows:

[0077] CAT1: 5'-GAATCCCGGGATGGAGAAAAAAATCACTGGA-3';

[0078] CAT2: 5′-ATAAGGATCCTTACGCCCCGCCCTGCCACTC-3′

[0079] Using the plasmid vector pCAT3-Control as a template, the CAT sequence was amplified by PCR; the PCR reaction conditions were 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s, 25 cycles; the product was cloned into the pMD18-T vector, and the enzyme was selected Correctly identified clones were sequenced.

[0080] CAT gene sequence:

[0081] ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTC...

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Abstract

The invention discloses a method for constructing a Dunaliella salina chloroplast transformation vector, in particular a method for constructing a dual-exchange dual-selection marker Dunaliella salina chloroplast transformation vector by using a light dependence free protochlorophyllide reductase chlN, chlB or chlL gene as a homological segment, chloramphnicol acetyltransferase (CAT) gene and a resistance gene bar of phosphinothricin (PPT) as a weedicide as screening markers and an atpA promoter and a rbcL terminator as expression elements. The Dunaliella salina is transformed through a gene gun method or electrization method and then subjected to two-step screening to realize homogenization, fixed point integration of an exogenous gene in a Dunaliella salina chloroplast genome is ensured, and finally the chloroplast transformation Dunaliella salina strain for stably expressing the exogenous gene is obtained. In the invention, the chlN, chlB or chlL gene is selected as the homological segment and can be used as an auxiliary screening marker of a transformant; and the chlorampenicol resistant CAT gene and the resistance gene bar of the PPT are selected to be used as screening marker genes together, thus the stable transformation strain can be obtained within shorter time.

Description

technical field [0001] The present invention relates to microalgae genetic engineering, specifically a method for constructing a Dunaliella salina chloroplast transformation carrier, by constructing a Dunaliella salina (hereinafter referred to as salina) chloroplast transformation carrier, transforming the salina, and then performing homogenization screening, and finally Realize the transformation of salina chloroplast. Background technique [0002] Although the traditional plant genetic engineering using the nucleus as the receptor of exogenous genes has matured and been widely used, with the deepening of research, people have gradually realized that there are a series of difficult problems in the genetic transformation of the nucleus genome: For example, due to the large nuclear genome and complex background, it is difficult to artificially control the integration site and integrated copy number of exogenous genes, resulting in low expression efficiency of exogenous genes ...

Claims

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Application Information

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IPC IPC(8): C12N15/65
Inventor 薛乐勋潘卫东侯桂琴李杰曲东京贾岩龙
Owner ZHENGZHOU UNIV
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