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Preparation method of T vector capable of cloning microalgae promoter and application thereof

A promoter and vector technology, applied in biochemical equipment and methods, microbial assay/inspection, introduction of foreign genetic material using vectors, etc., can solve problems such as hindering research work, impossibility of promoter regulation function, research, etc.

Inactive Publication Date: 2012-04-04
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) At present, there are no commercial microalgae cloning vectors, expression vectors or reporter vectors, which greatly hinders the development of research work;
[0005] (2) Many microalgae, including Haematococcus pluvialis, have not yet established a genetic transformation system, and it is impossible to study their functional genes through genetic engineering, and it is impossible to study the regulatory function of their promoters;

Method used

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  • Preparation method of T vector capable of cloning microalgae promoter and application thereof
  • Preparation method of T vector capable of cloning microalgae promoter and application thereof
  • Preparation method of T vector capable of cloning microalgae promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Construction of Microalgae Promoter Cloning T Vector

[0064] (1) Using pBle1 / pBle2 (pBle1: 5'-GTAGATATCATGGCCAGGTG-3', pBle2: 5'-GCGGGTACCTTAATTAAGCTTC-3') as primers, by PCR from plasmid pSP124 (preserved in our laboratory, purchased from American Algae Bank) The ble-3'RBCS2 terminator with a size of 970bp was amplified (94°C for 3min; 94°C for 1min, 58°C for 1min, 72°C for 1min, 25 cycles; 72°C for 10min), and cloned into pMD 18-T (TaKaRa Company) In the vector, the T-p124 plasmid was obtained and confirmed by sequencing. The ble-3'RBCS2 terminator was obtained by EcoRⅤ+KpnI double digestion of plasmid T-p124, which was inserted into the EcoRⅤ+KpnI site of the pSP124 vector, and finally the pB124 vector was obtained.

[0065] (2) Construction of pB vector: the pB124 vector was single-digested with Eam1105I to obtain a linear vector, and then the end of the vector was smoothed by T4DNAPolymerase, and the original Eam1105I site was mutated. The vector was ...

Embodiment 2

[0070] Example 2: Cloning of the promoter of the carotene ketolase gene from Haematococcus pluvialis

[0071] (1) Preparation of T carrier

[0072] The pB-0.45T and pRB-0.45T vectors were double digested with Eam1105I, separated by 1% agarose (mass volume ratio) gel electrophoresis, and large fragments were obtained, using the EZ-10 Spin Column DNA Gel Extraction Kit kit (TaKaRa Company ) reclaim the DNA fragments of the agarose gel, and the obtained DNA fragments can be used as the T carrier of T / A cloning;

[0073] (2) Insertion of the microalgae promoter fragment

[0074] Using pcrto5 and pcrto6 (pcrto5: 5′-CAGCTAGATGACTGCCTCAC-3′ pcrto6: 5′-CGTCTAGCTGTGCTATGGC-3′) as primers, a product with a size of 200 bp was amplified from the 091127-a3 plasmid by PCR (94°C 3min; 94°C 1min , 55°C for 1min, 72°C for 50sec, 5 cycles; 94°C for 1min, 58°C for 1min, 72°C for 50sec, 30 cycles; 72°C for 10min), the 5′bkt1(-200 / -1) promoter was obtained and named 5'bkt1-0.2.

[0075] T vect...

Embodiment 3

[0076] Example 3: Analysis of the promoter function of the carotene ketolase gene of Haematococcus pluvialis

[0077] (1) Genetic transformation of foreign genes

[0078] The genetic transformation of Chlamydomonas reinhardtii CC-849 (preserved in our laboratory and purchased from the U.S. Algae Bank) was carried out using the bead milling method. The specific steps are as follows: (1) CC-849 was placed under continuous light (about 90 μE / m 2 / s), cultured in TAP medium (self-prepared, see Table 1) to the logarithmic phase, the number of cells is about 1~2×10 6 Cells / mL can be fixed with Lugol's solution, and then counted by hemocytometer; (2) 5000rpm, 5min, 23°C centrifuge to collect CC-849; (3) Remove the supernatant and resuspend the pellet with sterilized TAP (not too violent), adjust the cell concentration to 2×10 8 cells / mL; (4) Pipette 300 μL of the suspension into a 1.5 mL test tube, which is pre-installed with 0.3 g of sterilized glass beads with a diameter of 0.4 m...

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Abstract

The invention discloses a preparation method of a T vector capable of cloning a microalgae promoter and application thereof. The preparation method comprises the following steps of: obtaining a spacer sequence by undergoing a PCR (polymerase chain reaction), and introducing a restriction enzyme recognition site; connecting the spacer sequence to a pSP124 vector with a mutated Eam1105I site, and constructing a pre-T vector respectively; introducing a ble gene to be served as a screening gene; performing enzyme digestion, electrophoretic separation and rubber cutting recovering to obtain a T vector, connecting a microalgae promoter fragment to an Eam1105I enzyme digestion site; and connecting the microalgae promoter to obtain a complete expression cassette, wherein the expression cassette consists of a promoter, an expression gene and a terminator. The invention further discloses application of the T vector capable of cloning the microalgae promoter to detecting the function of the microalgae promoter. The method is easy, and the technology is mature and reliable. The microalgae promoter is cloned rapidly by using the vector, and the function of the promoter is verified in chlamydomonas reinhardtii; and the T vector becomes a beneficial tool for research personnel of microalgae gene engineering.

Description

technical field [0001] The present invention relates to the field of microalgae genetic engineering, more specifically to a method for constructing a microalgae promoter cloning T vector, and also relates to a use of a microalgae promoter cloning T vector, which can be used for direct cloning of microalgae promoters and The function of the promoter was analyzed in Chlamydomonas reinhardtii, a method that allows rapid cloning and functional analysis of microalgal promoters. Background technique [0002] Promoter is a ubiquitous cis-acting element in gene regulation. It is a DNA sequence that RNA polymerase can recognize and bind to to initiate gene transcription. It is usually located upstream of the 5' end of the gene. It can determine the direction and efficiency of transcription, as well as the type of RNA polymerase, and guide the correct combination of RNA polymerase and template. Therefore, the functional sequence of the promoter is of great significance for the study ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12Q1/68G01N33/53
Inventor 王潮岗胡章立
Owner SHENZHEN UNIV
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