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Primer and method for augmenting flanking sequences of mutant genes inserted into hydrogen-producing microalgae (chlamydomonas reinhardtii)

A technique for amplifying the product, Chlamydomonas reinhardtii, applied in the field of primers for amplifying the flanking sequences of the insertion mutation gene of the hydrogen-producing microalga Chlamydomonas reinhardtii, which can solve the problems of many repeated sequences, difficulty in success, single gene and phenotype difficulty, etc. Achieve the effect of strong specificity, specificity improvement, simple and fast operation

Active Publication Date: 2013-09-04
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other model photosynthetic organisms, the genome sequence of Chlamydomonas reinhardtii has the characteristics of high GC content and many repetitive sequences, so the traditional gene cloning method is not easy to succeed
Therefore, it is difficult to study the relationship between a single gene and phenotype.

Method used

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  • Primer and method for augmenting flanking sequences of mutant genes inserted into hydrogen-producing microalgae (chlamydomonas reinhardtii)
  • Primer and method for augmenting flanking sequences of mutant genes inserted into hydrogen-producing microalgae (chlamydomonas reinhardtii)
  • Primer and method for augmenting flanking sequences of mutant genes inserted into hydrogen-producing microalgae (chlamydomonas reinhardtii)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the acquisition of various mutants

[0042] All kinds of mutants were obtained by random insertion method of algae CC400 using PSI103 (Kindle, Journal of Applied Phycology, Volume 6, Number 2, 231-238, 1990). The specific steps are as follows:

[0043] (1) Digest the PSI103 plasmid with restriction endonuclease KpnI to obtain a linearized plasmid;

[0044] (2) Transform the linearized plasmid into Chlamydomyces reinhardtii species CC400 (mt-) by the glass bead method, screen in TAP medium containing paromomycin, and obtain some mutants resistant to paromomycin, namely for the mutant library.

[0045] (3) The mutant strains with Fv / Fm less than 0.5 were screened in the mutant library by IMAGING-PAM, and 6 mutant strains were selected as the experimental materials of Example 3.

[0046] F0: initial fluorescence, initial fluorescence is the fluorescence output when the reaction center of photosystem II (PS II) is fully open, and it is related to the concent...

Embodiment 2

[0047] Embodiment 2, TAIL-PCR primer design

[0048] Specific primers Sp0 and Sp1 were designed at the 3' sequence end of the paromomycin gene, and a degenerate primer LAD-5-RMD227 targeting Chlamydomonas reinhardtii was designed. The NotI cleavage site was removed from the degenerate primer to reduce the cost of primer synthesis.

[0049] Each primer shown in Table 1 was synthesized. Among them, LAD1, LAD2, LAD3, LAD4 and LAD-5-RMD227 are degenerate primers.

[0050] Table 1 The sequences of primers used in PCR amplification

[0051] Primer name

[0052] V represents A, C or G; N represents A, G, C or T; B represents C, G or T; W represents A or T; S represents C or G;

Embodiment 3

[0053] Embodiment 3, application of the primers of Embodiment 2 to detect the flanking sequence of the insertion site in Chlamydomonas genomic DNA

[0054] 1. Genomic DNA extraction

[0055] The total DNA of Chlamydomonas reinhardtii species CC400(mt-) and the 6 mutants obtained in Example 1 were extracted using Tiangen Biochemical Technology (Beijing) Co., Ltd. Plant Total DNA Extraction Kit referring to the instructions of the kit.

[0056] Specific extraction steps: Take 1ml of algae liquid in a 1.5ml eppendorf tube, 7000rpm for 1 minute, remove the supernatant; add 700ul of GP1 solution containing 0.1% mercaptoethanol, 65°C for 20 minutes, during the period upside down several times; then add 700ul of chloroform, 12000rpm for 5 minutes, carefully transfer the obtained supernatant water phase to a new 1.5ml eppendorf tube, mix thoroughly; transfer the mixed liquid to the adsorption column CB3, 12000rpm for 30 seconds, discard the waste liquid, and put the adsorption column ...

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Abstract

The invention discloses a primer and method for augmenting flanking sequences of mutant genes inserted into hydrogen-producing microalgae (chlamydomonas reinhardtii). The invention provides a primer composition formed by DNA shown by a sequence 1 and a sequence 2 in a sequence table, degenerate primers and DNA shown in a sequence 8 in the sequence table; the degenerate primers are used for genome DNA of the chlamydomonas reinhardtii. By adopting the primer composition provided by the invention, insertion sites of paromomycin coding genes in the genome DNA of the chlamydomonas reinhardtii mutant can be obtained. The method provided by the invention has the advantages that the designed two specific primers are combined with the degenerate primers used by a TAIL-PCR (thermal asymmetric interlaced-polymerase chain reaction), and under the optimized PCR reaction condition, the high-efficiency and specific augmentation of the flanking sequences of the inserted the mutant genes can be achieved. The invention has an important significance in cloning the chlamydomonas reinhardtii gene in a high-flux manner and researching the gene functions.

Description

technical field [0001] The invention relates to a primer and a method for amplifying the flanking sequence of the insertion mutation gene of the hydrogen-producing microalga Chlamydomonas reinhardtii. Background technique [0002] Chlamydomonas reinhardtii is a model organism used to study the mechanism of photosynthesis for more than 50 years. The acquisition of the whole genome sequence information in 2007 provides a high-throughput molecular operation platform for studying the characteristics of various life activities and molecular regulation mechanisms of Chlamydomonas reinhardtii from the genome level. [0003] On the basis of the whole genome sequence information, how to determine the function of each gene and study the relationship between gene and phenotype is the main problem at present. Compared with other model photosynthetic organisms, the genome sequence of Chlamydomonas reinhardtii has the characteristics of high GC content and many repetitive sequences, so t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 黄芳赵磊杨浩萌陈梅
Owner INST OF BOTANY CHINESE ACAD OF SCI