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RNAi vector of chlamydomonas reinhardtii-related gene and construction method and application of RNAi vector

A rhine coat and gene technology, applied in the field of RNAi vectors, can solve the problems of unsatisfactory screening effect of positive clones and cumbersome construction steps, etc.

Active Publication Date: 2015-03-04
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is already a corresponding RNAi vector in Chlamydomonas, but its construction steps are relatively cumbersome, requiring multiple digestions and ligations to successfully construct, and the screening effect of positive clones after transformation is not ideal

Method used

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  • RNAi vector of chlamydomonas reinhardtii-related gene and construction method and application of RNAi vector
  • RNAi vector of chlamydomonas reinhardtii-related gene and construction method and application of RNAi vector
  • RNAi vector of chlamydomonas reinhardtii-related gene and construction method and application of RNAi vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] The construction of the RNAi carrier of embodiment 1 Chlamydomonas reinhardtii BBS1 gene

[0087] 1. Extraction of Chlamydomonas reinhardtii DNA

[0088] Under sterile conditions, take 5ml of liquid-cultured Chlamydomonas reinhardtii sample and centrifuge at 4000g for 5min, discard the supernatant. Add 600 μl of CTAB buffer, mix well, and incubate in a water bath at 65° C. for 1 hour. Add 10 μl RNAase and place at 37°C for 30min. Add an equal volume of chloroform and fully extract for several minutes. Centrifuge at 10000g for 5min, and transfer the supernatant to a new centrifuge tube. Add an equal volume of isopropanol to precipitate DNA, and place at -20°C for 10-30min. Centrifuge at 10000g for 5min and discard the supernatant. Add 1 ml of 75% ethanol to wash the precipitated DNA twice. Discard ethanol, dry DNA, add 50 μl ddH 2 O dissolves DNA.

[0089] 2. Selection and acquisition of spacer sequences

[0090] According to the published phosphoribulokinase se...

Embodiment 2

[0120] The method of this embodiment and embodiment 1 is basically the same, the difference is;

[0121] 10. Obtaining a partial cDNA fragment at another position of the BBS1 gene

[0122] Extract the total RNA of Chlamydomonas reinhardtii, reverse transcribe, and use its cDNA as a template to design a pair of primers:

[0123]

[0124] The above primers were amplified by PCR, and the PCR products were respectively connected to the pEASY-T1 vector and sequenced to form the pEASY-T1-BBS2.1 intermediate vector to obtain a cDNA fragment at another position of the BBS1 gene.

[0125] 11. Construction of pChlamy_RNAi_BBS2.1_F RNAi vector containing the forward target fragment of BBS1 gene

[0126] Digest the pEASY-T1-BBS2.1 intermediate vector plasmid containing the forward target fragment of the BBS1 gene, and connect it to the pChlamy_RNAi backbone vector that is also passed through Kpn I and Spe I to obtain the pChlamy_RNAi_BBS2.1_F RNAi vector containing the forward target ...

Embodiment 3

[0129] The construction of the RNAi carrier of embodiment 3 Chlamydomonas reinhardtii BBS4 gene

[0130] The method of this embodiment and embodiment 1 is basically the same, the difference is;

[0131] 10. Acquisition of partial cDNA fragment of BBS4 gene

[0132] Extract the total RNA of Chlamydomonas reinhardtii, reverse transcribe, and use its cDNA as a template to design a pair of primers:

[0133]

[0134] The above primers were used for PCR amplification, and the PCR products were respectively connected to the pEASY-T1 vector and sequenced to form the pEASY-T1-BBS4 intermediate vector to obtain the cDNA fragment of the BBS4 gene.

[0135] 11. Construction of pChlamy_RNAi_BBS4F RNAi vector containing the forward target fragment of BBS4 gene

[0136] Digest the pEASY-T1-BBS4 intermediate vector plasmid containing the forward target fragment of the BBS4 gene, and connect it to the pChlamy_RNAi backbone vector that is also passed through Kpn I and Spe I to obtain the p...

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Abstract

The invention discloses an RNAi vector of a chlamydomonas reinhardtii-related gene and a construction method and application of the RNAi vector. The RNAi vector contains a chlamydomonas reinhardtii chimeric composition type expression promoter HSP70A / RBCS2, an intron, 3'UTR and a hygromycin screening marker expression frame sequence, wherein the nucleotide sequence of the chlamydomonas reinhardtii chimeric composition type expression promoter HSP70A / RBCS2 is as shown in SEQ ID No. 1, the nucleotide sequence of the 3'UTR is as shown in SEQ ID No. 2, and the nucleotide sequence of the hygromycin screening marker expression frame sequence is as shown in SEQ ID No. 3. Partial cDNA fragments of a target gene are firstly constructed onto an intermediate vector pEASY-T1 by using a PCR technology, and then forward and reverse fragments of the target gene are successively connected onto a chlamydomonas reinhardtii RNAi skeleton vector by performing enzyme digestion twice, so that the construction steps of the vector are simplified, the construction time of the vector is shortened, the efficiency is improved; and the RNAi vector can be directly applied to the aspect of researching functions of other genes of chlamydomonas.

Description

technical field [0001] The invention relates to the field of RNAi vectors, in particular to an RNAi vector of genes related to Chlamydomonas reinhardtii and its construction method and application. Background technique [0002] Chlamydomonas reinhardtii belongs to the Chlorophyta, Volvox, and Chlamydomonaceae. It is a eukaryotic single-celled organism with a relatively ancient evolution, between higher plants and animals. Chlamydomonas reinhardtii has simple culture conditions, short growth cycle, high photosynthetic efficiency, and clear genetic background. It has many common characteristics with yeast cells and is known as "green yeast". Its biological characteristics are closely related to higher plants and animals, and it is currently the most widely used model organism for studying cilia. [0003] In recent years, most of our knowledge about cilia comes from research on Chlamydomonas reinhardtii, such as: our understanding of cilia structure, the discovery of the mecha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/66C12Q1/68
Inventor 陈利红李丽丽高利芬周俊飞李甜甜彭海张继方治伟章伟雄
Owner JIANGHAN UNIVERSITY