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Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression
Author(s) -
LópezPaz Cristina,
Liu Dianyi,
Geng Sa,
Umen James G.
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13731
Subject(s) - chlamydomonas reinhardtii , biology , transgene , chlamydomonas , selectable marker , genetics , gene , expression cassette , terminator (solar) , reporter gene , promoter , computational biology , gene expression , vector (molecular biology) , recombinant dna , ionosphere , physics , astronomy , mutant
Summary Chlamydomonas reinhardtii is a unicellular green alga that has attracted interest due to its potential biotechnological applications, and as a model for algal biofuel and energy metabolism. Despite all the advantages that this unicellular alga offers, poor and inconsistent expression of nuclear transgenes remains an obstacle for basic and applied research. We used a data‐mining strategy to identify highly expressed genes in Chlamydomonas whose flanking sequences were tested for the ability to drive heterologous nuclear transgene expression. Candidates identified in this search included two ribosomal protein genes, RPL 35a and RPL 23 , and ferredoxin, FDX 1 , whose flanking regions including promoters, terminators and untranslated sequences could drive stable luciferase transgene expression to significantly higher levels than the commonly used Hsp70A‐ RBCS 2 ( AR ) hybrid promoter/terminator sequences. The RPL 23 flanking sequences were further tested using the zeocin resistance gene sh‐ble as a reporter in monocistronic and dicistronic constructs, and consistently yielded higher numbers of zeocin‐resistant transformants and higher levels of resistance than AR ‐ or PSAD ‐ based vectors. Chlamydomonas RPL 23 sequences also enabled transgene expression in Volvox carteri . Our study provides an additional benchmark for strong constitutive expression of transgenes in Chlamydomonas, and develops a general approach for identifying flanking sequences that can be used to drive transgene expression for any organism where transcriptome data are available.