Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters

Abstract

In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family. Three promoters MeEF1A3, MeEF1A4 and MeEF1A5 were successfully isolated. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5′UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants.

Cowpea and abiotic stresses: identification of reference genes for transcriptional profiling by qPCR

“…Considering its fundamental role in the protein biosynthesis [ 37 ], the housekeeping gene EF1 - α has been used for normalization of qPCR data in some crop species, such as Vigna mungo [ 38 ], coffee [ 39 ] and potato [ 40 ], during salt stress. In the legume crop Caragana intermedia under osmotic, salt, cold and heat stress, the EF1 - α gene showed to be stable using geNorm, NormFinder, and BestKeeper algorithms [ 41 ].…”

Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa1 in rice

“…Therefore, OsLpa1.2 and OsLpa1.3 or their encoded proteins may function as competitors of OsLpa1.1 in transcription as genes involved in stress response (Staiger and Brown, 2013) or in catalysis through formation of homo-and hetero-dimer proteins. In addition, because the 5' untranslated regions (5' UTRs) could be a part of a promoter Suhandono et al, 2014), further studies are needed to reveal the spatiotemporal expression patterns of OsLpa1.2 and OsLpa1.3.…”

Isolation and characterization of “GmScream” promoters that regulate highly expressing soybean (Glycine max Merr.) genes

“…6B). promoters was constitutively expressed using transient expression in tobacco seedlings [62]. In 281 this study, we did not isolate and characterize all the soybean eEF1A promoters as only three 282 were selected for constitutive, high expression according to the criteria applied to the RNA-seq 283 analysis.…”

“…[65, 66] and cassava [62]. Parts of the Arabidopsis thaliana eEF1A promoter, along with its 286 native 5'UTR intron [56] have been used in the production of commercial Roundup-resistant 287 crops [Roundup Ready 2 Yield, 67] although the reason for its use was not specified.…”