Open Access

ARTICLE

Evolutionarily conserved untranslated regions facilitate the cloning of complete coding sequences of chondriogenes encoding NADH dehydrogenase subunits in higher plants

Jin G^1,2, XM Tang³, Y Niu⁴, XY Huang², T Chen², Q Huang², J Zhang², RY Zhou¹

1 College of Agriculture, Guangxi University, Nanning, Guangxi 530004, China
2 Guangxi Subtropical Crops Research Institute, Nanning, Guangxi 530001, China
3 Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China
4 Guangxi Academy of Specialty Crops, Guilin, Guangxi 541004, China
Address correspondence to: Prof. Ruiyang Zhou, ruiyangzhou@163.com. Dr. Xiangmin Tang, txm906@163.com. Mr. Qiang Huang, gxscrijg@163.com. Mr Tao Chen, 499481815@qq.com. Jin G and Tang X-M contributed equally to this work.

Phyton-International Journal of Experimental Botany 2017, 86(all), 137-142. https://doi.org/10.32604/phyton.2017.86.137

Abstract

In plants, the mitochondrial NADH dehydrogenase (complex I) is a large protein complex transferring electrons to ubiquinone. For the nine chondriogenes encoding complex I subunits (nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, and nad9), an efficient strategy for the cloning of complete coding sequences (CDSs) is important. Specific orthologous portions of untranslated regions (UTRs) were found based on multiple sequence alignments of chondriogene orthologues encoding complex I subunits in plant species. Based on the conservation of partial UTRs, a one-step PCR strategy was conceived for the cloning of CDSs of the nine chondriogene orthologues. Using this strategy, the five complete mitochondrial open reading frames (ORFs) , which encode mitochondrial NADH dehydrogenase subunits, nad1, nad2, nad6, nad7 and nad9 respectively, were cloned in three angiosperm species: kenaf (Hibiscus cannabinus), camphor tree (Cinnamomum camphora), and ramie (Boehmeria nivea). The fifteen cloned PCR products also included 5' and 3'-UTR partial sequences. Moreover, a potential C-U RNA editing site was identified in the start codon of kenaf nad9. In conclusion, the simple and efficient strategy avoids the use of time-consuming rapid amplification of cDNA ends (RACE) process, and facilitates the cloning mitochondrial complete ORFs whose 5' and 3' flanking UTR contain an orthologous region with some degeneracy in higher plant species.

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