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

Resumen. En plantas, la deshidrogenasa NADH de la mitocondria (complejo I) es un gran complejo de proteínas que transfiere electrones a la ubiquinona. Para los nueve genes mitocondriales que codifican las subunidades del complejo I (nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, y nad9), es importante una estrategia eficiente para el clonado de secuencias completas de codificación (CDSs). Se observaron porciones ortólogas específicas de regiones no traducidas (UTRs) basado en alineamientos de secuencias múltiples de ortólogos de genes mitocondriales que codifican subunidades del complejo I en las especies vegetales. Basado en la conservación de UTRs parciales, se determinó una estrategia PCR de un paso para la clonación de CDSs de los nueve ortólogos de genes mitocondriales. Usando esta estrategia, las cinco estructuras de lectura abierta mitocondriales (ORFs) , que codifican subunidades de la NADH deshidrogenasa mitochondrial (i.e., nad1, nad2, nad6, nad7, y nad9), se clonaron en tres especies de angiospermas: Hibiscus cannabinus, Cinnamomum camphora, y Boehmeria nivea. Los quince productos PCR clonados también incluyeron las secuencias parciales 5' y 3'-UTR. Más aún, se identificó un sitio de edición potencial C-U RNA en el codón de iniciación del nad9 de Hibiscus cannabinus. En conclusión, la estrategia simple y eficiente evita el uso del proceso de amplificación rápida de las terminaciones de cDNA (RACE) que consume mucho tiempo, y facilita la clonación del ORFs mitochondrial completo cuyos lados 5' y 3' del UTR contienen una region ortóloga con alguna degeneración en las especies de plantas superiores.

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.
RNA editing at the transcripts of nad1, nad2, nad5 and nad7 is essential for mitochondrion biogenesis, cell division, and plant growth (Kühn et al., 2011;Sosso et al., 2012;Li et al., 2014).To identify RNA editing sites in coding regions of the chondriogenes, suitable and efficient strategies are necessary to clone complete coding sequences (CDSs).To acquire complete open reading frames including start and stop codens, chondriogenes can be traditionally cloned by homologous amplification for internal fragments and rapid amplification of cDNA ends (RACE) technology or Genome Walking for 3' and 5' end unknown sequences (Liu et al., 2012).However, these procedures are complicated and laborious.In addition, the cloned sequences of chondriogenes did not convey complete CDS information when upstream and downstream primers cross at the start and stop codons, respectively.Therefore, the potential RNA editing sites could not be identified in primer regions.
Recently, high-throughput sequencing technology provides an efficient method to obtain the complete sequence of plant mitochondrial DNA.Several studies have shown that higher plant mtDNA is highly conserved in the coding sequences.Non-coding regions of plant mitochondrial genomes are extraordinarily dynamic with respect to DNA recombination (Handa, 2003).In the present study, orthologous UTR portions were found based on multiple sequence alignments of nine orthologous chondriogenes encoding complex I subunit.According to these results, a universal and efficient strategy based on the conserved UTR portion was presented to clone the complete coding sequences of chondriogenes encoding NADH dehydrogenase subunits in plant species by one-step cloning.

MATERIALS AND METHODS
Plants material.The kenaf cultivar 'P3' was grown in an experimental field at Guangxi Academy of Agricultural Sciences, Nanning, China (108° 14' E, 22° 50' N).The mature camphortree and ramie plants were maintained under natural conditions at the Guangxi Subtropical Crops Research Insti-tute, Nanning, China (108° 20' E, 22° 53' N).According to the APGIII system, a distance tree showed distant phylogenetic relationships between the three species and other plant species with sequenced mitochondrial genome.
Homology analysis of untranslated regions.The mitochondrial genome sequences of 20 species (Table 1) were obtained from the GenBank database.The twenty angiosperm species included both monocots and eudicots referencing the APGIII system.Based on the annotations of their mitochondrial genomes, the 3' and 5'-UTR in orthologous chondriogenes were analyzed by multiple alignment.A 200-bp sequence upstream of the start codon and downstream of the stop codon was referred as the 'putative UTR portion'.DNA-MAN software (Lynnon Corporation, USA) was used to obtain the results of multiple alignment of the nine orthologous chondriogenes encoding complex I subunits.Primer design for target genes.Based on multiple alignment, degenerate primer pairs were designed in conserved UTR regions of the five orthologous gene (nad1, nad2, nad5, nad7, and nad9) for the cloning of complete CDSs (Table 2).Primers of the target genes for RT-PCR were designed and analyzed using Primer Premier V5.0.

Facilitation of cloning of complete coding sequences of chondriogenes
Total RNA extraction and molecular cloning.Total RNA was extracted from the young leaf tissues of three plant species metioned above (Chang et al., 1993;Shi et al., 2016) and reverse transcribed with random hexamers using M-MLV transcriptase (Promega, USA).Taq DNA polymerase (Biomiga, USA), which is capable of adding a single 3'-A overhang to PCR products, was used for PCR amplification with cDNA templates and degenerate primer pairs specific to the target chondriogenes (nad1, nad2, nad5, nad7, and nad9) in a Tprofessional-96 thermal cycler (Biometra, Germany).PCR products were analysed on 1.2% agarose gels and the single target fragment was recovered and purified using a gel extraction kit (Biomiga, USA).The purified PCR products were subcloned into pUCm-T vector, sequenced, and assembled using DNAstar SeqMan software (Madison, WI, USA).

RESULTS
Sequence analysis of chondriogene orthologs.UTR orthologues of key chondriogenes in higher plants were analysed.According to the results of multiple sequence alignments (data not shown), orthologues of several chondriogenes in angiosperms showed evolutionary homology in partial 5' and 3'-UTRs.In addition to chondriogenes encoding complex I, some other genes showed evolutionarily conserved 5' or 3'-UTRs (Table 3).
The application of cloning strategy.The overall cloning strategy was illustrated with kenaf nad9 as an example (Fig. 1 and 2).The five mitochondrial genes (nad1, nad2, nad6, nad7, and nad9) of kenaf, camphor tree, and ramie, which encode corresponding NADH dehydrogenase subunits, were successfully cloned by using this strategy.Both nad1 and nad7 genes included introns, suggesting that this strategy could clone chondriogenes with introns.In higher plants, the sequences encoding complex I subunits show evolutionary conservation  Tabla 3. Ortología de varios genes mitocondriales en angiospermas.

Gene names 5'-UTR
The table includes the chondriogene of orthologous UTR ≥ 30 bp, with the sequence identity greater than or equal to 90%.√ indicate orthology, × indicate non-orthology.
Table 4. GenBank accession number of the fifteen cloned chondriogenes.

Plant species
Gene name GenBank no.Jin G et al., FYTON 86 (2017) in partial UTR, suggesting a crucial role in transcription.All of the 15 cloned genes were submitted to NCBI (Table 4).Notably, a potential C-U RNA editing site was found in the start codon of kenaf nad9 (Fig. 1 and 2).
The nucleotide sequences exhibited a high degree of similarity to the corresponding orthologues (data not shown).According to the APGIII system, ramie is a member of the Rosales order, and kenaf is a member of the Malvales order.Both Rosales and Malvales are ascribed to the eurosids I branch.However, the camphor tree is a member of the magnoliids, which have a distant phylogenetic relationship with Rosales and Malvales.These results suggest that the conservation of partial UTR in the cloned chondriogenes is a common phenomenon in angiosperm species, and the designed degenerate primers could be used for cloning complete ORFs of the corresponding genes in angiosperms.

DISCUSSION
Selective evolutionary pressure could result in the conservation of certain nucleotide sequences that may affect gene expression (Hardison, 2000;Mrowka et al., 2003).It was found that the 5'-UTRs of the orf25, atp6, and coxII mRNAs contain three conserved sequence blocks compared to plant mitochondrial mRNAs from both monocot and dicot species (Pring et al., 1992).The conservation of these non-coding regions may be under evolutionary selective pressure to remain relatively unchanged because it binds to transcription factor or miRNA (Binder et al., 1996;Toscano-Garibay & Aquino-Jarquin, 2012).
The results from the present study showed conserved 3'and 5'-UTR regions of the cloned chondriogenes in the molecular evolution of angiosperms.Based on the conservation of untranslated regions in chondriogenes, it is concluded that the orthologous UTR regions could play an essential role in the regulation of the expression of chondriogenes encoding complex I. Furthermore, a universal and efficient strategy was developed to clone complete coding sequences of chondriogenes encoding complex I by one-step homologous cloning.The cloning strategy will be useful for cloning mitochondrial complete ORFs whose 5' and 3' flanking UTR contain an orthologous region with some degeneracy among angiosperms.The feasibility of the strategy presented in the present study was verified by cloning of nad1, nad2, nad6, nad7, and nad9 genes in kenaf, ramie, and camphor trees.Orthologous UTRs in higher plant chondriogenes could be utilized as a new molecular marker system for the analysis of genetic diversity of maternal inheritance.
However, not all UTRs of chondriogenes remain conserved.For example, the UTR portion of orfB and cox1 in rice (Oryza sativa) show faster evolutionary speeds (data not shown), perhaps due to less evolutionary pressure.Despite some insufficiencies, the cloning strategy reported here could increase the efficiency of cloning chondriogenes encoding complex I subunits in plants, and help identify RNA editing sites in coding regions of mitochondrial complex I subunit genes.The strategy could also be applied to cloning chondriogenes encoding complex I in animals.

CONCLUSION
In angiosperm species, comparison of partial 3' and 5'-UTR regions in orthologous chondriogenes reveals some conserved aspects of the structure of UTRs.The conservative property facilitates the cloning of complete coding sequences of chondriogenes encoding NADH dehydrogenase subunits.

Fig. 1 .
Fig. 1.Illustrated map of cloning chondriogenes encoding complex I subunits, using nad9 from Hibiscus cannabinus as an example.Note: The two boxes indicate the primer sequences; ATG and TAA are underlined.A potential C-U RNA editing site was identified in the start codon.

Facilitation
of cloning of complete coding sequences of chondriogenes

Table 1 .
Plant species analyzed in this study and accession data.Especies vegetales analizadas en este estudio y datos de las variedades.

Table 3 .
Orthology of several chondriogenes in angiosperm plant.