Anthocyanin Biosynthesis Gene Analysis of Lonicera caerulea L. by RNAseq and DGE

Qingtian ZHANG Changyu LI Shutian FAN Yiming YANG Jun AI

AbstractFruit of Lonicera caerulea L. is a rich source of phenolic compounds such as phenolic acids as well as anthocyanins, proanthocyanidins and other flavonoids, which display potential healthpromoting effects. However, its genetics and genomic information is limited. Transcriptome and digital gene expression (DGE) profiles of fruit at four different development stages were compared. Related genes with anthocyanin biosynthesis were compared using realtime quantitative PCR (qRTPCR). RNASeq generated 6.26Gb clean reads, which was then de novo assembled into 45,656 UniGenes with a mean length 837 bp. A total of 7 928 unigenes were annotated into 32 known metabolic or signaling pathways in which a few primary, intermediate, and secondary metabolic pathways are directly related to fruit quality. The result of DGE and qRTPCR indicates that UFGT (comp20132_c0) may play a key role in honeysuckle coloration. This provides a reference for the study of complicated metabolism in nonmodel perennial species.

Key wordsLonicera caerulea L.; RNASeq; DGE; Transcriptome

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Received: May 3, 2018Accepted: August 16, 2018

Supported by Agricultural Science and Technology Innovation Project of China.

Qingtian ZHANG (1981-), male, P. R. China, assistant researcher, devoted to research about genetic breeding of fruit trees.

*Corresponding author. Email: aijun1005@163.com.

Lonicera caerulea L. (also known as haskap, blue honeyberry/honeysuckle) belongs to Caprifoliaceae family and comprises more than 200 species［1］. Blue honeysuckle berries are rich source of phenolic compounds such as phenolic acids as well as anthocyanins, proanthocyanidins and other flavonoids, which display potential healthpromoting effects. Chemopreventive, antimicrobial, antiadherence and antioxidant effects, among others, have been described for these compounds［2］. The anthocyanin content in honeysuckle is up to 657.10 mg/100 g［3］, and its anthocyanin composition was analyzed［4］. However, a comprehensive description of genes that expressed in L. caerulea fruit during the fruit development and maturation period remains unavailable. Based on genomewide expression profiles by sequencing, DGE is a powerful tool to identify and quantify gene expression on the whole genome level［5］.

This study has generated a large set of blue honeysuckle transcript sequences using the Illumina RNASeq method, which can be used to discover tissuespecific functions and the mechanisms of secondary metabolism. The dataset will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes［6］.

RNAseq and DGE Sequencing

Total RNA was extracted using modified CTAB method［7-8］, and RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

The eight DGE libraries prepared from the samples at four different development stages (5, 10, 15, and 20 d after flowering) were constructed using Sample Preparation Kit. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and pairedend reads were generated by Novogene company.

qRTPCR Analysis of Selected Genes

Sixteen unigenes were chosen for validation using qRTPCR. The qRTPCR was performed with the ABI 7500 Fast RealTime Detection System (Applied Biosystems) with the Ultra SYBR Mix (with ROX) (CWBIO, Beijing, China). The thermal profile for SYBR Green I RTPCR was 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 55 ℃ for 1 min. Each plate was repeated three times in independent runs for all reference and selected genes.

The reference gene (βACTIN) was used for normalization. The comparative CT method (2ΔΔCT method) was used to analyze the expression levels of the different genes.

Results and Discussion

In total, there were about 64.5 million raw reads generated. All the 45656 unigenes were annotated. Digital gene expression (DGE) method generates direct gene expression measurements, which avoids the inherent limitation of microarray analysis. Eight DGE libraries with one biological repeat corresponding to four developmental stages of blue honeysuckle were sequenced with 6.93 to 8.58 million raw tags per library (Table 1). The number of clean tags per library ranged from 6.90 to 8.52 million after filtering out the low quality reads. Of all clean tags, 94.03% to 95.03% were mapped to unigenes. 

The predicted biosynthesis pathways for the unigenes differed between the libraries, and most of the unigenes involved in these pathways were differentially expressed. In particular, we found that the unigenes predicted to be involved in anthocyanin biosynthesis significantly increased or decreased in different DGE libraries. The correlation between fruit anthocyanin content and the expression of anthocyanin biosynthetic genes has been studied in many crops, such as sweet cherry, grape and apple［9-11］. In this study, 15 key enzymeencoding candidate unigenes associated with anthocyanin biosynthesis in fruit were identified from the DEG data including PAL (3 unigenes), 4CL (1 unigene), CHS (2 unigene), CHI (1 unigene), F3H (2 unigene), DFR (3 unigenes), ANS (1 unigene) and UFGT (2 unigenes). These unigenes showed basically upregulated expression during the ripening process, particularly when the fruit turned red (stage 3 and stage 4) (Table 2). The result is consistent with our qRTPCR (Fig. 1) and previously reported results［9］. Most anthocyanin synthesis related genes in stage 4 exhibited higher expression than those in stage 3. This result indicates that the anthocyanin biosynthesis takes place rapidly during fruit coloring and ripening. Previous studies have shown that UFGT might play a regulatory role in anthocyanin biosynthesis at the transcriptional level and is a key enzyme gene in regulating anthocyanin synthesis in many plants, such as grape, malus and lychee［10-12］. This result indicates that UFGT (comp20132_c0) may play a key role in honeysuckle coloration.

It has been reported that transcription factors (TFs) potentially regulate anthocyanin biosynthesis. These TFs include R2R3MYB［13-14］, the basichelixloophelix (b HLH) and WD40 families［15-16］. The R2R3MYB TFs are known to be one of the most important gene in the activation of anthocyanin pigmentation in plants. We selected candidate transcription factors from the DEG data and through qRTPCR verification. The result reported that MYB transcription factors play a key role in regulating anthocyanin biosynthesis in blue honeysuckle.

Agricultural Biotechnology2019

Conclusions

Blue honeysuckle is an economically berry and widely known for its higher anthocyanin contents. However, the molecular mechanism on anthocyanin accumulation in blue honeysuckle is limited by insufficient genome information. On the basis of Illumina sequencing technology, we obtained 6.26 GB raw transcriptome data and generated 45 656 assembled unigenes, and 100% unigenes were annotated in seven public databases. The functional genes involved in anthocyanin biosynthesis were identified and studied, and their expression patterns were explored in the present study. This study demonstrates that the RNAseq and DGE techniques can be a rapid method to define the metabolic pathways in nonmodel perennial species. Furthermore, the transcriptome dataset information is a valuable contribution to accelerate research of gene expression, genomics, and functional genomics in blue honeysuckle.

References

［1］ NAUGEMYS D, ILINSKAIT S, DENKOVSKIJ J, et al. RAPD based study of genetic variation and relationships among Lonicera germplasm accessions［J］. Biologija, 2007 (53): 34-39.

［2］ PLEKHANOVA MN. Blue honeysuckle (Lonicera Caerulea L.) ——A new commercial berry crop for temperate climate: genetic resources and breeding［J］. Acta Hort (ISHS), 2000(538): 159-164.

［3］ YF W, XL X, L C, JW H. Total anthocyanins content in different species of Lonicera cearulea Linn. by pHdifferential spectrophotometry［J］. Food Research and Development, 2014(35): 75-78.

［4］ MYJAVCOVA R, MARHOL P, KREN V, et al. Analysis of anthocyanin pigments in Lonicera (Caerulea) extracts using chromatographic fractionation followed by microcolumn liquid chromatographymass spectrometry［J］. J Chromatogr A, 2010(1217): 7932-7941.

［5］ LI X, SUN H, PEI J, et al. De novo sequencing and comparative analysis of the blueberry transcriptome to discover putative genes related to antioxidants［J］. Gene, 2012(511): 54-61.

［6］ WEI H, CHEN X, ZONG X, et al. Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the red and yellow fruits of sweet cherry (Prunus avium L.)［J］. PLoS One, 2015(10): e0121164.

［7］ ZHAO W, XIA W, LI J, et al. Transcriptome profiling and digital gene expression analysis of Fallopia multiflora to discover putative genes involved in the biosynthesis of 2,3,5,4′tetrahydroxy stilbene2ObetaDglucoside［J］. Gene, 2014(547): 126-135.

［8］ FENG C, CHEN M, XU CJ, et al. Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNASeq［J］. BMC Genomics, 2012(13): 19.

［9］ LIU Y, SHEN X, ZHAO K, et al. Expression analysis of anthocyanin biosynthetic genes in different colored sweet cherries (Prunus avium L.) during fruit development［J］. Journal of Plant Growth Regulation, 2013(32): 901-907.

［10］ POUDEL PR, GOTOYAMAMOTO N, MOCHIOKA R, et al. Expression analysis of UDPglucose: flavonoid 3Oglucosyltransferase (UFGT) gene in an interspecific hybrid grape between Vitis ficifolia var. ganebu and Vitis vinifera cv. Muscat of Alexandria［J］. Plant Biotechnology Reports, 2008(2): 233-238.

［11］ LIU Y, CHE F, WANG L, et al. Fruit coloration and anthocyanin biosynthesis after bag removal in nonred and red apples (Malus xdomestica Borkh.)［J］. Molecules, 2013(18): 1549-1563.

［12］ ZHAO S, TUAN PA, LI X, et al. Identification of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation by transcriptome analysis of Lycium chinense［J］. BMC Genomics, 2013(14): 802.

［13］ LAITINEN RA, AINASOJA M, BROHOLM SK, et al. Identification of target genes for a MYBtype anthocyanin regulator in Gerbera hybrida［J］. J Exp Bot, 2008(59): 3691-3703.

［14］ FENG S, WANG Y, YANG S, et al. Anthocyanin biosynthesis in pears is regulated by a R2R3MYB transcription factor PyMYB10［J］. Planta, 2010(232): 245-255.

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