Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response

Abstract

The seed is the pharmaceutical and breeding organ of Cassia obtusifolia, a well-known medical herb containing aurantio-obtusin (a kind of anthraquinone), food, and landscape. In order to understand the molecular mechanism of the biosynthesis of aurantio-obtusin, seed formation and development, and stress response of C. obtusifolia, it is necessary to understand the genomics information. Although previous seed transcriptome of C. obtusifolia has been carried out by short-read next-generation sequencing (NGS) technology, the vast majority of the resulting unigenes did not represent full-length cDNA sequences and supply enough gene expression profile information of the various organs or tissues. In this study, fifteen cDNA libraries, which were constructed from the seed, root, stem, leaf, and flower (three repetitions with each organ) of C. obtusifolia, were sequenced using hybrid approach combining single-molecule real-time (SMRT) and NGS platform. More than 4,315,774 long reads with 9.66 Gb sequencing data and 361,427,021 short reads with 108.13 Gb sequencing data were generated by SMRT and NGS platform, respectively. 67,222 consensus isoforms were clustered from the reads and 81.73% (61,016) of which were longer than 1000 bp. Furthermore, the 67,222 consensus isoforms represented 58,106 nonredundant transcripts, 98.25% (57,092) of which were annotated and 25,573 of which were assigned to specific metabolic pathways by KEGG. CoDXS and CoDXR genes were directly used for functional characterization to validate the accuracy of sequences obtained from transcriptome. A total of 658 seed-specific transcripts indicated their special roles in physiological processes in seed. Analysis of transcripts which were involved in the early stage of anthraquinone biosynthesis suggested that the aurantio-obtusin in C. obtusifolia was mainly generated from isochorismate and Mevalonate/methylerythritol phosphate (MVA/MEP) pathway, and three reactions catalyzed by Menaquinone-specific isochorismate synthase (ICS), 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate (IPPS) might be the limited steps. Several seed-specific CYPs, SAM-dependent methyltransferase, and UDP-glycosyltransferase (UDPG) supplied promising candidate genes in the late stage of anthraquinone biosynthesis. In addition, four seed-specific transcriptional factors including three MYB Transcription Factor (MYB) and one MADS-box Transcription Factor (MADS) transcriptional factors) and alternative splicing might be involved with seed formation and development. Meanwhile, most members of Hsp20 genes showed high expression level in seed and flower; seven of which might have chaperon activities under various abiotic stresses. Finally, the expressional patterns of genes with particular interests showed similar trends in both transcriptome assay and qRT-PCR. In conclusion, this is the first full-length transcriptome sequencing reported in Caesalpiniaceae family, and thus providing a more complete insight into aurantio-obtusin biosynthesis, seed formation ...

De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis

“…Among the seven compounds, gluco-obtusifolin has the highest content in seed tissues (Fig 6 and Table 2). It is well known that aurantio-obtusin is the most significant active compound [8] and is distributed mainly in the seed [44]. However, we found that low levels of aurantio-obtusin were observed at the early and late developmental stages.…”

Single-Molecule Long-Read Sequencing of Avocado Generates Microsatellite Markers for Analyzing the Genetic Diversity in Avocado Germplasm

“…To date, several short-read next-generation sequencing (NGS) transcriptome databases have been developed for avocado mesocarp samples [54,55] and avocado mixed tissue samples [18,56]. However, both the number and length of the transcript sequences derived from these short-read NGS studies have hamperedtheirapplication ingenetics and molecular biology research [41]. One of the advances in sequencing technology has been the development of the long-read SMRT sequencing technique, which enables researchers to obtain a substantial number of full-length sequences from a cDNA library [32].…”

Prospects for the use of third generation sequencers for quantitative profiling of transcriptome

“…Наряду с этим, SMRT-секвенирование по протоколу Iso-Seq также используется для изучения альтернативного полиаденилирования [70][71][72] и детекции химерных (или "слитых") генов (fusion genes) [ В то время как SMRT-секвенирование per se или в сочетании с SGS широко используется для исследования альтернативного сплайсинга, альтернативного полиаденилирования, аннотации геномов и секвенирования транскриптомов de novo, оно практически не использовалось для количественного профилирования транскриптома. В работах, где транскриптомный анализ проводился с использованием гибридного секвенирования, сочетающего технологии SMRT и SGS, для количественного профилирования транскриптома использовались короткие "чтения", генерируемые SGS [62,[77][78][79][80][81]. Одна из причин того, что SMRT-секвенирование не применяется для количественного профилирования, состоит в использовании протокола Iso-Seq, который предполагает фракционирование кДНК по размеру и последующую раздельную амплификацию фракций, что может существенно исказить оценку количества транскриптов по количеству RoI.…”