Mehta Rucha Harishbhai, Ponnuchamy Manivel, Kumar Jitendra, Reddy Nagaraja Reddy Rama
ICAR-Directorate of Medicinal and Aromatic Plants Research (ICAR-DMAPR), Anand, 387310, Gujarat, India.
Funct Integr Genomics. 2017 Jan;17(1):1-25. doi: 10.1007/s10142-016-0523-y. Epub 2016 Oct 5.
De novo assembly of reads produced by next-generation sequencing (NGS) technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms. Senna (Cassia angustifolia Vahl.) is a drought-tolerant annual undershrub of Caesalpiniaceae, a subfamily of Fabaceae. There are insufficient transcriptomic and genomic data in public databases for understanding the molecular mechanism underlying the drought tolerance of senna. Therefore, the main purpose of this study was to know the transcriptome profile of senna, with special reference to drought stress. RNA from two different stages of leaf development was extracted and sequenced separately using the Illumina technology. A total of 200 million reads were generated, and a de novo assembly of processed reads in the pooled transcriptome using Trinity yielded 43,413 transcripts which were further annotated using NCBI BLAST with "green plant database (txid 33090)," Swiss Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Gene Ontology (GO). Out of the total transcripts, 42,280 (95.0 %) were annotated by BLASTX against the green plant database of NCBI. Senna transcriptome showed the highest similarity to Glycine max (41 %), followed by Phaseolus vulgaris (16 %), Cicer arietinum (15 %), and Medicago trancatula (5 %). The highest number of GO terms were enriched for the molecular functions category; of these "catalytic activity" (GO: 0003824) (25.10 %) and "binding activity" (GO: 0005488) (20.10 %) were most abundantly represented. We used InterProscan to see protein similarity at domain level; a total of 33,256 transcripts were annotated against the Pfam domains. The transcripts were assigned with various KEGG pathways. Coding DNA sequences (CDS) encoding various drought stress-regulated pathways such as signaling factors, protein-modifying/degrading enzymes, biosynthesis of phytohormone, phytohormone signaling, osmotically active compounds, free radical scavengers, chlorophyll metabolism, leaf cuticular wax, polyamines, and protective proteins were identified through BLASTX search. The lucine-rich repeat kinase family was the most abundantly found group of protein kinases. Orphan, bHLH, and bZIP family TFs were the most abundantly found in senna. Six genes encoding MYC2 transcription factor, 9-cis-epoxycarotenoid dioxygenase (NCED), l -ascorbate peroxidase (APX), aminocyclopropane carboxylate oxidase (ACO), abscisic acid 8'-hydroxylase (ABA), and WRKY transcription factor were confirmed through reverse transcriptase-PCR (RT-PCR) and Sanger sequencing for the first time in senna. The potential drought stress-related transcripts identified in this study provide a good start for further investigation into the drought adaptation in senna. Additionally, our transcriptome sequences are the valuable resource for accelerated genomics-assisted genetic improvement programs and facilitate manipulation of biochemical pathways for developing drought-tolerant genotypes of crop plants.
利用新一代测序(NGS)技术产生的reads进行从头组装,为获取非模式生物的表达基因序列提供了一种快速方法。番泻(Cassia angustifolia Vahl.)是豆科亚科苏木科的一种耐旱一年生小灌木。公共数据库中关于番泻耐旱分子机制的转录组和基因组数据不足。因此,本研究的主要目的是了解番泻的转录组概况,特别是干旱胁迫下的情况。从叶片发育的两个不同阶段提取RNA,并分别使用Illumina技术进行测序。共产生了2亿条reads,使用Trinity对合并转录组中的处理后reads进行从头组装,得到43413个转录本,这些转录本进一步使用NCBI BLAST与“绿色植物数据库(txid 33090)”、Swiss Prot、京都基因与基因组百科全书(KEGG)、直系同源簇(COG)和基因本体(GO)进行注释。在所有转录本中,42280个(95.0%)通过BLASTX与NCBI的绿色植物数据库进行比对得到注释。番泻转录组与大豆(41%)的相似性最高,其次是菜豆(16%)、鹰嘴豆(15%)和蒺藜苜蓿(5%)。在分子功能类别中富集的GO术语数量最多;其中“催化活性”(GO: 0003824)(25.10%)和“结合活性”(GO: 0005488)(20.10%)的代表性最为丰富。我们使用InterProscan在结构域水平上查看蛋白质相似性;共有33256个转录本针对Pfam结构域进行了注释。这些转录本被分配到各种KEGG途径。通过BLASTX搜索鉴定出编码各种干旱胁迫调节途径的编码DNA序列(CDS),如信号因子、蛋白质修饰/降解酶、植物激素生物合成、植物激素信号传导、渗透活性化合物、自由基清除剂、叶绿素代谢、叶片表皮蜡质、多胺和保护蛋白。富含亮氨酸重复激酶家族是最丰富的蛋白激酶组。孤儿、bHLH和bZIP家族转录因子在番泻中最为丰富。首次通过逆转录聚合酶链反应(RT-PCR)和桑格测序在番泻中证实了六个编码MYC2转录因子、9-顺式环氧类胡萝卜素双加氧酶(NCED)、L-抗坏血酸过氧化物酶(APX)、氨基环丙烷羧酸氧化酶(ACO)、脱落酸8'-羟化酶(ABA)和WRKY转录因子的基因。本研究中鉴定出的潜在干旱胁迫相关转录本为进一步研究番泻的干旱适应性提供了一个良好的开端。此外,我们的转录组序列是加速基因组辅助遗传改良计划的宝贵资源,并有助于操纵生化途径以培育耐旱作物基因型。