Project description:Clade A PP2C genes in different organs and responding to stresses were screened using qPCR. The BjuACTINs were regarded as house keeping gene.To analyze the gene expression patterns of clade A BjuPP2Cs genes under biotic stress, 14-day-old seedlings were treated with P. brassicae liquid (OD600=0.07) for 0, 0.25, 0.5, 1, 3, 5, 7, and 9 days; to analyze the gene expression patterns of clade A BjuPP2Cs genes under abiotic stresses, 5-day-old seedlings were treated with 50 μM ABA, 100 mM NaCl and 4℃ treatment for 3 h. To analysis the gene expression level in different growth stages of stem swelling, the samples D1 (the stems of 1-month-old seedlings, six leaf stage), D2 (the stems of 2-month-old seedlings, primary stage of stem swelling), D3 (the stems of 3-month-old seedlings, early stage of stem swelling), D4 (the stems of 4.5-month-old seedlings, fast-growing stage of stem swelling) and D5 (the stems of 5-month-old seedlings, last stage of stem swelling) of B. juncea var. tumida which were grown in the field were collected.
Project description:Oilseed mustard, Brassica juncea, exhibits high levels of genetic variability for salinity tolerance. To obtain the global view of transcriptome and investigate the molecular basis of salinity tolerance in a salt-tolerant variety CS52 of B. juncea, we performed transcriptome sequencing of control and salt-stressed seedlings. De novo assembly of 184 million high-quality paired-end reads yielded 42,327 unique transcripts longer than 300 bp with RPKM ≥1. When compared with non-redundant proteins, we could annotate 67% unigenes obtained in our study. Based on the mapping to expressed sequence tags (ESTs), 52.6% unigenes are novel compared to EST data available for B. juncea and constituent genomes. Differential expression analysis revealed altered expression of 1469 unigenes in response to salinity stress. Of these, 587, mainly associated with ROS detoxification, sulfur assimilation and calcium signaling pathways, are up regulated. Notable of these is RSA1 (SHORT ROOT IN SALT MEDIUM 1) INTERACTING TRANSCRIPTION FACTOR 1 (RITF1) homolog up regulated by >100 folds in response to stress. RITF1, encoding a bHLH transcription factor, is a positive regulator of SOS1 and several key genes involved in scavenging of salt stress-induced reactive oxygen species (ROS). Further, we performed comparative expression profiling of key genes implicated in ion homeostasis and sequestration (SOS1, SOS2, SOS3, ENH1, NHX1), calcium sensing pathway (RITF1) and ROS detoxification in contrasting cultivars, B. juncea and B. nigra, for salinity tolerance. The results revealed higher transcript accumulation of most of these genes in B. juncea var. CS52 compared to salt-sensitive cultivar even under normal growth conditions. Together, these findings reveal key pathways and signaling components that contribute to salinity tolerance in B. juncea var. CS52. We report transcriptome sequencing of two-weeks-old seedlings of B. juncea var. CS52 under normal growth conditions (CTRL) and in response to salinity stress (SS) using Illumina paired-end sequencing
Project description:Oilseed mustard, Brassica juncea, exhibits high levels of genetic variability for salinity tolerance. To obtain the global view of transcriptome and investigate the molecular basis of salinity tolerance in a salt-tolerant variety CS52 of B. juncea, we performed transcriptome sequencing of control and salt-stressed seedlings. De novo assembly of 184 million high-quality paired-end reads yielded 42,327 unique transcripts longer than 300 bp with RPKM ≥1. When compared with non-redundant proteins, we could annotate 67% unigenes obtained in our study. Based on the mapping to expressed sequence tags (ESTs), 52.6% unigenes are novel compared to EST data available for B. juncea and constituent genomes. Differential expression analysis revealed altered expression of 1469 unigenes in response to salinity stress. Of these, 587, mainly associated with ROS detoxification, sulfur assimilation and calcium signaling pathways, are up regulated. Notable of these is RSA1 (SHORT ROOT IN SALT MEDIUM 1) INTERACTING TRANSCRIPTION FACTOR 1 (RITF1) homolog up regulated by >100 folds in response to stress. RITF1, encoding a bHLH transcription factor, is a positive regulator of SOS1 and several key genes involved in scavenging of salt stress-induced reactive oxygen species (ROS). Further, we performed comparative expression profiling of key genes implicated in ion homeostasis and sequestration (SOS1, SOS2, SOS3, ENH1, NHX1), calcium sensing pathway (RITF1) and ROS detoxification in contrasting cultivars, B. juncea and B. nigra, for salinity tolerance. The results revealed higher transcript accumulation of most of these genes in B. juncea var. CS52 compared to salt-sensitive cultivar even under normal growth conditions. Together, these findings reveal key pathways and signaling components that contribute to salinity tolerance in B. juncea var. CS52.
Project description:Purpose: To identify high temperature, sailinity and drought-responsive miRNAs at genome wide level in B. juncea var varuna. Results: In this study four small RNA libraries viz. B. juncea control (BJC), B. juncea high temperature stressed (BJH), B. juncea salinity stressed (BJS) and B. juncea drought stressed (BJD) were prepared and sequenced. With the help of UEA sRNA workbench software package 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a very low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported so far in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of a few selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions. Conclusions: We have generated in this study, the first comprehensive abiotic stress influenced small RNA dataset in B. juncea. The combinatorial approach of NGS and computational methods led to the discovery of 51 conserved and 126 novel miRNAs. The present study provides a holistic view of B. juncea miRNAome under conditions of high temperature, salinity and drought. The catalogue of miRNA sequences, their expression and putative targets, generated in this study can be utilized to design crop improvement strategies in B. juncea and related species.