Expression Profiling Analysis Of Brassica Napus In Response To Drought Stress

Rapeseed（Brassica napus L.） is one of the most important oil crops, outranked only by soybeans. China is the largest producer of rapeseed, which is mainly produced in the Yangtze River region. Because of the spatial and temporal inhomogeneity of precipitation in the region, seasonal drought is the most important yield-limiting factors for dwarf and infertility rapeseed. Mining for drought resistance genes is an important work for rapeseed tolerance breeding. Identification of the drought-induced genes at the transcriptional level would be an effective method to target drought response genes and explore the mechanism of drought stress. With higher performance relative water content（RWC）, drought resistance index（DRI） and relatively lower electrolyte leakage in drought stress, drought resistant inbred lines Q2 has proved to be an important genetic resources for drought tolerant improvement in rapeseed. In this study, Q2 was taken as an experimental material in response to drought stress for 24 hours（air-dried）, root and leaf were extracted the total RNA to construct c DNA libraries. A high-throughput technology of RNA-Seq via Illumina Hi Seq was carried out in order to analyze the mechanism of rapeseed in response to drought stress and mine important drought induced functional genes as well as transcriptional regulatory elements. The main results are as follows:1. Transcriptome analysis of Brassica napus root and leaf in drought stress conditionsRNA-seq via the Illumina 2000, was used for generating an extensive map of Q2 root and leaf transcriptome under the air-dried stress conditions. A total of 12308861, 11763435, 11905805 and 12168994 clean reads were collected from root（drought）, root（Control, CK）, leaf（drought） and leaf（CK） samples, respectively. Multiple analyses were used to assess the quality of the sequence reads, and the 10 M sequence depth and full coverage of the expressed genes in genome from each samples indicated the RNA sequencing data high-quality.Clean reads were aligned to Brassica oleracea genome database. In total, 31,240 and 31,118 genes were identified in root and leaf, respectively. Using FDR ≤0.001 and | log2 Ratio | ≥1 as thresholds, a total of 6018 and 5377 genes were defined as differentially expressed genes（DEGs） in root and leaf, respectively. Among them, there were 2448 upregulated genes and 3570 down-regulated genes in root, 3770 up-regulated genes and1607 down-regulated genes in leaf, showing different expression patterns of DEGs in root and leaf might exist in response to drought stress.GO enrichment analysis indicated that most DEGs were mainly enriched in response to stress and stimulus in root and leaf; Blast2 GO analysis showed that upregulated genes were mainly enriched in response to stimulus and stress, while most down-regulated genes were enriched in cell and structure. However, the results were opposite in the leaf, suggesting a complementary division of labor by root and leaf in response to drought stress. KEGG enrichment analysis results showed that most DEGs were significantly associated with plant hormone signaling pathway and phenylpropanoid biosynthesis pathway. In addition, lots of transcription factors（TFs） were activated in root and leaf, including the MYB, b HLH, C2H2, AP2 / EREBP, NAC and so on. Among them, AP2 / EREBP and WRKY TFs played an important role in the differently regulated models of roots and leaves in response to drought stress. Transcription factors interacted with the cis-acting elements in the promoter of drought-induced genes to induce downstream genes expression, including ERD1, RAB18, RD29 B, RD20 A, JA-related genes and so on. Then the ion exchange of Cl-, NO3-, K+, Ca2+ in cell influenced the level of osmotic potential and then increased water uptake in root and induced stomata close in leaf to decrease transpiration water loss.2. Cloning of the five candidate genes in Brassica napus with homology based Brassica oleracea genes sequencesBased on the result of tanscriptome log2 Ratio and RPKM, five candidate genes were selected for further functional verification, including TMA7（Bra026774）, BAM3（Bra012676）, SERPIN1（Bra015607）, Peroxidase（Bra009221）, Unknown（Bra009013）. And these genes’ functions related to translation, B- amylase, serine protease inhibitors, peroxidases activity and unknown function, respectively. These five candidate genes CDS sequences were aligned to Brassica napus genome database to check the orthologous genes, then designed primers to clone and detect the sequences and detect the copies in Brassica napus. Finally, we confirmed 6 copies of TMA7, 2 copies of BAM3, 2 copies of SERPIN1, 6 copies of Peroxidase and 1 copy of Unknown gene in Brassica napus.3. Identification of the stress-related phenotypes for the mutantsStress testing suggested that candidate genes mutants — tma7（SALK₀44092C）, bam3（SALK₀41214C）, serpin1（SALK₀19150C）, peroxidase（SALK₁24744） and un-2（SALK₁21527C） showed higher rate of water loss of excised-leaf, earlier flowering, which were sensitive to drought stress. The un-2 mutant showed the highest rate of water loss of excised-leaf and seedlings wilted and crinkled faster than WT under dehydration condition.4. The expression and function analysis of candidate genesExpression patterns of Bnassica napus genes—Bna TMA7, Bna BAM3-C1, Bna BAM3-C2, Bna SERPIN1-C1, Bna SERPIN1-C2, Bna Peroxidase, Bnaunknown were investigated by q RT-PCR in different drought stress periods（0h, 3h, 6h, 12 h, 24 h, 36h）, suggested that the expressions of these genes were induced by drought stress in root. However, the results in leaf were not consistent with the findings in root, some genes were even suppressed during the stress condition. In addition, we detected the patterns of Bna SERPIN1-C2 promoter-driven GUS expression in seedlings. In unstressed plants, GUS expression was observed in root, leaf apex and leaf epidermal hair, hypocotyls. By contrast, drought treatment of seedlings enhanced the Bna SERPIN1-C2 promoter activity, especially the leaf vascular tissues.5. Function analysis of Bam3 geneBam3 encodes a beta-amylase targeted to the chloroplast. In the present study, three germs termed as Bna Bam3-C1, Bna Bam3-C2 and Ara Bam3 were cloned in B.napus and Arabidopsis, respectively. Overexpression of the three genes in Arabidopsis showed less rosette leaf during bolting and early-flowering under normal condition. Bna Bam3-C1-overexpressing and Ara Bam3-overexpressing plants were sensitive to osmotic stress. The transgene plants charactered higher biomass and decrement of growing period under drought stress, which were considered as drought avoidance.