Differential Alternative Splicing Genes In Response To Low Boron In Brassica Napus

Boron(B) is an essential micronutrient for plant growth and development. It is closely associated with the structure of plant cell wall, cell membrane function and carbohydrate metabolism. Oilseed rape(Brassica napus L.) is one of the main oil crops in China, which showed much more sensitive to low soil available boron than other species. In this study,the transcriptional profile of B-efficient cultivar Qingyou10 and B-inefficient cultivar Westar10 at low and normal B was used to study the differential alternative splicing events and reveal the important differential alternative splicing genes response to B deficiency in B-efficient and B-inefficient cultivars. The results are as follow.1. Assemble of the transcripts of B-efficient and B-inefficient cultivars at low and normal B conditions identified 135,036 expressed transcripts and 56,203 expressed genes,and each gene had two or more transcripts. A large number of new transcripts and genes were discovered by comparing the transcripts of assembling and the reference genome.GO analysis showed that the functions of the new genes were associated with the glucose metabolism, Ca2+ and H+ transport and ATPase activity.2. A total of 33.3% genes of B-efficient and B-inefficient cultivars showed alternative spliced at both B conditions. The order of ratio of four types of alternative splicing among all the splicing events was as follow, intron retention > 3’ alternative splice site > 5’ alternative splice site > exon skipping. The alternative splicing events of both cultivars occured at low B condition demonstrated much more than that at normal B condition; moreover, the alternative splicing events happened in Westar10 was significantly higher than in QY10 under B starvation, indicating B deficiency did much more harm to Westar10 than QY10. GO analysis showed that the genes that were not alternatively spliced related to protein folding and DNA repair housekeeping functions,while most of the genes that produce alternative transcripts were involved in the response to ion stress, cell wall modification and ion transport in plant membrane.3. 512 and 138 differential alternative splicing(DAS) genes were identified in QY10 and Westar10 under B deficiency, respectively, and the order in percentage of four types of alternative splicing of both cultivars were the same, which was as follow, intron retention > 3’ alternative splice site > 5’ alternative splice site > exon skipping. There existed 313 cultivar specific DAS genes function as SR splicing factors and transcription factors in QY10. KEGG pathway analysis of DAS genes suggested that, in QY10, DAS genes involved in glycolysis(BnaC05g18490D) and citrate cycle(BnaC08g29490D),starch and sucrose metabolism in root; pentose phosphate pathway and glycolysis(Bna C05g18490 D, Bna A06g20750D) in juvenile leaf; glycolysis pathway(Bna C05g18490 D, Bna A06g20750D) at low B. In Westar10, DAS genes were associated with ribosome in root, peroxisome and AMPK signaling pathway in juvenile leaf, starch and sucrose metabolism(Bna A02g29830D), amino sugar and nucleotide sugar metabolism(Bna C07g18010D) and pentose phosphate pathway(Bna C05g18490D) in old leaf.4. Combined with analysis of DE genes and DAS genes indicated that about 0.06%of DE genes showed alternatively spliced. GO analysis suggested that DAS and DE genes had different biological functions. For example, only DAS genes were annotated associated with m RNA process and co A metabolism; only DE genes were annotated involved in cell wall modification and carbohydrate metabolism; However, several DAS and DE genes had the same annotation in biological functions, e. g., glucose metabolism(Bna A06g17710D) and signal transduction(Bna A07g33860D). This indicated that the regulation of gene expression and alterternative splicing in oilseed rape under low B stress was not completely independent, and some genes, could be regulated by both transcriptionally and posttranscriptionally under B deficiency.5. Four splicing factors, BnaC06g14780 D, BnaA01g14750 D, BnaC01g41640 D and Bna A06g15930 D were identified in QY10 and Westar10 at low B. According to the expression levels of four splicing factors, Bna C06g14780 D was predicted to be an important splicing factor by the bioinformatic software. Further analysis showed that Bna C06g14780 D could interactive with other genes in response to low B, e. g. expansin genes(Bna A09g52970D) and cell wall modify gene(Bna A09g52970D). This suggested that oilseed rape could regulate the splicing of the genes tolertance to B deficieny through pre-m RNA of SR protein genes.