Small RNA And Degradome Sequencing Explore A Role Of MiRNAs And Their Targets In Regulation Of Seed Weight And Oil Content In Brassica Napus L

Oilseed rape(Brassica napus)is one of the most important oilcrop in the world,increase oil production of Brassica napus is the main objective of Brassica napus breeding,seed weight and oil content are the key factors of oil production in Brassica napus.In this study,two Brassica napus which have significant difference in seed weight and oil content,were used as the research materials.RNA extracted from immatured seeds at one to six weeks after fertilization was used to construct mixed libraries and perform small RNA and degradoome sequencing.Different expression analysis of mi RNAs and identification of mi RNA targets were used to explore candidate mi RNAs and targets involving regulation of seed weight and oil content.The main research results are as follows:(1)Using the platform of next generation sequencing,two RNA pools(G-42 and 7-9)of immatured seeds at 1 to 6 weeks after fertilization were used to identify mi RNAs.After filtering low quality reads,adapters,too long and too short sequences,18,277,880 and 20,079,520 clean reads were got,respectively.In G-42 and 7-9 RNA pools,92.80% and 93.56% of total small RNAs(s RNA)length distribution were ranged from 20 nt to 24 nt,respectively,and 21 nt and 24 nt mi RNA were the most.From the two total s RNAs samples,1,248 novel mi RNAs and 28 known mi RNAs were identified.And 300 novel mi RNAs and two known mi RNAs were differentially expressed between G-42 and 7-9.Through forecasting the target genes of this mi RNAs,503 candidate genes from the 181 mi RNAs of 1,276 total mi RNAs,465 of 503 have the function annotation were got.And 119 candidate genes from the 45 mi RNAs of 302 differently expressed mi RNAs were got,98 of 119 have the function annotation.7 candidate mi RNA target genes(including auxin response factor gene and pyruvate dehydrogenase complex ?subunit gene)may play important roles in in regulation of seed weight and oil content.(2)Using the next generation sequencing technology,the G-42 RNA pools of immatured seeds from one to six weeks after fertilization were used to construct degradome sequence library.After filtering low quality reads,adapter,too long and too short sequences,About 3.1×107 clean tags in G-42 RNA pools were got.The length of clean tags mainly focused on 20 nt and 21 nt.Cleaveland software was used to detect m RNA cleavage products and 64 cleavage of m RNAs belong to 57 candidated genes were identified,and 64 degradation locations were detected.14 candidate mi RNA target genes(including WPP protein gene?E3 ubiquitin protein ligase gene?zinc finger protein gene and pyruvate dehydrogenase complex ?subunit gene)may play important roles in in regulation of seed weight and oil content.(3)To verify the reliability of small RNA sequencing,QRT-PCR method was used to retest the expression level of the differentially expressed mi RNAs.The expression level of target genes of differentially expressed mi RNAs was also analyzed.And inverse expression pattern between mi RNAs and their targets indicate mi RNAs could negatively regulate expression level of their target genes.