The Functional Identification Of Long Non-Coding RNA Related To Cotton Fiber Initial

Long non-coding RNA is non-coding RNAs greater than 200 nt whose molecular structure is similar to mRNA,with a 5’ end cap and a 3’ end ployA tail.Long non-coding RNAs have many biological functions,such as signal molecule,decoy molecule,guide molecule,or scaffold molecule to regulate gene expression.Recently,with the development of high-throughput sequencing,the researches of long non-coding RNAs have advanced a lot.As one of the most important natural fiber,cotton fiber plays an important role in the national economy and the people’s livelihood;cotton fiber is trichome of the seed epidermis,mature fiber cellulose content is more than 90%,is one of the longest single cells.So it is a model cell to study cell differentiation and elongation,secondary wall deposition and cellulose synthesis.Until now there are few studies on how long non-coding RNA affects cotton fiber development.In 2015 Dr.Wang Maojun identified 11 lncRNA with high expression levels at different developmental stages of cotton fiber by analyzing cotton transcriptome data.Basing on Wang Maojun’s research,we selected 5 lncRNAs which predominantly expressed in fiber initiation stage to further research.Cotton RNAi transgenic lines were obtained and function elucidations of these lncRNAs were performed.The main results are as follows:1.Basing public database data,the expression patterns of 5 lncRNA expressed preferentially in the fiber initiation stage were further analyzed in upland cotton,which showed that the expression levels of 3 lncRNAs in 0DPA were significantly higher than in other fiber development stages and other tissues.Ghir_A01G011040 has the highest expression,and the FPKM values of 0DPA and 5DPA are 215.11 and 36.85,respectively.2.The transgenic cotton lines of these five lncRNA were obtained,the lines with single-copy insertion and down-regulated expression were screened.They are Ghir_A06G015820(1-1,1-2,1-32),Ghir_D08G008390(2-14,2-17,2-23),Ghir_A01G011040(4-14,4-22,4-37)etc.Measurements of mature fiber length by hand combing,we found that the fiber length of five lncRNA transgenic lines were shorter,and shorter trend of the Ghir_A01G011040 transgenic line was most significantly.3.The length of mature fiber of T3 generation of Ghir_A01G011040-RNAi was further determined,which showed that fiber shorter trend was consistent with T1 generation.Theresults of in situ hybridization showed that the hybridization signal of the transgenic line was significantly lower than that of the wild type,and the expression of Ghir_A01G011040 was on the outer epidermis of the ovule.4.The transcriptome of 0DPA ovules of three different transformation events of Ghir_A01G011040-RNAi and 2 controls(null and wild type)were analyzed.Total 1396 differential genes were screened,of which 744 were up-regulated and 652 were down-regulated.By GO analysis,it was found that these differentially expressed genes were classified into the pathways like methylation,response to karrikin,cellulose biosynthetic process,Golgi organization,nucleic acid binding etc.5.Twelve differentially expressed genes with expression changes greater than 1.5 folds were selected for qRT-PCR expression verification,and the qRT-PCR results were consistent with RNA-seq results.The twelve genes are the cell wall structural protein predictive gene Ghir_A07G016470,glutathione S-transferase DHAR2(Ghir_A05G036540),leucoanthocyanidin dioxygenase ANT17(Ghir_D08G019350)and Gibberellin-regulated protein 4 GASA4(Ghir_D09G001540),respectively.Our results show that lncRNA may regulate fiber initiation and elongation by regulating the corresponding metabolic pathways,but further research needs to be performed to elucidate how lncRNA regulates these pathways.