Transcriptome Sequencing Of Fiber Development And Functional Analysis On Expansin Family In Ramie

Ramie（Boehmeria nivea L.Gaud）is a perennial herbaceous flowering plant.Due to the significant shrink of ramie planting area,insufficient manpower in scientific research,and less research accumulation,the new cultivar breeding progress of ramie,which was mostly conducted through traditional breeding methods,went slowly.It is urgent to introduce current molecular breeding tools into ramie breeding processes to accelerate the progress.In this study,researches of ramie transcriptome sequencing regarding different fiber developmental status,cloning and expression profiling analysis of expansin gene family in ramie were conducted.The main results were as following:1.The total RNA extraction methods were further optimized,the high quality total RNA,which was utilized in the transcriptome sequencing,was isolated using the improved guanidinium-based protocol.Different parts of ramie bark samples were generated to represent different ramie fiber developing stages.After ligating to distinguishable adaptors,RNAs isolated from these samples were equally mixed and performed a full run sequencing on the Roche 454 FLX+ titanium platform.A total of 10,300,057 reads with average length of 457 bp were obtained.After assembling,the sequence lengths of 58,369 unigenes ranged from 90 bp-7641 bp,in which 51,025 sequences（87.42% of total unigenes）with lengths over 200 bp,and 8,052（13.75%）with lengths over 1000 bp.Finally,25,071 unigenes（7065 contigs and 10,886 isotigs）out of 58,396 unigenes（13,386 contigs and 44,983 isotigs）were efficiently annotated,corresponding to 6283 GO entries and 3076 KO entries.After analyzing multiple items,like the differentially expressed genes and GO entry enrichment,we assumed that the top part of ramie bark sample is special among all the four samples,which means the early developmental stage is more sensitive to molecular regulation and thus affecting ramie fiber development.Among the differentially expressed genes in the particular sample,the unigenes isotig01514₇7,isotig06919₇8,isotig06943₂5 and isotig07154₁9 from the cellulose synthase gene family,the isotig10345₂3,isotig01251₆ and isotig02054₁8 from the expansin gene family,and the HRX1MBH01BLWDW₉,isotig00610₂5,isotig01663₂2,isotig04660₈,isotig09773₁0 and contig04902₁9 from the XTH gene family,were up-regulated when comparing to the rest three samples,indicating these unigenes were probably involved in the pathways regulating ramie fiber development in the upper part of ramie stem bark sample.2.Using the homologous sequence cloning strategy,we cloned five expansin genes with full coding sequences from ramie,expression analyses were also conducted.After searching the three transcriptome libraries with keywords ‘expansin’ in annotation information,the number of cloned expansin genes was expanded to 16（12 of them belong to α-subfamily,the rest 4 belong to β-subfamily）,with 22（Gen Bank accession: KY748166-KY748187）full-length DNA sequences（each of 6 expansin genes has two clones with different sequences in ramie genome,which was respectively named as-1 and-2）.For sequence length variation of the 22 cloned sequences,exons varied from 107 bp to 485 bp,while the introns varied from 86 bp to 1540 bp;the lengths variation for c DNA was 732 bp to 831 bp,while for DNA was 1040 bp to 4259 bp.Afterwards,the expression analysis for the cloned expansin genes were conducted.Bn EXPA2,Bn EXPA3,Bn EXPA5 and Bn EXPA6 expressed generally higher than other expansin genes in the six samples（shoot apex,young leaves,mature leaves,the top,middle and bottom part of stem bark）of ramie,while Bn EXPA12 expressed most differently among these six samples.Bn EXPA3,Bn EXPA5,Bn EXPA10 and Bn EXPB2 expressed generally higher among the samples obtained from different ramie stem bark samples（bottom part at seedling stage,top and bottom parts at rapid growing stage,top,middle,and bottom parts at fiber mature stages）in the three growing seasons of ramie,Bn EXPA11 expressed extremely low in all samples and under advert conditions（Na Cl,high temperature and low temperature stresses）.At last,Bn EXPA1 was up-regulated under any of the three abiotic conditions,which was the only one like this in the expansin family of ramie.On the contrary,Bn EXPA4,Bn EXPA5,Bn EXPA12,Bn EXPB1 and Bn EXPB3 were down-regulated under the three advert conditions.These genes showing special expression patterns can be regarded as candidate genes in downstream experiments for further detailed analysis.All in all,there have the following highlights in this study:1.RNA isolation methods were further optimized and applied in the following transcriptome sequencing of ramie;2.It was the first time to use the sampling method stated here,which represented different fiber developmetal stages in ramie,for transcriptome sequencing;3.It was the first time to use the high-throughput sequencing platform to illuminate the molecular mechanisms of ramie fiber development;4.Five expansin genes were cloned utilizing the homologous sequence cloning strategy;5.Expansin gene cloning was expanded by combining multiple transcriptome sequencing data;6.A bunch of expansin genes were presumably included in the pathways corresponding to advert conditions or fiber developmental processes of ramie.In a word,the molecular mechanisms of ramie fiber developmemt were preliminarily studied,which provided experimental data and operational mimic for downstream studies.