Identification And Analysis Of MicroRNA In Gingo Biloba L. And Ginkgo Biloba Var. Epiphylla Mak

Ginkgo biloba L. is a type of the relict spermatophyte found in China thathas survivedsince the4Ice Age. There were obvious morphological variation occured in seeds and leavesof Ginkgo biloba along the long evolutionary process. In1891, Shirai discovered the firstGinkgo biloba var. epiphylla Mak. tree of the wolrd in Japan. The Ginkgo biloba var.epiphylla which is a special germplasm of Ginkgo family has great ornamental and scientificvalues. MicroRNAs (miRNAs) are noncoding RNAs of approximately21nt that regulate geneexpression in plants post-transcriptionally, either by endonucleolytic cleavage or translationalinhibition. Increasing evidence indicates that miRNAs play a major role during development,signal transduction and other biological processes. Identification and functional analysis ofmiRNA in the Ginkgo biloba and Ginkgo biloba var. epiphylla will further promote theirstudy of the evolution and function might further disclose the molecular mechanism ofepiphylla ovules.In order to reveal the variation of miRNA during the Ginkgo biloba and Ginkgo bilobavar. epiphylla, we use the method of combining high-throughput sequencing technology andbioinformatics to characterize, identify and do expression analysis of sRNA cDNA library.The results obtained as follows:(1)We pooled18-28nt RNA from Ginkgo biloba and Ginkgo biloba var. epiphylla, andconstructed two small RNA libraries. Solexa sequencing generated a total of46,191,036High-quality sequence sequences, including24,227,844sequences from Ginkgo biloba smallRNA library and21,963,192sequences from Ginkgo biloba var. epiphylla small RNA library.After get rid of the5’ adaptor, contaminant and low quality reads,23,798,499(3,059,786unique) and21,717,220(2,585,993unique) clean sequence in the Ginkgo small RNA librariesand Ginkgo biloba small RNA library, respectively.(2)Processing and analyzing the clean reads above. The majority of the small RNAs are20-24nt in size (approximating to90%), and the21nt size class is predominant(approximating to66%), it’s the typical length of miRNA. The distribution characteristics isconsistent with the other small RNA library of gymnosperms, but it is varies significantlybetween angiosperms and gymnosperms. This is mainly due to the DCL enzyme whichinvolved in the miRNA generation process. The abundance of miRNA and siRNA (12.5%and25.85%in Ginkgo biloba;13.73%and29.39%in Ginkgo biloba var. epiphylla)in small RNAlibraries indicated that the complex miRNA and siRNA regulatory mechanism exist inGinkgo. (3) After analyzing all the small RNA reads in Ginkgo biloba,2,974,600plant miRNAshomologues with20,262unique reads were identified, and belonged to444miRNA families.After further complex analysis and rigorous screening,69conserved miRNAs comprising24families were identified in Ginkgo biloba. In the meantime,2,981,348plant miRNAshomologues with18,685unique reads were identified, and belonged to391miRNA families.Finally,82conserved miRNAs comprising28families were identified in Ginkgo biloba var.epiphylla after strict screening and manual conformed.(4)10novel miRNAs were identified in the two small RNA libraries, including onecommon novel miRNA.2novel miRNAs were identified in Ginkgo biloba, meanwhile9novel miRNAs were identified in Ginkgo biloba var. epiphylla, including1miRNA*Whichcorresponds to a miRNA. The10pre-miRNAs of novel miRNAs could folden into hairpinsecondary structure, the miRNAs are20-23nt in size, their minimal folding free energy wereless than-19.5kcal/mol, these are all in line with the typical characteristics of miRNAs.(5) The algorithm miRUwas used to identify potential mRNA targets for miRNAs inGinkgo biloba and Ginkgo biloba var. epiphylla.161putative target genes with variousfunctions were predicted for22miRNAs families, which were involved in varied aspects ofenvironmental responses and developmental processes in plants.37putative target genes withvarious functions were predicted for10novel miRNAs, The potential target genes of10novelmiRNAs in Ginkgo biloba and Ginkgo biloba var. epiphylla showed that17target ESTsencoded functional protein, and20target genes encoded unknown proteins.(6) Compare the known miRNA expression between two samples to find out thedifferentially expressed miRNA. The result showed that the expression level of MIR156,MIR164, MIR168, MIR169, MIR390, MIR397, MIR399, MIR408, MIR535, MIR1030inGinkgo biloba var. epiphylla were significant higher than in Ginkgo biloba, while theexpression level of MIR159, MIR160, MIR394, MIR828in Ginkgo biloba were significanthigher than in Ginkgo biloba var. epiphylla.The abundance of small RNAs in Ginkgo biloba L. and Ginkgo biloba var.epiphylla Makmight indicate a long evolutionary history of post-transcriptional gene expression regulation,and differential expression of small RNAs between the two Ginkgos might further disclosethe molecular mechanism of epiphylla ovules.