Small RNA Sequencing Of Seedling, Identification And Expression Of MicroRNAs With Target Genes In Japanese Larch (Larix Leptolepis)

Japanese larch (Larix leptolepis) is one of the most important coniferous species withfast-growing speed. It is widely used for forestation and production of timber in north China.Some major progress has been achieved upon selective breeding and cross breeding in larch.However, the progress can not meet the significant needs of fine tree varieties development,wood yield improvement and productivity enhancement. Apart from the complicated geneticbackground and long growth cycle, the lack of important breakthroughs in basic theory andmolecular genetic research for coniferous trees has hampered the development of geneticimprovement progress. MicroRNA (miRNA), generally21–24nt in length, spatiotemporallyregulates gene expression at transcriptional and/or post-transcriptional level. MiRNAs andtheir target genes involve in morphogenesis of organs, signal transductions and biotic/abioticstress responses, playing an important role in plant growth and development, nutrienthomeostasis. It is that the central role that miRNA networks play in the control of keyargonomic traits makes them appealing biotechnological targets for the production of varietieswith improved performance. The study on the miRNA identification and function in larch isvery important, not only enriching the information resource of miRNA, but also providing anew idea for the genetic improvement of conifers.A pooled library was constructed with equal amounts total RNA (10μg) of needle, stem,and root from a two-year-old larch seedling in the present study. Solexa sequencing combiningwith bioinformatics methods were applied for screening and processing the miRNAs whichcould regulate the growth and developement of Japanese larch. The psRNATarget onlinesoftware was used to predict and analyze miRNAs target genes. Quantitative real-time PCR(qRT-PCR) technology was emploied to analyze the differential expression of miRNA duringthe different periods of seed germination (0d,1d,5d,9d,28d) and different stages of leaf andstem in plant growth (90d,1.5a,5a,10a,25a and50a). RACE technology was conducted todetect the cleavage sites in three predicted target genes. The major results are as the following: (1) Small RNA library construction and Solexa sequencing for screening candidatemiRNA. Sequencing of sRNA was implemented using Illumina GAIIx platform. A total of27,042,027raw reads were obtained, and7,466,370clean mappable sequences were generatedafter filtering and removing contaminent, low quality, known other RNA sequences. Thesesequences were submitted to NCBI-Gene Expression Omnibus (GEO) database (GEO Number:GSE34805). Among them, a total of3,612,795sequences were21nt in length, approximatelyto48.39%of the total number of sRNA population, demonstrating that21nt sRNA in size wasthe main type. The query sequences were matched to the database of miRBase (17.0)(http://www.mirbase.org) and the larix transcriptome ESTs,308candidate miRNAs wereobtained. Solexa sequencing provided large amounts of candidate miRNA sequenceinformation for next miRNA identification.(2) One hundred and seventy-four miRNAs from85miRNA families had been screenedusing BLAST and secondary structure prediction. There were77miRNAs from48conservedmiRNA families (47miRNAs were21nt in length,51miRNAs had uracil in which5′-endfirst base),97miRNAs from37novel families (72miRNAs were21nt in length,47miRNAshad uracil in which5′-end first base). Among them,20miRNA families were commonconserved in land plants;12miRNA families were conifer-specific. The expression detectionof34conserved and6novel miRNAs were validated by qRT-PCR. The knowledge of miRNAsequence characteristic in L.leptolepis will contribute to expand the miRNA information ofconifer trees.(3) Two hundred and thirty-six mRNAs were predicted to be targets of85miRNAfamilies by bioinformatics. Target gene prediction and GO classification results indicated that85miRNAs targeted236target genes, which involved in all kinds of biological processesincluding transcriptional regulation, growth and development, protein transport, stressresponses, hormone signaling, electron transport and so on.(4) The expression profiles of29miRNAs reduced during the early period of seedgermination,8miRNAs demonstrating a regular expression change pattern during the processof plant growth and development. The expression profiles of29miRNAs were analyzed by qRT-PCR during seed germination (0d,1d,5d,9d and28d) and plant growth (90d,1.5a,5a,10a,25a and50a) in larch. The results shown that all miRNAs were highly expressed in dryseed stage (0d) than subsequent stages, and the expression levels of13miRNAs (44.83%) allwent down gradually as a whole along with the progression of seed germination. The relativeexpression level of miR894was the highest, while the one of miR408was the lowest at acertain stage during seed germination. The expression levels of8miRNAs presented gradualupward or downward trends with increasing plant age. And5a and25a were the turning pointsof miRNAs differential expressions, which revealed that the two periods may be the criticalperiods of miRNA regulating the larch growth. The spatiotemporal and differentialexpressions of miRNAs may play important roles during seed germination and plant growth.(5) The stabilities of12candidate internal reference genes were evaluated by geNorm andNormFinder softwar. The expressions of the tree candidate internal gene, APm,EF1and eIF,were most stable among different organs and developmental stages (somatic embryogenesis,seed germination and plant growth stage), providing optimum internal reference genes formiRNA target genes expression analysis using qRT-PCR.(6) The predicted target genes of miR172, miR397and miR398were AP2L1, JR147962(Laccase) and JR143920(Phytocyanin) respectively. The target genes were found to becleaved most between10thand11thbase of complementary region of miRNAs and their targetgenes. The significant negative linear relationship between miR172and its target gene (AP2L1)was minus0.960(P=0.002).In conclusion,174miRNAs from85families were identification, and8miRNAs mightbe involved in the regulation of growth and development of L.leptolepis. The significantnegative linear relationship between miR172and its target gene (AP2L1) was minus0.960.AP2L1is a crucial regulator being of great value for functional molecular studies duringconifer tree seed germination, growth and development. These results expand the number ofknown conifer miRNAs and provide a foundation for research on the expression patterns andfunctions of these miRNAs during individual development in conifer tree.