Identification Of MiRNAs From Brassica Napus And Expression Regulation Of Bna-miR1140

MicroRNAs (miRNAs) are non-coding RNAs of approximately21nucleotides that havebeen identified as important regulators of gene expression in both animals and plants. In plants,miRNAs not only post-transcriptionally regulate their targets but also interact with each other inregulatory networks to affect many aspects of development, such as developmental timing,senescence, leaf morphogenesis, reproductive development, and modulation of root architecture.miRNAs are also reported to be involved in plant responses to biotic and environmental stresses.miRNAs are in the core position of the gene expression regulation network, and effectively inhibitits target genes encoding the proteins, or inhibit the expression of target genes via otheradjustment mechanism, resulting in the phenomenon of gene silencing. In the past decades, theplant miRNA research has been made great progress and Significant achievements.As adicotyledons, oilseed rape (Brassica Napus) is one of the most important oil crops,its miRNAhas also become a hot research field of molecular biology today. The studies about miRNAgenetic control mechanism involved in the regulation of rapeseed development can improve thetheoretical basis of the developmental biology of higher plant. Meanwhile, the clone andidentification of miRNA gene involved in the regulation rapeseed development, combined withmolecular breeding means to foster the ideal rape varieties for increasing yield of rapeseed andreducing the rapeseed production costs are very practical significance.So far, the reports about studies of rapeseed miRNAs and its targets are much less, andmiRBase currently lists48miRNAs forming17miRNA families in Brassica napus. Here, wedescribe the high-throughput sequencing analysis of sRNAs from a cultivated variety of B. napus,cv Westar, using the Illumina Solexa platform. The sRNAs library was prepared for Solexasequencing from greenhouse cultivated rape plants, new miRNAs and target genes of knownmiRNAs and newly discovered miRNAs have been identificated. In order to provide a theoreticalbasis for rape breed improvement and rape miRNAs regulatory mechanism, the expressionregulation mechanism for the Bna-miR1140of Brassica unique miRNA families has also beenstudied. The methods are as follows, Leaves, petiole, stalk, roots and shoot apices from onemonth-old seedlings were collected and total RNA from different tissues was extracted usingTrizol (Invitrogen). sRNA and degradome cDNA libraries for Solexa sequencing were constructedfollowing the Illumina protocol. The purified cDNA library was sequenced on an IlluminaGAIIx(Biological Information Technology Co., Ltd., Hangzhou LC). Bioinformatic analysis ofthe sequencing data has been carried to identify known and new miRNAs and its target genes;Stem–loop RT primers, universal reverse primer and miRNA-specific forward primers for20sequenced miRNA sequences were designed according to Varkonyi-Gasic to validate and measurethe levels of B. napus miRNA by qRT-PCR. The primers for the precursor of Bna-miR1140 sequence were designed on the basis of the miRNAbase and its full-length was obtained by PCRamplification of Rape genome database, Then the35S:: Bna-miR1140plants over expressionvector constructed infected the cotyledons of wild-type rapeseed varieties by the Agrobacteriummediated. The phenotype of positive plants with the Bna-miR1140transformed was analyzed; Thesequence of the Bna-miR1140upstream1.5kb was analyzed via plantCARE software, finding thekey elements of promoter, such as the TATA-box, G-box et al., and then design primers for it andamplify the1.5kb fragment from rapeseed genomic DNA and constructed miR1140pro::GUSplant expression vector. The tissue of positive lines with miR1140pro::GUS transformed wasanalyzed by GUS staining to analysis the Bna-miR1140expression specificity.Through the experimental analysis, the results as follows:1. Here,41new conserved and67brassica-specific candidate miRNAs, including20miRNA*sequences were firstly identified. The sequencing results were further confirmed using stem-loopquantitative RT-PCR. The data will be updated to incorporate future miRBase updates. Ourapproach leads to the prediction of several conserved and specific brassica miRNA targets in theavailable EST and genomic databases.113mRNA targets of conserved brassica miRNAs and84mRNA targets of new brassica-specific miRNAs were identified. Validated miRNA targets in B.napus are potentially involved in diverse biological processes, including phase transitions,flowering, hormone signaling, photosynthesis, metabolism and biotic and abiotic stress resistance.Our findings will be a useful resource toward tracing the evolution of small RNA-based regulationin Brassica napus and related species. Most importantly, this study will serve as a foundation forfuture research into the functional roles of miRNAs and their target genes in this important oilcrop.2. The precursor of Bna-miR1140gene cloned from Rape is197bp, and the plant overexpression vector35S::Bna-miR1140was constructed successfully. After disseminating thepetiole of rapeseed cultivars Westar via agrobacterium mediated transformation method. At Last,14T0generation Rape positive strains of over expressed35S::Bna-miR1140have been identifiedby the PCR method. There were5strains performed specific plant type, they had double maininflorescence and significantly increased the number of branches, nine positive strains consistentwith the wild-type rapeseed phenotype. After investigating the phenotype separation of five T1generation lines with specific plant type, and, of which there are four strains, their plant typevariation genetic followed3:1Mendel segregation law by the chi-square test. As for the otherpositive strains of over-expressed the35S::Bna-miR1140were not performed variation, they maynot express the35S::Bna-miR1140or transformed copy numbers are few, the real reasons must befurther validated by Northern blotting and other molecular experimental means in future. By phenotypic analysis, initially speculated the Bna-miR1140may participate in the regulation ofbranching and development of oilseed rape.3. The1351bp fragment upstream of Bna-miR1140gene has been isolated, and the cis-actingelements of the promoter sequence were analyzed by plantCARE online software, the promotercontains TATA boxes binding RNA polymerase and CAAT boxes which are necessary fortranscription, because it play important role in the regulation of gene transcription efficiency. ThemiR1140pro::GUS plant expression vector was further constructed. Similarly, five positive T0rape strains transformed miR1140pro:: GUS were got, which have been transformed byagrobacterium-mediated infection rapeseed petiole of cultivars Westar. The GUS staining analysisshowed that the1.5kb region of upstream miR1140precursor sequence have promoter function bystaining each tissue of positive rapeseed strains, which able to drive the expression of GUS in rape.The miR1140pro::GUS transgenic rapeseed roots, stems, leaves, leaf axillary (shoot apexpoints Health organization, SAM), petiole, flower, pods and other tissue were carried GUSstaining analysis, the results showed that GUS only expressed in petioles and leaf axillary, thatconsistent with the results of the Bna-miR1140vivo expression, which is further corroborated theBna-miR1140regulation rape branching and development.Summing up the results of the study, we found that the results of the Bna-miR1140vivoexpression and miR1140pro:: GUS expression patterns are consistent, proving Bna-miR1140play a role in rape branching development process; By solexa sequencing technology, many newconservative and specific miRNAs have been identified, finding that the target gene ofBna-miR1140are glycosyltransferase and the response to the adjustment factors.