Cloning And Identification Of MicroRNAs From C-Type Cytoplasmic Male Sterile Line And Its Maintainer In Maize

As a class of negative regulators in eukaryote organism,miRNA(abbreviated to "miRNA")regulations almost reach to all cellular levels of organism.It has only been about six years from the first plant miRNA was found in 2002,a great deal of research findings about plant miRNAs have been reported.However the identified plant miRNAs are still far from saturation,and understanding about miRNA founction is being faced with some formidable problem.Being a non-model plant,Zea mays fall behind Arabidopsis and Oryza sativa on miRNA research accordingly.And only 96 identified miRNAs from Zea mays were submitted to miRNA database so far.For this reason,cloning and characterization of miRNA in maize is being a hot academic domain in the future.In the research,miRNAs were abstracted and cloned from C-type cytoplasmic male sterile line 48-2 and its maintainer in maize,and target genes of miRNA were subsequently predicted.As a result,some conclussions were made concerning relationship between miRNA regulation and C-type cytoplasmic male sterility in maize by assay of miRNA content differentiation and relative function of miRNA targets.The findings of the research are as follows:1.Eighty one sequences sized from 18 to 26nt were cloned,of which the majority has the length ranging from 20 to 24nt.The sequences,excluding tRNA, rRNA and degraded fragments,were analyzed by m-fold program for stem-loop.The results showed that ten small RNAs could form stem-loop when joined with flanking sequence in genomes.2.The lengths of the ten sequences range from 21 to 24nt,and five of the sequences began with "U" from 5' terminatio.The trend is similar to what have been reported in other plant miRNAs.By "blast" genomes of other plants including rice, wheat and Arabidopsis,conservation of miRNA was identified among them.As a result,six miRNA sequences exhibit conservation to a large extent.On the other hand, there is a trend that these miRNAs were more conservative among maize,rice and wheat than between maize and Arabidopsis. 3.By RNA-tailing and primer-extension RT-PCR,the ten small RNAs were detected in both anthers of line C48-2 and anthers of line N48-2,which indicates their reality in maize.In according to above reasons,the ten sequences were identified to be new microRNAs in plants,and attributed respectively to 10 new miRNA families.4.Then targets were respectively predicted for 9 of the miRNAs by a series of methods including using of miRU program,searching CSRDB and NCBI,analyzing fuctional domain of amino acid by "Pfam".Resultantly,the targets are involved in 3 such transcription factors as MADS-box,3 important enzymes ralative to anther metabolism,named respectively NADH dehydrogenase,cytochrome P450 monooxygenase and dihydroflavonol-4-reductase,1 zein-alpha,1 integral membrane protein and 1 antioxyen molecular chaperones.Among them,NADH dehydrogenase, cytochrome P450 monooxygenase and dihydroflavonol-4-reductase have been confirmed to participate in the process of energy or substance metabolism of anther, and be tightly related to plant male sterility.As to MADS-box genes,they are regarded as a class of transcription factors concorning plant male sterility,and have got close attention in recent year.5.The contents of the ten miRNA in anthers of both C48-2 and N48-2 were assayed by real-time PCR.The result showed the miRNA contents in N48-2 was significantly larger than that in C48-2 at tetrad stage.In addition,3 of the ten miRNAs also displayed distinct expression at monocaryon stage between the two lines.Namely, the content of miR-1 in line C48-2 was smaller than that in line N48-2,but it is the adverse case for miR-6 and miR-10.6.By analyzing both distinction of miRNA contents between sterile line C48-2 and N48-2 and function of target genes,the relationship between miRNA regulation and cytoplasmic male sterility in maize got explaination to some extent,as follows:First,from tetrad stage to monocaryon stage,expression of the ten miRNA in line N48-2 downregulated,which is essential for fertility of line N48-2 probably.On the other hand,it may be the result of normal development for anther.By the regular downregulation of the ten miRNA in line N48-2,it can be conclude they get involved in the progress of anther development.Second,as to miR-1,miR-2 and miR-6,their regulation is responsible for male sterility both at tetrad stage and in monocaryon period for expression disparity between the two lines is exhibited at both the two stages.With regard to miR-3, miR-4,miR-5,miR-7,miR-8,miR-9 and miR-10,pathways of C-type cytoplasmic male sterility mediated by their regulation occur dominately in tetrad period;Third,miRNA regulation may be the reason of male sterility,namely,dramaticly abnormal contents of miRNAs in sterile line make significantly different conents of mRNA of important enzymes and transcription factors ralative to anther metabolism, lead to disorder of substance and energy of anther subsequently,and result in abnormal development of pollen at last;Finally,inordinate of miRNAs in male sterility has the chance to be the result from male sterility.In other words,in the effect of male sterility,expression of some protein is disturbed,including enzyme and transcription factors responding for biosynthesis of miRNA.Accordingly,normal expression of miRNAs is inhibitted. Furthermore,the gene regulated by miRNAs fails to normal expression.