Function Analysis Of Transcription Factors (DISCLl6and DIAP2) And Identification Of MiRNAs From Dendracalamus Latiflorus

Ma bamboo (Dendrocalamus latiflorus) is one of the most widely cultivated bamboospecies with high economic value in the south of China, which has a variety of uses for food,timber and afforestation. The regulation of gene expression in plant precisely occurred at thelevels of transcription and translation. Research on bamboo mechanism of gene expressionregulation is of great value for deeper understanding of the phenomenon of bamboo life andbetter development and utilization of bamboo resources. Transcription factor (TF) andmiRNAs, which involved in the gene regulation at transcription and post-transcription levelsrespectively, are two important ways of controlling gene expression in plants. In this paper,two plant-specific transcription factors (SCL6and AP2) from ma bamboo were studied. At thesame time, isolation and identification of miRNAs from leaves of ma bamboo were carried outthrough deep sequencing, the expression of some novel miRNAs were further analyzed byusing qRT-PCR. The main research results are as follows:(1) Two plant-specific TF genes DlSCL6and DlAP2were cloned from ma bamboo leavesusing RT-PCR and RACE methods. The cDNA of DlSCL6is2006bp in full length including124bp in5’ UTR,266bp in3’ UTR, and an ORF of1611bp which encodes536amino acids.The cDNA of DlAP2is1731bp in full length and consists of a5’ UTR of81bp, a3’ UTR of186bp and an ORF of1464bp which encodes487amino acids.(2) Expression vectors of sense and antisense DlSCl6gene were constructed, and transformedinto Arabidopsis thaliana mediated by Agrobacterium tumefaciens respectively. Then thetransgenic plants were identified by RT-PCR method. Phenotype observation showed: thattransgenics plants with sense DlSCL6gene had prolonged vegetative growth period, delayedblooming, stronger stem, and more rosette leaves, while the transgenics of antisense DlSCL6geneshowed opposite phenotypes: such as ealier precocious flowering, weaker stem and less rosetteleaves. All of these phenotypes are similar to the phenotypes of ham deficient mutant in A.thalianaand petunia. All of this results indicated that DlSCL6has similar function with HAM genes in A.thaliana and petunia, and maybe play important roles in the maintenance of bamboo stem apicalmeristem (SAM).(3) Overexpression vector of DlAP2gene was constructed, and transformed intoA.thaliana by Agrobacterium-mediated method. The transgenic plants were selected byHygromycin B, and DlAP2was proved expression at transcription level by RT-PCR method.The transgenic plants bloomed earlier than that of wild type, which indicated that DlAP2maysplays a role as blooming promoter.(4) The miRNA library was constructed with leaves of ma bamboo using Illumina kit.11513607raw reads were obtained using Solexa high-throughput sequencing technology. Afterremoving the sequences of low quality,5’end of pollution,3’ end joint deletion, insertion,polyA and less than18nt sequence,10593305clean reads was left. Bioinformatics methodswere used to forecast possible miRNAs, and total165candidate miRNA was identified, whichincuded84conversed miRNAs (54mature miRNAs and30star miRNAs) belongs to17converse miRNA families and84novel miRNAs (76mature miRNAs and5star miRNAs).Based on two data sets of CDS for moso genome and ESTs for ma bamboo,176potentialtargets for81novel miRNAs were forecasted.(5) Highly specific stem-loop reverse primers were designed according to the miRNAssequences, and30higher expression novel miRNAs were detected by qRT-PCR methods.Highly specific dissolution curves indicated that these miRNAs were really exist in ma bamboo.Further analysis for the expression of these miRNAs in leaves under different light conditionsshowed that dla-miRC18, dla-miRC27and dla-miRC27-3p were upregulated significantlyunder high light tress (1200μmol/m2/s1), in which dla-miRC18was the hightest one with up to15times compared to that of control, whilt dla-miRC1, dla-miRC19and dla-miRC28weredownregulated, decreased by62%-76%. Under dark conditions for24h, only dla-miRC1anddla-miR22were significantly upregulated, about4times of the control, contrarily dla-miRC5,dla-miRC17and dla-miRC29downregulated significantly (decreased by50%-72%). All theresults above indicated that these miRNAs might play important roles in genes regulationinvolved in light signal transduction and light stress. The expression of DlSCL6and DlAP2in Arabidopsis thaliana showed that both DlSCL6and DlAP2gene play roles in the control of transition from vegetative growth to reproductivegrowth, which provides some reference for the mechanism study on bamboo blossom.81newmiRNAs were identificated from bamboo leaves, and the expression changes of30novelmiRNAs under different light conditons were studied, which lays a foundation for further studyon their functions.