| Creating new germplasm resources by transgenic methods is a fundamental way to broaden the maize germplasm resources which is relatively strait in China. The conventional way of cultivating new transgenic variety of maize consists of several critical steps including genetic transformation, self-crossing for homozygosis, crossing combination, and back crossing, among which the process from self-crossing transformants to acquiring the transgenic elite inbred lines by backcrossing usually takes at least3to6years.The infeasibility of using embryonic callus as the transgenic recipient material directly, which is attributed to the low frequency of maize embryonic callus introduction being affected by genotype significantly in the majority of elite inbred lines of maize, severely hinders the rapid development of breeding for transgenic maize in China. By regulating plant gene expression through ways such as directing gene splicing or suppressing transcription, miRNA, a class of endogenous non-coding RNA, involves widely in every stage of plant growth and development, as well as responds to the adversity stress.In this study, the maize inbred lines18-599R with the strong ability of embryonic culturing were used as test materials, many candidate miRNAs involved in the process of embryonic callus formation in immature embryos of18-599R under the2,4-D induction were screened based on the initial research combining Solexa high-throughput sequencing with bioinformatics analysis methods; by combing the RT-PCR with miRNA overexpression technology, expression of important candidate miRNAs were detected and the function of target genes were verified; information of target genes of miRNA involved in the formation of embryonic callus was acquired through the high-throughput Degradome Sequencing for target genes. And the final results are as follows;1.78differentially expressed miRNAs from25known conservative miRNA families were screened out by Solexa high-throughput sequencing. From the control to the last3stages (Stage Ⅰ,Stage Ⅱ and Stage Ⅲ) of callus induction,45miRNAs accounting for the57.69%of differentially expressed miRNAs were up-regulated, while23miRNAs accounting for the29.49%of total were down-regulated; in addition,10miRNAs accounting for the12.82%of all were up-regulated as well as down regulated. 2.13miRNAs detected in this study were of significant sequence conservation with members of8known maize miRNA families including zma-miR167、zma-miR172、 zma-miR319、zma-miR393、zma-miR396、zma-miR397、zma-miR408and zma-miR528, the further bioinformatics analysis confirmed that they are novel members of those miRNA families.3.7new miRNAs belonging to4miRNA families named zma-miR701, zma-miR702, zma-miR703, zma-miR704, respectively, with various levels and types of differentially expression during the formation of callus, were identified by high-throughput sequencing. Those new miRNAs with the length ranging from21to22nt and3’in the stem of secondary structure, are of relatively low refolding free energy and emblematical secondary structure.4. According to the cluster analysis, expression models of those sequencing-detected differentially expressed miRNAs of know families were categorized into four groups including down-regulating model, inconspicuously differential expression model, up-regulating model, significantly up-regulating model. Based on the further analysis for the4expression models, we presume that Stage II(the stage of the formation of primary callus) may be the most important stage during the formation of embryonic callus, and closely related to the formation of embryonic callus of maize.5.14of the differentially expressed miRNAs detected by sequencing were verified by RT-PCR, showing that10were significantly up-regulated while the rest4zma-miR156a, zma-miR167a, zma-miR1671and zma-miR827were down-regulated. In addition, the correlation coefficient of the results of qRT-PCR verification and sequencing is above0.94, indicating the high accuracy of sequencing results in this study.6.213target genes sliced under the induction of22known miRNA conservative families were identified by the degradome sequencing. These target genes involved in the regulation of processes including plant growth and development, transcriptional regulation, stress responses, hormone signaling, plant metabolic pathways can be categorized into three types:cell composition, molecular function and biological process. Their target gene functions involve transcription factors including SBP, TCP, GAMYB, ARF, F-box and GRAS.7. The hormone signaling pathway was identified being significantly correlated with the formation of embryonic callus by KO analysis for the results of degradome sequencing. 14target genes were differentially expressed in this pathway under the negative regulations of4miRNAs including zma-miR160, zma-miR167, zma-miR393, zma-miR394, respectively.8. According to the phenotype identification for the embryonic callus induction rate of immature embryo of T1generation transgenic positive plants, the induction rate of transgenic plants overexpressing zma-miR159a or zma-miR393a is significantly or extremely significantly lower than that of wild-type controls. There is a difference between the results and the expected transgene results... |
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