ABSTRACT
miRNAs are important regulators of plant gene expression. There are few studies on the regulation of miRNAs in Lonicera edulis. We used high-throughput sequencing technology to analyse miRNAs in L. edulis, aiming to identify miRNAs and elucidate their function in L. edulis. In the present study, we employed the high-throughput sequencing technology to profile miRNAs in L. edulis. A total of 51,819,072 small RNA tags with sizes ranging from 18 to 30 nt were obtained, indicating that L. edulis have a large and diverse small RNA population. Bioinformatic analysis identified 507 mature miRNAs, and 16 predicted novel miRNAs that are likely to be unique to L. edulis. Three miRNAs related to anthocyanin biosynthesis were locked by gene ontology (GO) analysis and target gene analysis. The selected three miRNAs are relatively high in the expression of L. edulis. Some of the previous studies have studied these types of miRNAs involved in the anthocyanin metabolism pathway in fruits. Among them, expression profiles of three conserved miRNAs were validated by stem loop qRT-PCR. Further, the potential target genes of conserved and novel miRNAs were predicted and subjected to GO annotation. Enrichment analysis of the GO-represented biological processes and molecular functions revealed that these target genes were potentially involved in a wide range of metabolic pathways and developmental processes. In particular, different families of miRNAs can directly or indirectly regulate anthocyanin biosynthesis. In recent years, the research on miRNAs has become more and more clear, but the research on miRNAs involved in the regulation of anthocyanin synthesis of L. edulis is still lagging. This study provides a useful resource for further elucidation of the functional roles of miRNAs during fruit development and ripening
ABSTRACT
Insects pests and weeds are the main factors that reduce the yield of sugar beet. Genetic engineering breeding is an effective method to breed insect-resisitant and herbicide-resisitant sugar beet. A transformation system for foreign genes in sugar beet chloroplast was established. The expression of the foreign genes can confers resistance in transgenic sugar beet plants to insects pests and weeds. The chloroplast transformation vector pSKARBt/bar, which carries Bt cry1Ac gene and bar gene expression cassettes, was constructed by using molecular method. The Bt gene expression cassette contained the 3.5 kb Bt cry1Ac gene under the control of psbA promoter and terminator cloned from sugar beet chloroplast genome. The bar gene expression cassette contained the bar gene, 16 S promoter and terminator cloned from sugar beet chloroplast genome, The atpB and rbcL gene cloned from sugar beet chloroplast genome were used as homologous fragment, the bar gene was the selective marker. Plasmid pSKARBt/bar were transformed into the petioles of sugar beet with particle bombardment method. The petioles were planced onto the shoot-inducing selection medium which contained spectinomycin (20 mg/L), 6-BA (1.5 mg/L) and NAA (0.2 mg/L) at first. And when the green shoots regenerated, the green shoots were transfered into the shoot-propagation medium for optimal shoot development which contained spectinomycin (20 mg/L) and 6-BA (0.5 mg/L) and NAA (1.0 mg/L) one subculture at 20-day intervals, and then the shoots were transfered into the shoot-propagation medium for optimal shoot development with herbicide (PPT 10 mg/L) several subcultures. The shoots were transfered into the root-induction medium with herbicide (PPT 10 mg/L) and the transgenic plants were obtained at last. The transgenic sugar beet plants were testsed by PCR and Southern blot. The results showed that the Bt gene and bar gene had been transferred into the chloroplast genome of sugar beet. The transgenic plants had tolerance to both PPT and bioassays testsed. The insecticidal activity (the mortality of larvaes was 33%～80%) and herbicide resistance of the transgenic plants indicated that the relevant protein had been expressed already in sugar beet. The study showed that the bar gene can also be used as an efficient selective marker gene besides antibiotic resistant markers in plant transformation. Efficient transformation system in sugar beet chloroplast had been established.