Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

BACKGROUND: Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. METHODS: Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. RESULTS: In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. CONCLUSION: We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Molecular mechanism of ethylene stimulation of latex yield in rubber tree (Hevea brasiliensis) revealed by de novo sequencing and transcriptome analysis.

BACKGROUND: Rubber tree (Hevea brasiliensis) is an important industrial crop cultivated in tropical areas for natural rubber production. Treatment of the bark of rubber trees with ehephon (an ethylene releaser) has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. Deciphering the enigma is of great importance for improvement of rubber tree for high yield. RESULTS: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 h (E8) and 24 h (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. CONCLUSIONS: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.

Transcriptome sequencing and analysis of rubber tree (Hevea brasiliensis Muell.) to discover putative genes associated with tapping panel dryness (TPD).

BACKGROUND: Tapping panel dryness (TPD) involves in the partial or complete cessation of latex flow thus seriously affect latex production in rubber tree (Hevea brasiliensis). Numerous studies have been conducted to define its origin and nature, but the molecular nature and mechanism of TPD occurrence remains unknown. This study is committed to de novo sequencing and comparative analysis of the transcriptomes of healthy (H) and TPD-affected (T) rubber trees to identify the genes and pathways related to the TPD. RESULTS: Total raw reads of 34,632,012 and 35,913,020 bp were obtained from H and T library, respectively using Illumina Hiseq 2000 sequencing technology. De novo assemblies yielded 141,456 and 169,285 contigs, and 96,070 and 112,243 unigenes from H and T library, respectively. Among 73597 genes, 22577 genes were identified as differential expressed genes between H and T library via comparative transcript profiling. A majority of genes involved in natural rubber biosynthesis and jasmonate synthesis with most potential relevance in TPD occurrence were found to be differentially expressed. CONCLUSIONS: In TPD-affected trees, the expression of most genes related to the latex biosynthesis and jasmonate synthesis was severely inhibited and is probably the direct cause of the TPD. These new de novo transcriptome data sets provide a significant resource for the discovery of genes related to TPD and improve our understanding of the occurrence and maintainace of TPD.