Comparative Proteomics Of Tuberous Root In Cassava(Manihot Esculenta Crantz)during Root Development

Cassava (Manihot esculenta Crantz), originated from the Amazon Basin of South America, is an important tropical starch-rich crop and also one of a concernful food and economic crops in tropical and subtropical regions. The tuberous roots of cassava contain as high as32%of starch content which has great economic value. Therefore, it’s crucial for us to study the molecular mechanism of starch synthesis and accumulation during the tuberous roots development, which is of significant importance for improving quality and breeding new cassava varieties with higher cassava starch using of biological engineering, and provides theoretical guidance for agricultural product.In this study, we have established the technology system of proteomics using SC8cassava as material. To explore the change patterns of protein during root bulking development, we analyzed the root proteomes in different development stages of cassava SC8by comparative proteomics, and focused on the group of proteins involved in starch synthesis, sugar metabolism, which was aimed at searching for the key proteins in regulating the starch synthesis. The main results were presented as the following:1. Light microscopy observation on cross-sections of different tissues found that leaf stockade tissue and sponge tissue contained abundant proteins, stem phloem and epidermal cells had corpuscular proteins, but fibrous and tuberous roots contained a few proteins, especially root xylem. Simultaneously, starch particles were found in raycells of secondary phloem and xylem of fibrous and tuberous roots.2. To set up the technology system of proteomics in cassava, high quality proteins have been obtained from different tissues in cassava by BPP method, and high resolution and repeatability gel maps of1-DE and2-DE using GAP staining method were also presented in this study, and these methods demonstrated good mass compatibility. The simple protein in-gel digest method had high efficiency in digestion, and we could acquire the pruducets with little impurity. Finally, we identified93credible proteins by mass spectrometry analysis.3. Light microscopy observation on cross-sections of cassava root at different development stage found that there are six protoxylem bundles in developing tuberous root. With cambial activity, xylem parenchyma were strongly divided at2month after planted (MAP) leading to root diameter increased. The tuberous root was largely swollen at2-6MAP. Starch began to accumulate in parenchyma cells of secondary phloem and secondary xylem, and then starch content increased gradually. Granular proteins accumulating in cortex and extern phloem cells were observated during the whole development process. However, the granular proteins disappeared gradually at maturation stage.4. During the later stage and mature period of tuberous root development, the protein content in tuberous root was decreased firstly, then increased, and finally stabilized. The protein content wasn’t significantly changed.5. Comparated with2-DE maps in different roots development, we found154proteinswith good separation and obviously expressional changes by MALDI TOF and MALDI TOF/TOF MS identification, among which about94proteins were credible verified corresponding to80proteins. These proteins could be devided into11categories, such as carbohydrate transport and metabolism, posttranslational modification, protein turnover, chaperones, energy production and conversion, et al.6. Blast2Go analysis found that the differences expression proteins distribution in27of metabolic pathways, among which starch synthesis and sugar metabolism, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, methane metabolism, glutathione metabolism, pyruvate metabolism and vitamin C metabolism were the main pathways. The lots of proteins involved in sugar metabolism suggested sugar signal regulation pathway may play important roles in the process of roots development and starch accumulation.7. Identified some key enzymes related to starch synthesis, such as AGPase small subunit, starch phosphorylase, and several proteins involved in signal transduction, for example14-3-3family protein, cyclophilin et al., which provided informations for verifying the roles of these proteins in the process of root development and starch accumulation.In this study, proteomic technologies for different tissues in cassava were primarily established, and then systematic comparison analysis of the tuberous root proteome in cassava SC8during development was perfomed. This work provided some interesting information for further research on tuberization process and starch accumulating regulation.