| Cassava (Manihot esculenta Crantz), an important cultival, belongs to the Manihot genus in the Euphorbiaceae family. Cassava is native to the Amazon region of South America, and an important root crop in the tropical, subtropical, African and Latin America. The root is the main starch-storing organ of cassava, the major source of food for8billion people in the tropical developing countries, and the important raw materials for the production of starch and bioethanol in Tropical Asia. However, the research on cassava root starch metabolic and accumulation is still rare currently, so the further study of regulation mechanism of its starch accumulation is needed. What’s more, study of the function and expression of the genes correlated with cassava root starch metabolism can provide basis for in-depth understanding of the regulation mechamisms of starch metabolism in cassava roots and the promotion of cassava breeding.On the basis of previous comparative proteomics studies, we used SC8cassava as material in this study to analyze the expression profiles of several genes, which could be involved in the regulation of starch metabolism at different developemental stages of cassava root by molecular biology methods. Results are listed as follows:1. Ten differentially expressed protein genes from cassava root at different developmental stages were analyzed by RT-PCR and qRT-PCR technology. Results showed that:eukaryotic transcription initiation factor5A1,14-3-3protein, ascorbic acid peroxidase APX2, Cu/Zn-superoxide dismutase, S-adenosylmethionine synthase and malate dehydrogenase, genes were significantly upregulated during the storage root thickening(120-210days after planting).14-3-3protein may be involved in signal regulation or directly involved in starch biosynthesis; as the only methylation donor, s-adenosylmethionine synthetase may be involved in the synthesis of lignin as the only methylation donor, related to root development of cassava; malate dehydrogenase may be involved in the synthesis of starch by gluconeogenesis.2. Based on the peptide sequences identified by mass spectrometric, we identified several proteins, which may be involved in the regulation of starch metabolism, and took RACE (Rapid Amplication of cDNA End) method to clone the related gene sequences. The full length of malate dehydrogenase gene was1175bp with an open reading frame of999bp, encoding a protein with332amino acids. The full length of UDP-D-apiose/UDP-D-xylose synthase gene was1661bp with an open reading frame of1167bp, encoding a protein with389amino acids. Partial sequences of ADP-glucose pyrophosphorylase small subunit gene and starch phosphorylase1precursor gene were obtained.3. A plant expression vector for14-3-3protein were constructed and transformed into Arabidopsis by Agrobacterium-mediated method. We obtained several lines of transformants.4. Prokaryotic expression vectors for14-3-3protein, malate dehydrogenase and UDP-D-apiose/UDP-D-xylose synthase were constructed. The recombinant plasmids were transformed into the E.coil BL-21by a chemical transformation. Target proteins were induced by IPTG and indentificated by SDS-PAGE. We used the His purification columns to obtain three purified proteins, which provided materials for antibodies preparing and Western Bloting.5. Comparisons of the soluble sugar and starch content of SC8, SC5and SC124cassava varieties, disclosed that:the tapioca starch content of SC8was most abundant, followed by SC5and SC124. The soluble sugar content was related to the growth characteristics of the varieties. Then we analyzed the gene expression differences of ADP-glucose pyrophosphorylase and starch phosphorylase1precursor at different developing stages of cassava root by qRT-PCR technology, and we found ADP-glucose pyrophosphorylase small subunit gene mainly expressed in early developmental stage of SC124and storage root thinckening stage of SC5and SC8, and the starch phosphorylase1precursor gene low expressed in late root thinckening stage and mature stage.Based on the comparative proteomics results on the early developmental stages of cassava roots, this study identified several functional genes which could be related to cassava root starch storage and metabolism, coloned the cDNA sequences of these genes, and several expression vectors for further gene function verification experiments were constructed. Furthermore, the expression characteristics of these genes in different cassava varieties were studied. The results provided basis for further studies on the function mechamisms of these genes, provided references for further studies on the regulation mechamisms of starch storage and metabolism in cassava roots and for the cassava molecular breeding studies. |
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