The Primary Study On Photosynthesis In Aspect Of Physiology, Structure And Transcriptome In Leaf Of Cassava(Manihot Esculenta)

Cassava is one of the top six food crops, which is known as "the king of starch" in the world. Cassva cultivated variety has a high photosynthesis efficiency and high starch characteristics. However, cassava is still a lack of theories on the mechanism of high photosynthesis. Photosynthesis is a complex processe of life and the basis of material production. Studying on the mechanisms of high photosynthesis efficiency in cassava can not only for the rich theories of cassava research, but also provide a theoretical basis for cassava breeding and germplasm screening. Therefore, this study selected cassava cultivars Arg7(Manihot esculenta) and the wild species of W14,(Manihot esculenta, subsp. Flabellifolia), which have significant difference in the starch content of the tuber roots, as test materiala, and investigated the characteristics of physiology and structure of leaves, the photosynthesis-related key gene expression levels to explore the reasons for the differences of two cassava photosynthetic efficiency, as well as the mechanism of high photosynthesis efficiency of the cultivated type cassava. The main results were as follows:1. The net photosynthetic efficiency of cassava cultivar was higher than the wild species and both showed higher PEPC activity in their leaves. But the cultivar one showed higher activity of PEPCase and Rubisco which were corresponding for carbon fixation in leaves. SuSy activity performance was on the opposite way.Tracing the Pn and several photosynthesis key enzyme activities of the two cassava species during the whole growth period showed that, from the120d to270days after planting, the Pn was always higher in the function leaves of Arg7than in W14and the two highest difference was10μmol· m-2s-1. The activities of Rubisco, PEPCase and AGPase were always higher in functional leaves of Arg7than in W14, while the activity of SuSy was higher in W14. The peak activies of the4enzymes were at about180days in the two cassava species. The function leaves of Arg7and W14showed high activity of PEPCase. The stems of the two cassva species showed a higher SuSy activity. Chlorophyll content is always higher in W14.2. The vascular cells surrounding the leaf veins were more developed in Arg7than in W14and showed similar arrangement as "Kranz" structure. There were obvious palisade tissue and spongy tissue in Arg7leaves. Through the analysis of the leaves structure, we found that the vascular cells surrounding the leaf veins were more developed in Arg7than in W14and the vascular cells were extending to the region of the palisade tissue. But both of Arg7and W14had palisade tissue and spongy tissue in leaves just like other kindsc of C3plants. It was worth noting that the ultrastructure of leaves showed similar "Kranz" structure in Arg7. Plasmodesmata were present between the mesophyll cells of two cassava species. Chloroplast was well-developed in Arg7than in W14. In addition, both of Arg7and W14had starch granules in chloroplasts, and the starch granules in Arg7were smaller than in W14, and the chloroplast Arg7within the starch granules.3. The immunofluorescence location showed subcellular distribution and characteristics of the semi-quantitative of Rubisco and PEPCase. PEPCase showed specific localization in mesophyll cells of Arg7.Immunofluorescence localization of Rubisco showed no differences between Arg7and W14, but result of PEPCase showed specific localization in mesophyll cells of Arg7, while the rest distributed in other mesophyll cells.4. We constructed a full-length cDNA library from cassava, obtained5013ESTs, assambled1259unigenes from the sequence data. Finally, we found22clones related to the genes which encoding the enzymes in the pathway of photosynthesis and stach metabolism.We constructed a full-length cDNA library from leaf and root tissues of the Manihot esculent a cultivated variety Arg7and the W14strain of its ancestor, Manihot esculenta subsp. flabellifolia. The library, which comprises four sub-libraries, contains32640recombinant clones. We sequenced6176randomly selected clones and obtained5013ESTs (NCBI access number JK733886-JK738898), as well as1259unigenes. Overall323(25.76%) of the unigenes were assigned to114different pathways, with some unigenes assigned to more than one pathway of which8unigenes related to photosynthesis and5unigenes related to stach metabolism. Finally, we found22clones related to the genes which encoding the enzymes in the pathway of photosynthesis and stach metabolism in our library.5. We obtained a large number of genes expressed in leaves of cassava and found27genes related to photosynthesis. However, no significant differences were found in the expression of genes in the two cassava species. Six RNA samples were extracted from functional leaves, respectively. The original sequencing data were assembled into131256unigenes. Transcriptome analysis showed that the unigenes of functional leaves from the two manihot species were annotated into27genes encoding photosynthesis-related enzymes, and six samples form the two manihot species were annotated into44genes encoding starch-metabolizing enzymes.6. The full-length cDNA of phosphoenolpyruvate carboxylase gene was cloned for the first time in cassava. Real-time quantitative expression analysis revealed that expression level of pepc was higher in the leaves of cultivar type. The analysis of the cis-actiing element of these two pepc promoters shwed that the cultivated type had specific corresponding optical components. The full-length cDNA of phosphoenolpyruvate carboxylase gene was cloned using Nest-PCR method in cassava cultivar Arg7(Manihot esculenta) and the wild species W14(Manihot flabilifollia), respectively, for the first time. The two cDNA sequences accession numbers in GenBank were JN387053and JN387052, respectively. Both of the two cDNA sequences were2945bp in length including a2895bp-ORF, predicting to encode a protein with964amino acids and containing the typical conserved sequences/domains of pepc gene. Cassava PEPC had the highest homology level with the PEPC of Ricinus communis and Jatropha curcas. Bioinformatics analysis showed pepc cDNA sequence homology of up to98%and the PEPCase amino acid sequence homology of up to99%between two cassavas. The highest pepc expression level was observed in the leaves of W14and Arg7, followed in the fibrous roots and tuberous roots. The lowest expression level was observed in the stems. The one-day dynamic expression analysis showed a higher pepc expression level in Arg7leaves than in W14before16:00while it was higher in W14after16:00. The promoters of pepc were isolated from Arg7and W14, and the analysis of the cis-actiing element of these two promoters shwed that there were two light response components:LAMP and GA-the motif Specific existed in Arg7.How to further study the mechanisms of cassava high photosynthetic efficiency, and its relationship between starch efficient accumulations were discussed in this paper.