Expression Patterns Analysis Of MePho And MeSBE Gene Family In Cassava (Manihot Esculenta)under Diverse Environment

Starch branching enzyme (SBE) is one of key enzymes involved in starch biosynthesis in higher plants and have important functions in the synthesis of amylopectin. Research found that SBE tended to from protein complexes through protein interaction with other enzymes involved in starch synthesis. There are two kinds of starch phosphorylase in plants, the plastidal Pho and cytosolic Pho, they catalyze the following reversible reaction:(1,4-a-D-glucosyl)n+a-D-glucose-l-P(?)(1,4-a-D-glucosyl)n+1+Pi. Studies in potato and rice find that Pho functions in starch biosynthetic pathway. Genome-wide sequencing and transcriptome analysis finds that Pho and SBE are strongly expressed in roots from different growth stages, however, the exact mechanism and their functions under different environment conditions are unknown. In this study, the systematic study of Pho and SBE might shed light on starch biosynthesis in cassava. Results are as follows:1. A1980bp fragment of MePhold is cloned which shares87%,86%,86%and84%identities with the known Ricinus communis Pho, Populus trichocarpa Pho,Populus euphratica PhoL and Vitis vinifera PhoL respectively. Conserved protein domain analysis of the cloned sequence finds that the fragment contained the characteristic domains of starch phosphorylase such as the Phosphorylase, Glycogen_phosphorylase domains. The full-length gene sequence of MeSBE2.2is cloned. The fragment is2053bp and shares91%,86%,86%,81%and79%identities with Ricinus communis SBEII, Theobroma cacao SBE2.2, Vitis vinifera GBE2, Dioscorea esculenta SBEII and Zea mays SBEIIb respectively. Conserved protein domain analysis of the cloned sequence finds that the fragment contained the characteristic domains of starch branching enzyme.2. A1737bp upstream promoter sequence fragment of MePhold is cloned and cis-acting elements analysis identified ABA, JA, heat-shock and drought responsiveness elements. A2153bp upstream promoter sequence fragment of MeSBE2.2is cloned and the putative regulatory cis-acting elements analysis find that the fragment contains heat-shock responsiveness element, drought, GA, SA responsiveness elements.3. Six SBE gene members and seven Pho gene members are identified by blasting the cassava genome database using the published Arabidopsis and Ricinus communis SBE and Pho sequences. Phylogenetic analysis divided the MeSBEs into dicot family A, dicot family B, dicot family C and the new group (NG). MePhos are divided into two types:the plastidial type (PhoL) and the cytosolic type (PhoH).4. Tissue-specific expression analysis results show that the plastidial type MePholc and MePhold have the highest expression levels in root stele while MePholb has the highest expression level in leaves; the cytosolic MePho2has higher expression levels in leaves, petiole and stem, other isoforms are expressed with low abundance in all tissues tested. MeSBE2.2is mainly expressed in leaves, stem and root. MeSBE2.1, MeSBE4and MeSBES have higher expression levels in stem while MeSBE3is strongly expressed in stem and root. MeSBEl is expressed with low abundance in all tissues tested.5. During the tuberous root development, the expression levels of the plastidial phosphorylase MePholc and MePhold were relatively higher; the transcription activity of MeSBE2.1, MeSBE2.2and MeSBE3gradually increased as the plants aged while other gene members remain unchanged.6. Various abiotic stress treatments show that the expression levels of MePho2and MePholc are significantly up-regulated under drought, salt "and exogenous ABA treatment, indicating that they might have functional roles in starch biosynthesis in cassava under stress conditions. MeSBE2.2and MeSBES is significantly up-regulated under salt, drought, exogenous ABA, JA and SA treatment.7. The relative values of SBE and Pho gene members are studied, which revealed that SBE and Pho gene members exhibit high R value under various stress treatments, especially between MeSBE2.2and MePholc. The relative value of MeSBE2.2and MePholc under salt, drought, exogenous ABA, high-temperature and low temperature treatment are0.978,0.998,0.946,0.763and0.889respectively, which indicates that MeSBE2.2and MePholc might function together through protein-interaction in starch accumulation in cassava under abiotic stress conditions.