Cloning,Structure Evolution And Expressing Analysis Of The Hexokinase Family Genes In Cassava(Manihot Esculenta Crantz)

Cassava is one of the three main tuber crops (cassava, sweet potato, potato) and the world’s sixth largest food crops in the world. Cassava is a kind of C3plants which possesses highest photosynthetic rate, and has high sucrose synthesis ability. There is a large storage of starch in its root tubers. Sucrose synthesized in photosynthesis is catalyzed into hexoses by invertase or sucrose synthase and transported to sink tissues. Hexokinase can irreversiblely phosphorylate hexoses and form hexose phosphate, which is one of the key enzymes during carbohydrate metabolism and sugar signal transduction in plants. Therefore, the research on cassava hexokinase gene family might conducive a further understanding of sucrose metabolism and sugar signal transduction during starch accumulation in cassava tubers.In this research, based on the information of cassava genome database, all the predicted cassava hexokinase family genes have been isolated. The evolutionary history of cassava hexokinase gene family has been summarized. In order to explore the roles of cassava hexokinase family members in the source and sink organs, their promoters, gene expression and enzyme activity were analyized. The results are as follows:1. Based on the information of cassava genome database,7cassava hexokinase family members have been predicted and named as MeHXK1-7. There accession numbers are as follows:MeHXKl (KJ417433)、MeHXK2(KJ417434)、MeHXK3(KJ417435)、MeHXK4(KJ417436)、MeHXK5(KJ417437)、MeHXK6(KJ417438) MeHXK7(KJ417439). The CDS regions of six hexokinase genes (MeHXK1-6) have been isolated from the cassava variety SC8by RT-PCR.2. Cassava hexokinase can be divided into two types, A (MeHXKl) and B (MeHXK2-7). The prediction of3D protein structure shows that, MeHXKl-5are constituted by two different size of domains (N-terminal constitute a small domain, C-terminal constitute a large domain), and the catalytic residues and the substrate binding site are located in the cleft between the two domains. MeHXK6has a small domain resulting from a lost of its C-terminal sequence.3. Intron-exon structure analysis declares that the structure of the first exon of all the cassava hexokinase family members is similar. Besides, MeHXKl has seven exons, MeHXK2,3,4and5have nine exons, MeHXK6has six exons, and MeHXK7has five exons.4. The bioinformatic analysis suggests that MeHXK1,2,5have a phosphorylation function and also play a role in sugar signal transduction. However, MeHXK3,4,6,7only have the function of sugar signal transduction. 5. Promoter sequence analysis shows that cassava hexokinase genes are involved in regulating the photosynthesis, responsing to hormonal signals and stresses.6. The phylogenetic analysis of hexokinase gene family in cassava, castor and Arabidopsis shows that their common ancestor (LCA1) has five original hexokinase genes (HXK Ⅰ-Ⅴ), belonging to Group Ⅰ-Ⅴ. HXKII and Ⅲ encodes HXK proteins, and HXK Ⅰ, Ⅳ and V encode HKL proteins.7. Tissue specific analysis of cassava hexokinase gene expression showed that the expression of MeHXK2was higher than other MeHXKs in all the detected tissues of leaves, stems, root phloem, male and female flowers. The expression of MeHXK1was higher, but it was lower than MeHXK2, which mainly expressed in leaves, root phloem, male and female flowers. The expression of MeHXK3was at a relatively low level in all the detected tissues. MeHXK4had a relatively high expression in female flowers; and for MeHXK5, the relatively high expression was in male and female flowers; for MeHXK6was in female flowers In addition, the expression of MeHXK7had not been detected in any tissues; and. the expression of MeHXKl,2,5in all tissues was higher than that of MeHXK3,4,6, which indicated that the expression levels of these two kinds of hexokinase genes were variety in organs..8. The expression of MeHXKs in leaves was increased as the development of root tuber,. The expression of MeHXK1and MeHXK2was increased rapidly in root maturity stage (225days and270days after planting) and the late period of root enlargement stage (180days after planting). MeHXK4.5,6had a highest expression in the late period of root maturity stage (270days after planting). In root phloem, the expression of MeHXK1,2was higher than the other MeHXKs, which MeHXK2had the highest expression especially in root formation stage (90days after planting) and the late period of root enlargement stage (180days after planting). The expression of MeHXK2was higher than the other MeHXKs in root xylem. especially in root formation (90days after planting). The above results showed that in sink organs, MeHXKs mainly activated in root formation and starch rapid accumulation stages, and in source organs, MeHXKs mainly activated in the late period of root development; and MeHXK1,2were the key genes in hexose phosphorylation and sugar signal transduction in cassava.9、The detection of hexokinase activity in leaves indicated that the hexokinase activity in root initial formation stage was lowest, and in root enlargement stage was highest. In root maturity stage, the hexokinase activity was decreased. However in tubers, the activity of the hexokinase was going down as the root tuber maturity. Generally, the hexokinase activity in leaves was much higher than in root tubers in root enlargement and root maturity stages, which indicated that the hexokinase played a significant role during the sucrose metabolism in cassava leaves.