Cloning And Primary Functional Identification Of MeSTP7 Sugar Transporter Gene In Cassava (Manihot Esculenta Crantz)

Cassava (Manihot esculenta Crantz), which is an important tropical crop with high starchy roots,can either be used as food or as "energy plants" for the ethanol producting,thus drought and low-temperature stress restrict the high yield of cassava seriously. In plants, monosaccharides play roles as osmotic protective agent, antioxidants and signal molecules to participate in various stress response,the transporting and distribution of monosaccharides is highly dependent on the transport capacity of sugar transport proteins(STPs), the role of STPs in plant stress responses is still unclear. The key genes which response stress were identified from 20 genes encoding STP of cassava, and the study of its function was helpful to comprehensively understand the physiological and biochemical response mechanism of cassava under stress environment. In this study, real-time PCR was used to analyze the expression pattern of MeSTPs ; Subcellular localization and yeast heterologous expression analysis was used to analyze the function of MeSTP7. In addition,a highly efficient cassava mesophyll protoplast isolation and transient expression system were established, which provided a powerful tool for the further researches of subcellular localization and gene function of cassava. The main results are as follows:1. The expression patterns of 20 MeSTPs were compared under drought and low-temperature. It was found that the expression of 5 MeSTPs did not change significantly,9 MeSTPs were down-regulated, and 2 MeSTPs were up-regulated under drought stress.The expression of MeSTP7 changed significantly, and the expression level of MeSTP7 was 2.6 times and 4.1 times at 12 h and 48 h. It was found that the expression of 5 MeSTPs did not change significantly, 9 MeSTPs were down-regulated, and 2 MeSTPs were up-regulated under low-temperature stress.2. As for the tissues expression, MeSTP7 is mainly expressed in leaves, but low in roots and stems. With the plant developping, the expression level of MeSTP7 showed an increase in leaves and decrease in roots. In addition, MeSTP7 was found to have similar expression pattern in KU50 and CAS36, but the expression of MeSTP7 is higher in CAS36 than that in KU50, suggesting that MeSTP7 was involved in the accumulation of monosaccharides.3. MeSTP7 has an open reading frame of 1 590 bp, encoding 529 amino acids with a molecular weight of 58.24 kDa and an isoelectric point of 9.09 containing the Sugar_tr domain. MeSTP7 belonged to major facilitator superfamily (MFS) and the hydrophobicity prediction showed that it owes 12 transmembrane domains. Analysis the MeSTP7 promoter sequence revealed that it has lots of drought response-related cis-acting elements such as MYB binding site (MBS), heat shock response element (HSE) and abscisic acid response element (ABRE).4. An efficient mesophyll protoplast isolation and transient gene expression in cassava was established. The enzyme mixture containing 1.6% cellulase R-10 and 0.8%macerozyme R-10 was the best mixture in terms of both yield (4.4 × 107 protoplasts/g FW)and vitality (92.6%) of protoplast. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. The subcellular localization of MeSTP7 was studied by this transient expression system and a heterologous Arabidopsis thaliana gene expression system, the results showed that MeSTP7 locates on the cell membrane.5. The functional characterization identification of MeSTP7 in yeast mutant strain showed that MeSTP7 had the ability to transport glucose, mannose, fructose and galactose,and was sensitive to uncoupling agent CCCP. This result indicates that MeSTP7 has a broad spectrum of monosaccharide absorption properties, and its transport depends on the transmembrane proton power potential established by H+-ATP.In summary, MeSTP7 promoter contains multiple stress-related cis-acting elements;MeSTP7 having the ability to transport multiple monosaccharides, locates to the plasma membrane, suggesting that it may involve in the accumulation of soluble sugar in cassava.The expression of MeSTP7 was up-regulated by drought stress, indicating that MeSTP7 may be involved in the drought stress response of cassava.