Beta-amylase in sweet potato tuberous root: gene expression , immunolocalization and biochemical studies

• Main Authors: Ling, Thai-yen, 林泰元
• Other Authors: Jong-Ching Su
• Format: Others
• Language: en_US
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/69171855710306919863

博士 === 國立臺灣大學 === 農業化學系 === 86 === b-Amylase (a-1,4-glucan maltohydrolase; EC 3.2.1.2) is known to occur in some species of microorganisms and higher plants. b-Amylase can convert soluble starch to maltose and b-limit dextrin. The physiological function of b-amylase in some plants is not clear because the enzyme and its substrate are spatially separated. Although b-amylase in spinach is localized in the chloroplast, in pea, wheat and Arabidopsis thaliana, it occurs in the vacuole where no starch is found. Recently, b-amylase was identified as a phloem specific protein in Streptanthus tortuosus (Brassicaceae). b-amylase was found as a major protein in the tuberous root of sweet potato. It was also a specific non-competitive inhibitor of starch phosphorylase (SP) which was localized in the amyloplast. The active b- amylase is homotetramer having a molecular weight of 200,000. It is encoded by a nuclear gene, and no transit- or signal-peptide is found in the deduced amino acid sequence. Due to these fact, although we had tentatively identified earlier that the "phosphorylase inhibitor" co-localized in the amyloplast together with the inhibited enzyme, more precise information about the subcellular location of b-amylase is need to elucidate the physiological role it may play in sweet potato. The cDNA encoding 1535bp b-amylase cDNA was cloned by RT- PCR/TA-cloning techniq using a sweet potato tuberous root [Ipomoea batatas (L.) Lam. cv. Tainong 57] total RNA preparation as the template. The specific primers were deduced from the cDNA of b-amylase (Yoshida, 1991): [sense primer (66-95): 5''''- TGCAAAATAACCAACAATGGCTCCAATCCC-3''''] and [anti sense primer: (1571-1600): 5''''- GAGGGTGATCTAGAGCTGAGCAATCAAACG-3'''']. Five clones containing full-length b-amylase cDNA (named as p11, p14, p17, p19 and p20) were obtained. The partial DNA sequencing results indicated that three of them (p14, p19 and p20) were in the sense-direction and the other in the antisense-direction. The full length b-amylase DNA sequence of p20 were compared to that of the reported sweet potato (-amylase cDNA (Yoshida, 1991). On comparison, it has been found that there are differences in nucleotide sequences at seven positions, and that the deduced amino acid sequences are also different at four sites. The major mutation site is the deletion of nucleotide 849 and the insertion of a thymidylate between 873 and 874, causing a frame-shift and the nucleotides 1281 and 1282 are switched from "GC" to "CG" and thus the amino acids "KL" were changed to ":NV". The protein sequence of p20 were also compared with those of sweet potato (-amylases available in the databank. It suggests that p20 is similar to (AMYB_IPOBA) which also contains the "frame-shift region" and "nucleotides-switched regions" but do not have the other variants. Other mutations in p20 are nucleotide exchanges as follows: the adenylate at 1108 is changed to thymidylate, the adenylate 1260 is altered to thymidylate and the adenylate 1543 is changed to guanylate. An expression vector under the control of a highly efficient T7 promotor and translational signal was used for constructing a plasmid harboring the b-amylse cDNA (b-amylase/pET-16b). An Escherichia coli strain BL21(DE3) that was transdormed by the construct over-expressed a fusion protein (His-BA). It had an N-terminal histidine decapeptide fused to a full length b-amylase up induction with isopropyl b-D-thiogalactoside. For the estimation of the amount of (-amylase produced under different expression conditions, four expressed condition were set up for 30℃ /IPTG: 0.025 mM, 0.1 mM and 0.4 mM and 37℃/IPTG:0.025mM. The expressed 55-kDa protein were purified by metal-chelate affinity chromatography. The results indicated that the protein expressed in 30℃ /IPTG: 0.025 mM had highest specific activity and in 37℃ /IPTG:0.025mM.had the highest amount of protein. The activity staining pattern of expressed b-amylase in native-PAGE showed that expressed b- amylase had different migration rate when samples of different dilutions were run in electrophoresis. When a higher concentration of expressed b- amylase was used, the protein polymerized to a tetrameric form without addition of other factors, and it will be in dimer and monomeric form in a higher dilution. The enzyme kinetic studies revealed that expressed b- amylase could also be a non-competitive inhibitor to the starch phosphorylase but the inhibitory activity of expressed b-amylase was lost after treated with HgCl2. The results of immunohidtochemistry at light microscopic level indicated that b-amylase was located only in amyloplast s and in the cell walls of the cells containing amyloplasts but not in other loci. In very young roots (5- 10 day after tip emergence), a small number of amyloplast in the cells distributed radically between vascular bundle. In roots of 15-20 days after tip emergence, not only did the amyloplasts radiate out between the phloem, but also increased in number to form a circular band surrounding vascular bundles. The electron microscopic results also showed that labeling of amyloplasts and some cell walls were very specific; immuno- golds particles with 15 nm diameter were hardly ever observed in the cytoplasm and other organelles. No specific immuno-gold labeling was detected in parallel experiments using preimmune serum as the primary antibody. There results indicated that the sites of sweet potato b-amylase gene expression are highly specific at both tissue and subcellular level.