| Alpha-amylases are able to cleaveα-1-4 glycosidic bonds that are present in the inner part of amylase or amylopectin chains, they are one of the most important enzymes, being widely applied in sugar, brewage and fermentation industry. Chinese sucker (Myxocyprinus asiaticus) is endemic to China or even Asia. It has been listed as CategoryⅡspecies under national key protection in China. It is also well known for the large body, fast growth,delicious fleshiness, tame temper. Fish amylase is one of the major carbohydrases, which is produced by pancreatic cells located in a diffuse mesentery surrounding the digestive tract, it's secretion affects the digestive ability of fish. It is one of the most important digestive enzymes. Many experiments have showed that the synthesis of amylase is not induced by food but it is the life phenomenon of fish development, amylase synthesis relates to the days post hatching. In order to better understand the structure ofα-amylase of Myxocyprinus asiaticus, theα-amylase mRNA in Myxocyprinus asiaticus was cloned and sequenced, its nucleotide and deduced amino acid sequence and the predicted protein structure were analysed, the expression ofα-amylase in Myxocyprinus asiaticus was also investigated to know how the fish makes use of the food and secretes and reservesα-amylase. Main results are as follows:1.Full-length cDNA sequence of a -amylase from Myxocyprinus asiaticusTwo special primers were designed after multiple comparison amongα-amylase genes in many kinds of teleosteans. Alpha-amylase middle segment sequence of Myxocyprinus asiaticus was acquired through amplification on the templet of mRNA from its hepatopancrea. Two 3'cDNA ends primers used in the amplification ofα-amylase genes were designed. The sense primer was designed on the base of the middle segment sequence of Myxocyprinus asiaticus,and the antisense primer was provided by TaKaRa 3' -Full RACE Core Set Ver.2.0 kit, then the 3'cDNA ends sequence ofα-amylase gene was got. According to the middle segment sequence and the 3'cDNA ends sequence,αspecial reverse transcription primer and three pairs of special primers on 5'cDNA ends were designed, and then 5'cDNA ends sequence was acquired. Finally, combined above three sequences,the cDNA sequence ofα-amylase in Myxocyprinus asiaticus was known: its full-length was 1633bp, including a 5' untranslated region of 24bp, translated region of 1539bp which encoded a protein precursor of 512bp and a 3' untranslated region of 70bp.2. Deduced amino acid sequence analysis of Myxocyprinus asiaticusα-amylase geneThe full length of Myxocyprinus asiaticusα-amylase cDNA was 1633bp, it included a 5' untranslated region, an open reading frame composed of 512 amino acids, a stop codon (TAA) and a 3' untranslated region which contained a polyadenylation signal and a poly (A) tail. The open reading frame was 1539bp in length, encoded 512 amino acids, it was composed of an signal peptide (15 amino acids), three active site residues, four residues involved in calcium binding (Ash 115, Arg 173, Asp 182, and His 216), three residues of the chloride binding site (Arg 210, Ash 313, Arg 352) and ten Cysteine residues involved in disulfide bonds.3.Homology analysis of Myxocyprinus asiaticusα-amylase geneThe cDNA sequence of Myxocyprinus asiaticusα-amylase gene demonstrated high similarity with other vertebrates. Comparison of the ORF of Myxocyprinus asiaticusα-amylase to that of other fishes such as Danio rerio, Lates calcarifer, Pseudopleuronectes americanus, Salmo salar by the DNAMAN soft shows 88.50%, 78.30%, 76.80%, 72.51% nucleotide consistency, and comparison of the full-length cDNA shows 86.10%, 75.26%, 75.17%, 70.73% consistency.Amino acid sequence of Myxocyprinus asiaticusα-amylase from cDNA similarity is higher than nucleotide. Comparison of the amino acid sequence of Myxocyprinus asiaticusα-amylase from cDNA to that of other fishes such as Danio rerio, Lates calcarifer, Pseudopleuronectes americanus, Salmo salar, shows 89.65%,82.03%,79.88%,74.80%, and to that of other species such as Xenopus laevis,sus scrofa,Homo sapiens shows 76.39%,74.33%,71.88% amino acid similarity. 4. Alpha-amylase structure prediction of Myxocyprinus asiaticusIt is predicted theα-amylase gene encodes a peptide chain of 512 amino acids, with Mw of 56910.87 Da and pI of 8.49. In the composition of amino acids, the content of glycine is the highest which up to 11.72%, and asparagine, valine, leucine, serine are 8.98%,7.42%,6.45%,6.05% respectively, and histidine is relatively lowest, accounting for 2.34%. The proportion of acidic amino acid and basic amino acid is 9.38% and 12.69% respectively. Its secondary structure mainly contains a-helix, B-turn, random and extended strand, and random is the most important one. 5. Multiple tissue expression ofα-amylase in Myxocyprinus asiaticusA semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to estimate tissue-specific expression ofα-amylase gene in ten different tissues of the Myxocyprinus asiaticus. Two pairs of special primers were designed for amplification of tissue expression. Amplification ofβ-actin cDNA was performed in order to confirm the steady-state level of expression of a housekeeping gene allowing an internal control forα-amylase gene expression. Primers were designed from goldfishβ-actin mRNA sequence.A strong preferential expression ofα-amylase gene was observed in the hepatopancrea tissue, pyloric intestine, mid intestine, distal intestine, trace ofα-amylase gene expression was observed in the heart.
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