Molecular Cloning And Expression Analysis Of Cathepisn D And Alpha Amylase Gene In Pinctada Maxima And Pteria Penguin

The pearl oyster, Pinctada maxima, is widely distributed in tropic and subtropic oceans and seas of China, Australia and South-East Asia. It is an economically important species that produces top-quality pearls. Pteria penguin is distributed in coastal areas of Guangdong, Guangxi, Hainan, and Taiwan in China, the south of Kyushu in Japan, Ryukyu Islands and the Philippines. It is mainly used in the cultivation of blister pearls and large round pearls.Two genes, cathepsin D and alpha amylase, were cloned respectively from P. maxima and P. penguin by homologous cloning approach. Sequencing and tissue expression analysis were carried out subsequently. This study made a foundation for the function of cathepsin D and alpha amylase. Results are as follows:1. The full length of pearl oyster Pinctada maxima cathepsin D (named as pmCTSD) was determined for the first time through homologous cloning and rapid amplification of cDNA ends (RACEs) approaches. The full length cDNA contained a 5'UTR of 38 bp, an open reading frame (ORF) of 1 170 bp encoding 390 amino acids and a 3'UTR of 534 bp. The deduced amino acid residues comprised a signal peptide of 18 aa, a pro-sequence of 29 aa and a mature protein of 343 aa, with an estimated isoelectric point of 7.57 and molecular mass of 41.9 ku. Homologous analysis indicated that pmCTSD shared 64% ~ 70% identity in terms of amino acid sequence with other known members in CTSD subfamily. The reconstructed phylogenetic tree demonstrated that pmCTSD was closely related to that of Chlamys farreri. The tissue expression profile showed that pmCTSD expressed in all the tissues examined including adductor muscle, gonad, hepatopancreas, mantle and gill, with higher expression level in gonad and hepatopancreas.2. The full length cDNA of cathepisn D cloned from P. penguin (named as pgCTSD) was 1,767bp with a 5'UTR of 38 bp, an ORF of 1,176 bp encoding 392 amino acids and a 3'UTR of 553 bp. The predicted amino acid sequence contained a signal peptide of 18 aa, a pro-sequence of 29 aa and a mature protein of 345 aa, with an estimated isoelectric point of 8.04 and molecular mass of 42.3 kDa. It was characterized with two aspartyl proteases active sites (Val84-Val95, Ala272-Gly283) and two N-glycosylation sites (Asn124, Asn237). pgCTSD had a high identity with P. maxima, and shared 59%~75% identity with other organisms. NJ tree showed a closest relationship with Chlamys farreri and clustered with Pinctada maxima. The tissue expression profile suggested that pgCTSD expressed in all the tissues including adductor muscle, gonad, hepatopancreas, mantle and gill, with higher expression level in hepatopancreas and lower level in adductor muscle. Compared to the blank group, the expression level of pgCTSD increased significantly in adductor muscle and gonad, but decreased in hepatopancreas, mantle and gill after injection of PBS for 6h. Compared to the control group, the expression level of pgCTSD in LPS group increased in adductor muscle and decreased in gonad and hepatopancreas significantly, obvious changes were not observed in mantle and gill; after 6h challenge with Vibrio harveyi, the expression profile of pmCTSD showed a little different with LPS group, the expression level of pgCTSD increased in gill and decreased in mantle but not changed much in gonad. The information generated in the present study is helpful for further studies on the functions of pgCTSD.3. The full length of alpha amylase cDNA (named as pmAMY) was obtained for the first time with a 5′UTR of 25 bp, an open reading frame (ORF) of 1554 bp encoding 518 amino acids and a 3′UTR of 153bp. The estimated isoelectric point and molecular mass of deduced amino acid residues were 7.63 and 57.7 kD. Sequences analysis suggested that pmAMY contained a signal peptide of 16 aimino acids (MLLIVCSIAFFHSVYG), 8 cysteine residues (Cys46,Cys104,Cys157,Cys176,Cys392,Cys398,Cys464,Cys476) , 3 active site residues (Asp213,Glu249,Asp314), 4 calcium binding residues (Asn118,Arg174,Asp183,His217), 3 chloride binding sites (Arg211,Asn312,Arg350) and 4 conserved sequences (Ile111-Val116,Val207-Ala215,Phe247-Val251,Val308-Asn315). pmAMY shared 79% the highest identity with Crassostrea gigas and 57%~62% with other organisms. We obtained the alpha amylase gene of P. maxima with 3 exons and 2 introns. The first exon was 117 bp encoding 39 amino acids. The second was 152 bp encoding 50 amino acids and the third was 1, 288 bp encoding 429 amino acids. The first intron was 846 bp and the second was 162 bp. Both of the two introns sequences began from GT and ended as AG, which was consistent with common sequences of intron splice sites.4. The full length cDNA of alpha amylase from P. penguin (named as pmAMY) was 1, 670 bp, containing a 5′UTR of 18 bp, an open reading frame (ORF) of 1569 bp encoding 523 amino acids and a 3′UTR of 83bp. The estimated isoelectric point and molecular mass of deduced amino acid residues were 8.7 and 62.4 kD. Sequence analysis suggested that pmAMY contained a signal peptide of 20 aimino acids (MRTFFLTFVCVLVLHSTVYG), eight Cysteine residues (Cys50,Cys108,Cys162,Cys181,Cys397,Cys403,Cys469,Cys481) , three active site residues (Asp218,Glu254,Asp319), four calcium binding residues (Asn122,Arg179,Asp188,His222), three chloride binding sites (Arg216,Asn317,Arg355) and four conserved sequences (Ile115-Val120,Val212-Ala220,Phe252-Val256,Val313-Asn32). pgAMY shared the highest identity (83%) with C. gigas, 82.8% with P. maxima and 57%~67% with other organisms. The cysteine residues were conserved among species as well as active site residues, calcium binding residues and chloride binding sites. In the starvation experiment, the expression of pgAMY in the control group was the highest with 2-ΔΔCT value of 1. The expression of pgAMY dropped to 43% of that of the control group at 1 week (P <0.5). The expression of pgAMY at 4 week was not change much compared to 1 week with the expression of 51% of the control group. After refeeding for 1 day, the expression of pgAMY decreased to 39% of the control group slightly compared to 4 week. After soaking with different salinity for 24h, the expression of pgAMY in salinity 33 was the highest, the salinity 40 the second with 2-ΔΔCT values of 3.52 and 3.46. With the decreasion of salinity from 30 to 15, the expression of pgAMY also decreased with 2-ΔΔCT values of 2.68, 2.01, 1.81, 1.01 respectively. The expression levels of pgAMY in salinity 25 and 40 decreased with time, the 2-ΔΔCT values were 2.01, 1.92, 0.44 and 3.46, 1.39, 0.92 at 24h, 48h, 76h..