Acta Academiae Medicinae Sinica
Acta Academiae Medicinae Sinica ›› 2018, Vol. 40 ›› Issue (2): 201-210.doi: 10.3881/j.issn.1000-503X.2018.02.010
• Original Articles • Previous Articles Next Articles
HUANG Xiaomei1, SUN Long2, WU Xiaobing2(
)
Received:2017-07-14
Online:2018-04-28
Published:2018-05-03
CLC Number:
HUANG Xiaomei, SUN Long, WU Xiaobing. Molecular Cloning,Sequence Analysis,and Expression of Pepsinogens C Gene from Adult Alligator Sinensis Stomach[J].Acta Academiae Medicinae Sinica, 2018, 40(2): 201-210.
Table 1
Primers used for PCR amplification of the pepsinogen C gene of Alligator sinensis"
| 引物代码Primer codes | 引物序列Primer sequences | 实验技术Experimental technique |
|---|---|---|
| F | 5’-TCTTGCCCTGGTGTGTCTC-3’ | RT-PCR |
| R | 5’-CTGGTTCCACCGATGACAA-3’ | RT-PCR |
| 5’-GSP1 | 5’-CACAGGTTGGAAGAGC-3’ | 5’RACE |
| 5’-GSP2 | 5’-CAGGAAGTTCTGGGGAGG-3’ | 5’RACE |
| 5’-GSP3 | 5’-TCCCAATGCTGATCTCAC-3’ | 5’RACE |
| 3’-GSP1 | 5’-TCACCCAGGAGGCTTATTGGCAAA-3’ | 3’RACE |
| 3’-GSP2 | 5’-CAAGGCATTGTGGACACTGGGACT-3’ | 3’RACE |
| PGCF | 5’-TCTTGCCCTGGTGTGTCTC-3’ | qPCR |
| PGCR | 5’-CTGGTTCCACCGATGACAA-3’ | qPCR |
| RPL8F | 5’-GGTGTGGCTATGAATCCTGT-3’ | qPCR |
| RPL8R | 5’-ACGACGAGCAGCAATAAGAC-3’ | qPCR |
Fig 1
1.0% agarose gel electrophoresis of 5’-RACE(A) and 3’-RACE(B) products M:DNA marker;1:RACE products"
Fig 2
The cDNA sequences and deduced amnio acid sequences of PGC of Alligator sinensisPGC:pepsinogen C;3’-,5’-untranslated regions are shown as lowercases;coding region is shown as uppercases,where the upper sequence indicates the nucleotide and the lower shows the amino acids;the region of putative signal peptide is underlined;a sterisk(★) indicates stop codon;putative polyadenylation signal(AATAAA) is marked with a thin line box;two aspartic acid residues associated with the active site motifs(D90,D275) are marked with a thin round frame;the six cysteine residues(C103,C108,C266,C270,C309,and C343) involved in forming three disulphide bonds characteristic of pepsinogen are indicated with a box of rough lines"
Fig 3
The predicted three-dimensional structure of the PGC from Alligator sinensis(B) with the three-dimensional structure of 2PSG as a template(A) Asp:aspartic acid;Cys:cysteine"
Table 2
Homology of the amino acid sequence of Alligator sinensis pepsinogen C to those of other organisms in GenBank"
| 物种名称 Species name | GenBank登录号 Accession number in GenBank | 氨基酸序列相似度 Amino acid identity(%) |
|---|---|---|
| Alligator mississippiensis PREDICTED | XP_006278997.2 | 98 |
| Crocodylusporosus PREDICTED | XP_019402206.1 | 90 |
| Thamnophis sirtalisPREDICTED | XP_013911358.1 | 55 |
| Gallus gallus | BAA76893.1 | 76 |
| Anasplatyrhynchos PREDICTED | XP_005023488.1 | 78 |
| Columba livia PREDICTED | XP_005510476.1 | 51 |
| Xenopuslaevis | BAB20797.1 | 71 |
| Cynopspyrrhogaster | BAM46127.1 | 71 |
| Epinepheluscoioides | ABX44850.1 | 67 |
| Salvelinusfontinalis | AAG47643.1 | 58 |
| Thunnusorientalis | BAG48265.1 | 57 |
| Homo sapiens | AAA60074.1 | 71 |
| Callithrix jacchus | BAA90872.1 | 66 |
| Mus musculus | AAH99409.1 | 64 |
| Oryctolaguscuniculus | BAB11756.1 | 64 |
Fig 4
Comparative analysis of putative amino acid sequences of PGC cDNA in different species*indicates the conserved residues;amnio acid subsititution leading to strong or no conservation of physiology chemical properties are indicated by:and.;two aspartic acid residues associated with the active site motifs are marked with a box;the six cysteine residues involved in forming three disulphide bonds characteristic of pepsinogen are indicated with a gray background"
Fig 5
Rooted the neighbor-joining tree based on the amino acid sequences of PGC from the vertebrates Phylogenies were determined with MEGA 6 software by bootstrap analysis using neighbor-joining(1000 replicates);scale bar equals 0.05(neighbor-joining) unit of Kimura’s two-parameter distance"
Fig 6
The location of PGC in the stomach tissues of Alligator sinensis A.gastric mucosa tissue(HE,×400);B.gastric mucosa was positive for PGC(immunohistochemical staining,×400)"
Fig 7
PGC mRNA expression in various tissues of Alligator sinensis by quantitative real-time polymerase chain reaction"
Fig 8
The mRNA levels of PGC in the stomach of Alligator sinensis at different life stages by quantitative real-time polymerase chain reaction aP<0.05 compared with data in January,March,May,and November"
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