Cloning and sequence analysis of the coding sequence of β-actin cDNA from the Chinese alligator and suitable internal reference primers from the β-actin gene.
β-Actin is an essential component of the cytoskeleton and is stably expressed in various tissues of animals, thus, it is commonly used as an internal reference for gene expression studies. In this study, a 1731-bp fragment of β-actin cDNA from Alligator sinensis was obtained using the homology cloning technique. Sequence analysis showed that this fragment contained the complete coding sequence of the β-actin gene (1128 bp), encoding 375 amino acids. The amino acid sequence of β-actin is highly conserved and its nucleotide sequence is slightly variable. Multiple alignment analyses showed that the nucleotide sequence of the β-actin gene from A. sinensis is very similar to sequences from birds, with 94-95% identity. Ten pairs of primers with different product sizes and different annealing temperatures were screened by PCR amplification, agarose gel electrophoresis, and DNA sequencing, and could be used as internal reference primers in gene expression studies. This study expands our knowledge of β-actin gene phylogenetic evolution and provides a basis for quantitative gene expression studies in A. sinensis.
College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China.
H N
HN
Zhu
College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China szzhang@mail.ahnu.edu.cn.
S Z
SZ
Zhang
Alligator Research Center of Anhui Province, Xuanzhou, China.
Y K
YK
Zhou
Alligator Research Center of Anhui Province, Xuanzhou, China.
C L
CL
Wang
College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China.
X B
XB
Wu
D000199:Actins / Q000235:genetics*; D000489:Alligators and Crocodiles / Q000235:genetics*; D000818:Animals; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D010802:Phylogeny
D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary