Molecular Cloning and Tissue-specific Expression of Cu/Zn and Mn-superoxide Dismutase in the Three-keeled Pond Turtle, Chinemys reevesii

Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.

Molecular Cloning and Characteristics of Catalase cDNA from Chinese Soft-shelled Turtle(Pelodiscus sinensis)

A catalase cDNA was cloned from the liver of the Chinese soft-shelled turtle （Pelodiscus sinensis） using reverse transcription-polymerase chain reaction （RT-PCR） with degenerate primers. Both 3＇-and 5＇-untranslated regions were isolated by the rapid amplification of cDNA ends method （RACE）. Analysis of nucleotide sequence revealed that the catalase cDNA clone consisted of 2173 bp with an open reading frame of 1587 bp encoding a protein of 528 amino acids. The calculated molecular mass of the mature protein is 59.8 kDa with an estimated pI of 6.84. The peroxisomal targeting signal SNL at the C-terminal and two putative N-glycosylation sites NLSV and NVSQ were found in the catalase. Sequence comparison showed that this catalase, deduced by the amino acid sequence, had high similarity and identity with those of vertebrates recorded in GenBank. Four functional domains and conserved amino acids responsible for binding heme and NAPDH including four essential residues were observed. The 3-D homology model of the turtle catalase was predicted by SwissModel based on the relative domains of bovine catalase structure （PDB ID： 3rgp）. The mRNA expression and enzyme activities in liver, brain, spleen, kidney, heart, gut, lung and muscle were investigated, and the results showed that the mRNA and enzyme activities of catalase in these tissues were species-specific.

Effect of Dietary Vitamin C on the Antioxidant Defense System of Hibernating Juvenile Three-keeled Pond Turtles (Chinemys reevesii)

Juvenile three-keeled pond turtles(Chinemys reevesii) were fed diets supplemented with vitamin C(Vc) at doses of 0(basal diet, Vc0), 100(Vc100), 200(Vc200), 500(Vc500) and 2500(Vc2500) mg/kg diets at 28°C for 4 weeks, respectively. Then, the water temperature was gradually reduced to 10°C, and the turtles were induced into hibernation. Liver tissue samples were collected at three time points: start of hibernation(T1), 4 and 6 weeks’ hibernation(T2 and T3). A control group fed with the basal diet was set to parallel the whole treatment process, but reared at 28°C constantly. The results showed that hibernation mildly affected the antioxidant system and the influence varied with hibernating time. Hepatic malondialdehyde content of the Vc100 group was significantly lower than that of the other groups at T1. At T2, hepatic MDA in the groups of Vc500 and Vc2500 decreased significantly, while no clear differences were found among all groups at T3. The activities of antioxidant enzymes showed a positive correlation with dietary Vc dose before hibernation. After hibernation, total antioxidant capability was not affected by Vc. Superoxide dismutase activity became similar in different groups at T2, but decreased in higher Vc groups(≥ 200 mg/kg) at T3. Glutathione peroxidase and glutathione-S-transferase activities decreased significantly with dietary Vc supplementation(≥ 100 mg/kg) at T2, but recovered at T3. The result indicates that under normal rearing condition, low dietary Vc supplementation(< 100 mg/kg) might be beneficial to the antioxidant defense system. The effect of dietary Vc on the antioxidant defense system differed during hibernation.

Effects of acute cold exposure on oxidative balance and total antioxidant capacity in juvenile Chinese soft-shelled turtle,Pelodiscus sinensis

Acute cold exposure may disturb the physiological homeostasis of the body in ectotherms.To date,there has been no information on the effects of cold exposure on homeostasis of reactive oxygen species(ROS)or antioxidant defense response in the Chinese soft-shelled turtle,Pelodiscus sinensis.In this study,P.sinensis juveniles were acclimated at 28℃,transferred to 8℃ as cold exposure for 12 h,then moved back to 28℃ rewarming for 24 h.We measured the ROS level and total antioxidant capacity(TAC)in the brain,liver,kidney and spleen at 2 and 12 h cold exposure,and at the end of the rewarming period.Malonaldehyde(MDA)and carbonyl protein were used as markers of oxidative damage.Turtles being maintained simultaneously at 28℃ were used as the control group.Cold exposure did not disturb the ROS balance in all 4 tissues,while rewarming raised the ROS level in the brain and kidney of P.sinensis.Cold exposure and rewarming decreased the TAC in the brain,liver and spleen but did not change the TAC in the kidney.MDA and carbonyl protein levels did not increase during the treatment,indicating no oxidative damage in all 4 tissues of P.sinensis.Our results indicated that extreme cold exposure did not impact the inner oxidative balance of P.sinensis,but more ROS was produced during rewarming.P.sinensis showed good tolerance to the harsh temperature change through effective protection of its antioxidant defense system to oxidative damage.This study provides basic data on the stress biology of P.sinensis.

Division of Chinese soft-shelled turtle intestine with molecular markers is slightly different from the morphological and histological observation

The Chinese soft-shelled turtle(Pelodiscus sinensis)is a commercially important species in Asian countries.Knowledge of its nutritional requirements and physiology is essential for determining the appropriate content of the feed for this animal.However,the lack of functional characterization of the intestine of this turtle limits the understanding of its absorption and utilization of nutritional materials.To solve this problem,this work utilized anatomical and histological methods to characterize 9 segments sampled along the anterior-posterior axis of the intestine.Furthermore,9 genes,which have been well documented in the intestine division of mammals and fish,were employed to functionally characterize the 9 sampled segments.Our results suggest that regions covering from the starting site to S3(position at 29.9%of the total length from the starting of the intestine)are the equivalent of mammalian dedumonen,and those covering S4(40.2%)and S5(65.4%),posterior to S8(92.7%),are the equivalent of the mammalian ileum and the large intestine,respectively.As to the region spaning S6(81.3%)and S7(87.3%),its functional equivalent(small intestine or large intestine)may be variable and depends on the functional genes.This molecular characterization in relation to the division of the intestine of Chinese soft-shelled turtle may contribute to the understanding of the nutritional physiology of the turtle,and promote Chinese soft-shelled turtle production.