Prokaryotic Expression of Antimicrobial Peptide CATH PR1–2 from the Skin of Paa robertingeri in Escherichia coli

The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin （CATH） PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low （3195.88 and 2838.34 Da, respectively）. Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid pET-32a. This reconstructed plasmid was then transfected into the expression vector E. coli BL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37℃ for 4 h. The antimicrobial activity assay using Staphylococcus aureus （Song） and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method.

A New Species of the Asian Leaf Litter Toad Genus Leptobrachella(Amphibia,Anura,Megophryidae)from Chongqing City,Southwest China

Herein we describe a new species,Leptobrachella yunyangensis sp.nov.,from Yunyang County,Chongqing City,China,based on a combination of molecular and morphological data.Phylogenetic analysis based on the mitochondrial 16 S rRNA gene and six nuclear genes indicated that this new species represented an independent evolutionary lineage.The uncorrected genetic distance between the new species and its closest congener species,L.oshanensis,was 5.4 % for 16 S rRNA.The new species can be distinguished from its congeners by a combination of the following characters:(1) moderate body size(SVL28.3-30.6 mm in males);(2) rough dorsal skin,with sparse large granules and tubercles and short longitudinal ridges on the shoulder;(3) distinctly discernible tympanum with a diameter smaller than that of the eye(TMP/EYE ratio:0.51) and a distinct black supra tympanic line present;(4)an internasal distance almost equal to interorbital distance(IND/IOD ratio:1.05);(5) flanks with several dark spots arranged longitudinally in two rows;(6) distinctly visible supra-axilla ry,femoral,pectoral,a nd ventrola teral glands;(7) bicolored iris,with the upper 1/3 of the iris being copper orange and the lower 2/3 a light silvery grey;(8) relative finger lengths of Ⅰ <Ⅱ=Ⅳ <Ⅲ and relative toe lengths of Ⅰ <Ⅱ <Ⅴ <Ⅲ <Ⅳ;(9) absence of webbing and lateral fringes on fingers,and toes with rudimentary webbing and narrow lateral fringes;(10) heels overlapping when the thighs are positioned at right angles to the body;(11) tibiotarsal articulation reaching to the anterior corner of the eye when the leg is stretched forward;(12) ventral surfaces of the throat,chest,and belly greyish white with purple-brown speckling;(13) lacking distinct blackish dorsolateral markings;and(14) transverse dark brown bars on the surfaces of limbs and digits.

Description of a new species of the genus Rana (Anura: Ranidae) from western Guizhou, China, integrating morphological and molecular genetic data

The diversity of the brown frog genus Rana may be underestimated as the high similarity of morphological characters.In this study,a new species of Rana from Guizhou Province,China is described,namely Rana zhijinensis Luo,Xiao&Zhou,sp.nov.Molecular phylogenetic analyses clustered the new species into the R.japonica group of Rana and significant morphological characters can be distinguished from the 11 recognized species of the R.japonica group.This description increases the number of recognized Rana to 57 species and the R.japonica group to 12 species,and increases our knowledge of the diversity of the genus Rana.

A new species of the genus Tylototriton (Amphibia: Urodela: Salamandridae) from the Eastern Dalou Mountains in Guizhou, China

A new newt species,Tylototriton daloushanensis Zhou,Xiao&Luo,sp.nov.,is described from Guizhou,China,based on phylogenetic and morphological analyses.Phylogenetic analyses based on mitochondrial 16S and ND2 genes show that the new species is an independent lineage closely related to T.maolanensis,T.broadoridgus,T.dabienicus,T.anhuiensis,and T.wenxianensis.Morphologically,the new species is distinguished from its congeners by a combination of the following morphological characters:(1)large body size(SVL 64.7–83.6 mm in male and 70.5–100.3 mm in female);(2)head longer than wide,prominent bony ridges present;(3)snout rounded in dorsal view;(4)tail length less than snoutvent length in both males and females;(5)dark black body coloration,except for bright orange cranial region,palms and soles,vent region,and ventral ridge of tail;(6)relative length of toes Ⅲ>Ⅳ>Ⅱ>Ⅰ>Ⅴ;(7)fingertips reaching to between the eyes and nostrils when the forelimbs are stretched forward;(8)distal tip of the limbs greatly overlapping when the fore and hind limbs are pressed along the trunk;(9)presence of gular fold;(10)vertebral ridge slightly segmented,slightly flattened rib nodules,indistinct interspaces between rib nodules;and(11)lacking orange color markings on posterior parotoids and rib nodules.The found of the new species suggests that the species diversity of the genus Tylototriton may be underestimated.