探究大熊猫核糖体蛋白基因RPL35A及其编码蛋白的特点,以进一步研究其编码蛋白的功能,为深入开展大熊猫分子生物学研究及大熊猫基因保护等积累科学资料;本研究以大熊猫骨骼肌为材料,采用RT-PCR和降落-PCR方法克隆RPL35A基因的cDNA基因组序列,IPTG法诱导基因在BL21细胞系中的表达;大熊猫RPL35A基因的cDNA序列为337 bp,编码110个氨基酸;该基因的基因组序列长1 275 bp,包含2个外显子和1个内含子;RPL35A基因在BL21细胞系中的表达产物与组氨酸标签蛋白融合形成18 kDa的融合蛋白;该基因的cDNA序列、基因组序列中的外显子片段及其编码的蛋白序列与已报道的人、牛、小家鼠、褐家鼠等物种的对应序列同源性很高,蛋白质理化性质和功能位点基本相同,这说明该基因及其编码蛋白在进化过程中非常保守,可用于分子进化方面的研究,在物种进化关系研究中具有一定的参考意义和研究价值;同时,该基因可在原核生物中表达出具有活性的产物,可进一步开展纯化和蛋白质功能的研究.

以生物学为例,浅析高等师范院校生物学教学语言的要求与特点.教师的教学用语应该是科学与艺术结合的典范,教师要根据教学内容、课程特点及教学阶段,做到教学语言的科学性、确切性、可接受性、机动性和丰富性,不断促进高等师范院校生物学教学的持续快速发展.

The cDNA fragments ofhnRNPA2/B1 were cloned from the giant panda and black bear using RT-PCR method, which were, respectively, 1029bp and 1026bp in length encoding 343 and 341 amino acids. Analysis indicated the cDNA cloned from the giant panda encoded variant B1 while the cDNA cloned from black bear encoded variant A2.Analyzing the hnRNPA2B1 peptide of the giant panda and black bear, 76 glycine residues and 86 glycine residues were, respectively, found, and moreover, most glycine are concentrated in the latter halves of the hnRNPA2B1 peptides. Functional sites prediction also showed many N-myristoylation sites existed in the glycine-rich domain, which is probably related to the role of telomere maintenance.From base bias and substitution analysis, we can conclude that the ORF of hnRNPA2/B1 biased G while hated C, and transition of the third site did not achieve the level of saturation.Orthology analysis indicated that both the nucleotide sequence and the deduced amino acid sequence showed high identity to other 26 hnRNPA2/B1 sequences from mammals and nonmammals reported. These sequences were used to construct phylogenetic trees employing the NJ method with 1000 bootstrap, and the obtained tree demonstrated similar topology with the classical systematics, which suggested the potential value of hnRNPA2/B1 in phylogenetic analysis.This report will be the first step to the study function of hnRNPA2/B1 in the giant panda and black bear, and will provide a scientific basis to disease surveillance, captive breeding, and conservation of the endangered species.

利用大分子序列差异构建分子系统树已成为现代系统分类和生物进化研究工作者的基本方法和工具,掌握建树方法对该领域的研究工作具有十分重要的意义。构建分子系统树的软件很多。就DNAMAN6.0和MEGA5.1联合建树的简易方法进行阐述,帮助初学者建树。

The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance.

ATP6V1F encodes a component of vacuolar ATPase mediating acidification. The cDNA and the genomic sequences of ATP6V1F were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription polymerase chain reaction and touchdown-polymerase chain reaction, respectively. The cDNA fragment cloned is 364 bp in size, containing an open reading frame of 360 bp encoding 119 amino acids. Alignment analysis indicated that both ORF and the deduced amino acid sequence are highly conserved. The length of the genomic sequence of the Giant Panda is 2225 bp, including two exons and one intron. Topology prediction showed that there is one protein kinase C phosphorylation site, two Casein kinase II phosphorylation sites, and one N-myristoylation site in the ATP6V1F protein. The ATP6V1F gene was overexpressed in Escherichia coli indicating that ATP6V1F fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 17 kDa polypeptide, which was according with the predicted protein and also could be used to purify the protein and study its function.

From March 2008 to February 2009,we used Night trapping method to investigate the ecology of Suncus murinus in Zhanjiang Rormal University.The results showed that the means capture rate of Suncus murinus was 15.40%,the peak was in August.The amphoteric ratio was 1.25∶1.Whole year average pregnancy rate of Suncus murinus was 32.41%,the average enbryo was 3.5.Density and species proportion of Suncus murinus were influenced by some factors,such as environmental condition,climate and poison.Pregnancy rate of Suncus murinus was closely related to climate.

[Objective] The research aimed to understand structure differentiation of Penaeus vannamei.[Method] With Penaeus vannamei as the test objects,the appendage of Penaeus vannamei was separated and its structure was observed.[Result] The results indicated that there were 19 appendages in Penaeus vannamei.The morphological features were diverse in different parts of the appendage.Because the different function of appendage,the morphological features were also diverse.[Conclusion] Different living styles of different species of shrimp caused various differentiation of appendage.

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.

[Objective]The purpose was to clone and analyze mitochondrial NADH dehydrogenase subunits 5 and 6(ND5 and ND6) genes of Ursus thibetanus spp.mupinensis.[Method]The primers were designed according to the reported information related to ND5 and ND6 gene sequence of part Ursid.The coding sequences of ND5 and ND6 genes were cloned in the total DNA in musculature of mupinensis successfully for the first time by applying PCR technique and their sequence characteristics were analyzed and compared with that of 9 reported Ursid species.[Result]This cloned sequence included 2 genes of ND5 and ND6,their total length was 2 456 bp and they coded 781 amino acids and had very high homology with ND5 and ND6 genes of the 9 Ursid species.The molecular weights of proteins coded by ND5 and ND6 genes of this cloned sequence were 68.262 1 and 19.038 6 kDa resp.and their isoelectric points were 9.19 and 4.25 resp.Their molecular weights and isoelectric points were very close to that of the 9 Ursid species.[Conclusion]The ND5 and ND6 genes and their coded proteins of mupinensis had very high similarity and functional consistency with that of the 9 Ursid species,especially high functional consistency with that of Selenarctos thibetanus G.Cuvier.

RPS28 is a component of the 40S small ribosomal subunit encoded by RPS28 gene, which is specific to eukaryotes. The cDNA and the genomic sequence of RPS28 were cloned successfully from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily and the cDNA of the RPS28 gene was also overexpressed in Escherichia coli ER2566 and immunoblotted. The cDNA of the RPS28 cloned from Giant Panda is 216 bp in size, containing an open reading frame of 210 bp encoding 69 amino acids. The length of the genomic sequence is 790 bp, which was found to possess three exons and two introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos Taurus, Mus musculus, Rattus norvegicus and Sus scrofa (92.4, 92.4, 87.1, 86.7 and 89.5%, respectively) as determined by Blast analysis.. The amino acid sequence encoded by RPS28 gene of the Giant Panda shared a high homology (100%) with those of Homo sapiens, B. Taurus, M. musculus, R. norvegicus and S. scrofa. Primary structure analysis revealed that the molecular weight of the putative RPS28 protein is 7.841 kD with a theoretical pI 10.70. Topology prediction showed there is one ribosomal protein S28e signature site, three protein kinase C phosphorylation site and two casein kinase II phosphorylation site in the RPS28 protein of the Giant Panda. The RPS28 gene can be really expressed in Escherichia coli and the RPS28 protein fusioned with the N-terminally GST -tagged protein gave rise to the accumulation of an expected 34 KDa polypeptide. The expression product obtained could be used for purification and study of its function further.

RPS20 is a component of the 40S small ribosomal subunit encoded by RPS20 gene, which is conserved between eukaryotes, prokaryotes and archaebacteria. The cDNA and the genomic sequence of RPS20 were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively. Both sequences were analyzed preliminarily and the cDNA of the RPS20 gene was also overexpressed in Escherichia coli BL21. The cDNA of the RPS20 cloned from Giant Panda is 392 bp in size, containing an open reading frame of 360 bp encoding 119 amino acids. The length of the genomic sequence is 1205 bp, which was found to possess 4 exons and 3 introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Pongo abelii, Macaca fascicularis, Mus musculus, Bos taurus and Rattus norvegicus are 93.1, 92.5, 92.2, 91.1, 90.6 and 90.0% respectively. The amino acid sequence encoded by RPS20 gene of the Giant Panda shared a high homology (100%) with those of H. sapiens, Mac. fascicularis, Mus musculus, B. taurus and R. norvegicus, except for P. abelii (99.88%). Primary structure analysis revealed that the molecular weight of the putative RPS20 protein is 13.373 kD with a theoretical pI 9.95. Topology prediction showed there is one ATP/GTP-binding site motif A, one ribosomal protein S10 signature site, 5 protein kinase C phosphorylation sites and three Casein kinase II phosphorylation sites in the RPS20 protein of the Giant Panda. The RPS20 gene can be really expressed in E. coli and the RPS20 protein fusioned with the N-terminally GST-tagged form gave rise to the accumulation of an expected 39 kDa polypeptide.

Mitochondrial ATP synthase F0 subunit 8(ATP8) and subunit 6(ATP6) gene in muscular tissue of Asian black bear Sichuan subspecies(Ursus thibetanus mupinensis) are amplified for the first time through PCR technology with primers designed according to ATP8 genes and ATP6 genes of some other mammals reported ,which are cloned,sequenced and analyzed preliminarily.The result shows that the full length of the PCR product is 942bp which contains ORF 842 encoding the ATP8 and ATP6 genes with a 43bp overlap between them.The ATP8 sequence is 204bp encoding a protein with 67 amino acid residues whose molecular weight is 7.9KD and isoelectric point is 10.35.The ATP6 gene is 682bp encoding a protein with 226 amino acid residues whose molecular weight is 24.8KD and isoelectric point is10.63.Both of them are highly homologous to those of other mammals reported.Construction of phylogenetic tree based on nucleotide sequence manifests that the Asian black bears with Sichuan subspecies have closer relationship to the American black bears.

The cDNA of acidic ribosomal phosphoprotein P1(RPLP1)was cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily. The result shows that the cDNA fragment cloned is 449 bp in size, and it contains an open reading frame of 344 bp encoding 114 amino acids. The deduced protein sequence shows that the protein is composed of 114 amino acids and its estimated molecular weight is 11.566 kDa with a pI of 4.4. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of giant panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the giant panda.

The cDNA of RPS15 was cloned successfully for the first time from the Giant Panda using RT-PCR technology, which was also sequenced and analyzed preliminarily. The cDNA fragment is 442bp in size, containing an open reading frame (ORF) of 438bp encoding 145 amino acids. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence show a high homology to those of human and other mammalian species reported. Topology prediction shows there are two cAMP and cGMP-dependent kinase phosphorylation sites, one Ribosomal Protein S19 signature site, four N- myristoylation sites and five Casein kinase C phosphorylation sites in the RPS15 protein. Further analysis indicates that the expression sequence of RPS15 and the protein encoded are highly homologous to those of human and other mammals reported. The study is of significance to provide truthful and scientific data for studying the Giant Panda (Ailuropoda melanoleuca) at molecular level, especially for cloning and further researching the complete sequence of RPS15.

RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first report on the RPS25 gene from the Giant Panda. The data will enrich and supplement the information about RPS25, which will contribute to the protection for gene resources and the discussion of the genetic polymorphism.

Mitochondrial Cytochrome C Oxidase subunit Ⅲ gene(mtCOXⅢ) in muscular tissue of Asiatic black bear Sichuan subspecies(Ursus thibetanus mupinensis) was amplified for the first time using PCR technology with primers designed according to mtCOXⅢ genes of some mammals reported,which was cloned,sequenced and analyzed preliminarily.The mtCOXⅢ sequence of Asiatic black bear Sichuan subspecies is 784 bp containing ORF 783 bp and codes 261 amino acid residues whose isoelectric point is 6.47 and molecular weight is 29840.6 Da.The mtCOXⅢ gene is highly homologous to some mammals reported.Further analysis indicates that the position,the type and the quantity of the function sites of cloned mitochondrial Cytochrome C Oxidase subunit Ⅲ are almost same to some mammals reported.

Mitochondrial ATP synthase (F1Fo-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, using RT-PCR combined with in silico cloning, we isolated and sequenced the cDNA encoding the inhibitor protein of the giant panda (Ailuropoda melanoleuca). The deduced protein sequence showed that the protein is composed of 106 amino acids and the estimated molecular weight of the ATPIF(1) protein is 12.32 kDa with an isoelectric point (pI) of 10.17. Alignment analysis revealed that the deduced protein sequence shares 66%, 78.3%, 66%, 72.6%, 77.4%, and 78.3% homology with that of Mus musculus, Pan troglodytes, Rattus norvegicus, Bos taurus, Macaca mulatta, and Homo sapiens, respectively. Topology prediction showed that there are three protein kinase C phosphorylation sites, one amidation site, three N-myristoylation sites, one casein kinase II phosphorylation site, and one tyrosine kinase phosphorylation site in the ATPase inhibitor. In particular, amino acids in the region between 39 and 72, which is the minimum sequence showing ATPase inhibitory activity, were highly conserved in the protein.

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16,868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%).