Genetic and Protein Sequence Analysis of rpS29 from Ailuropoda melanoleuca(giant panda)

Jie Deng，Xiang Ding，Wanru Hou，Jie Zhong，Li Luo，Bo Song，Ting Wang，Yiling Hou

(Key Laboratory of Southwest China Wildlife Resources Conservation;College of Life Science China West Normal University，Nanchong 637009，China)

1.INTRODUCTION

A ribosome is the component of a biological cell that creates proteins from all amino acids by reading information of mRNA.It consists of a small 40S subunit and a large 60S subunit，and together these subunits are composed of 4 RNA species and approximately 80 structurally distinct proteins［1］.A-bundant researches shows that many ribosomal proteins are related to the various extra ribosomal activities，including regulation of cell proliferation，DNA repair，transcription，and RNA processing［2－3］.

Ribosomal protein S29，as a component of the 40S subunit，belongs to the S14P family of ribosomal proteins，and is located in the cytoplasm.By comparison，a high similarity was found in 40S amino acid sequences between Senegalese sole and Atlantic halibut，but rpS29 gene exhibited different expression patterns in tissues，with the lowest levels in brain［4］.It is reported this gene is related to squamous cell carcinoma and its expression level was significantly higher in tumors than in normal samples［5］.

Giant panda，regarded as a national treasure of China，is one of the oldest species，belonging to the endangered mammals because its population has been declining and it is now rare and limited to several provinces of China.Recently，the ribosomal protein genes have been cloned from giant panda，such as rpL34，rpS26，rpS12［6－7］，however，there is no report about rpS29 gene of this species.

In this study，the cDNA and genomic sequences of rpS29 gene was cloned from giant panda and the structural characteristic of this gene was analyzed.

2.MATERIALS AND METHODS

Muscle tissues were collected from a dead giant panda at the Wolong Conservation Center，Sichuan，China and kept in liquid nitrogen.Total RNA was isolated using RNA Extraction Kits，dissolved in DEPC water，and kept at －70℃.

The PCR primers were designed by Primer Premier 5.0，according to the mRNA sequence of rpS29 gene from H.sapiens(NM_001030001)，B.taurus(NM_174804)，M.musculus(NM_009093)and R.norvegicus(NM_012876)，as follows:

rpS29-F:5'-GTTGGGCGTCTGAAGGCAAG-3';

rpS29-R:5'-CCCATCAAGGTCGT-3';

The expected fragments of PCR products were harvested and purified from gel using a DNA harvesting kit，and then ligated into a pET28a vector at 22℃ for 12 hours.The recombinant molecules were transformed into E.coli complete cells(JM109).Plasmid DNA was isolated and digested by Nde I and Sac I to verify the insert size，then sequenced by Huada Zhongsheng Scientific Corporation(Beijing，China).

The rpS29 gene was amplified using Touchdown-PCR.The fragment amplified was also purified，ligated into the clone vector and transformed into the E.coli competent cells.Finally，the recombinant fragment was sequenced by Sangon.

The sequence data were analyzed by GenScan software.Homology analysis compared with the other species was performed using Blast 2.1;ORF of the DNA sequence was searched using ORF finder software.The rpS29 cloned was deduced using Predict Protein software.Multiple sequence alignment was performed by DNAMAN 6.0.The prediction of protein functional sites and biochemical characteristics was depended on ExPASy Proteomics Server.

3.RESULTS

A cDNA fragment of about 200 bp was amplified from giant panda，and its exact length is 205 bp by electrophoresis(Fig.1).Blast research showed that the cDNA sequence shares a high homology with the rpS29 from some other mammals，including H.sapiens，B.taurus，M.musculus and R.norvegicus.Basing on the high level of identity，it could be concluded that the cDNA encoding rpS29 protein has been cloned from giant panda(Genbank accession number:HQ318031).The cDNA sequence contains a 20 bp of the 5’－untranslated sequence，a 14 bp of 3’－unstranslated region and an 171 bp of open reading frame(ORF)，with 21.0%A，24.9%C，30.2%G and 23.9%T，encoding 56 amino acids.

A DNA fragment of about 1600 bp was amplified.The length of the cloned DNA fragment is 1598 bp，with three exons and two introns.A comparison between the cDNA sequence and the DNA fragment sequence of the rpS29 amplified from giant panda revealed that the cDNA sequence is in full accord with five fragments in the DNA fragment，which confirms that the amplified DNA fragment is the genomic sequence of the rpS29 from the giant panda(Genbank accession number:HQ318032)(Tab.1).

Fig.1 Nucleotide and putative amino acid sequence of rpS29 cDNA from A.melanoleuca.* for stop codon

Primary structure analysis showed that the molecular weight of the putative rpS29 protein of giant panda is 6.68KD，with a theoretical pI of 10.63.Further study found the protein is composed of 2 negatively charged residues(Asp)，14 positively charged residues(Arg7，Lys4 and His3)and 40 uncharged residues.Among all the amino acids，the content of Arg(12.50%)is the highest，while Trp，Ala，Pro and Val(1.79%)listed for the lowest.Topology prediction shows that there are two protein kinase C phosphorylation sites，two N-myristoylation sites，one ribosomal protein S14 signature site in the rpS29 protein of giant panda(Fig.2).

Fig.2 Comparison of rpS29 amino acid sequences among the different species

The coding sequence and deduced amino acid sequences of rpS29 in giant panda were compared with those of mammals mentioned previously，the results revealed that the coding sequence shows a high degree of homology to these mammalians(Tab.1).Among all the mammalians compared，the degree of homology between giant panda and B.taurus is the highest(94.74%).However，the degree of homology between giant panda and H.sapiens is only 75.00%.Further analysis showed that homologies for amino acid sequences are 82.09%，100%，100%and 100% respectively，but their isoelectric points(pI)are all the same(Tab.2).

Table 1 Compare of rpS29 genomic sequence among 5 mammal species

Table 2 Comparison of nucleotide，amino acid sequences and physicochemical property between A.melanoleuca and other 4 mammal species

4.DISCUSSION

It is reported that rpS29 gene is the more suitable reference gene than ACTB and GAPDH in expression studies of porcine stomach.Uechi et al.knocked down 21 RP genes in zebrafish and found that 19 rpS resulted in the development of morphants with obvious deformities.He concluded that each knocked-down ribosome protein gene that affected the morphogenesis of the brain produced a different pattern of abnormality.Thus，to study ribosomal proteins is important to understand the biological development.

In this study，we cloned genomic sequence and cDNA clone encoding rpS29 from the giant panda.The genomic sequence of rpS29 is 1598 bp in size.A comparison among giant panda and other four mammals of the nucleotide sequences of the genomic and cDNA sequences indicated that their genomic sequence of rpS29 possesses three exons and two introns.Further study indicated that the genomic，the introns，the 5’－untranslated sequence and the 3'－untranslated sequence are different in length.The variations in lengths of the introns determine the lengths of the rpS29 gene.From the alignment analysis of the cDNA sequence of rpS29 gene and the deduced amino acid sequence between giant panda and other four mammals，it could be conclude that giant panda shares high homology in nucleotide sequence with those mammals，among which，the giant panda shares the highest homology in nucleotide sequence with B.Taurus of 94.74%，meanwhile，the degree of homology for amino acid sequences among them is high up to 100%(Tab.2).

Through comparing the coding sequenc of rpS29 for giant panda with H.sapiens，B.taurus，M.musculus，and R.norvegicus，it was found that their functional sits have the same species，positions and numbers，but variable site exists among them.Analysis indicated that Only H.sapiens has the change at site 59(L→D)(Fig 2).However，the variation of site has no effect on the s function of rpL21 protein.Physical and chemical analysis showed that the molecular weight of the putative protein among the six mammalians is very close，with exactly identical theoretical pI.

In summary，the complete coding sequence of rpS29 gene has been cloned using RT－PCR technology successfully.The characteristics of genomic sequence and cDNA clones encoding ribosomal proteins would be beneficial in the study of ribosomal biogenesis and would allow the elucidation of structure，organization，and regulation of genes encoding ribosomal proteins in eukaryote.

5.ACKNOWLEDGEMENTS

This work is financially supported by Application Foundation Project in Sichuan Province(2009JY0061)and Youth Fund Project of Educational Committee of Sichuan province(09ZB088).

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