Abstract: The complete coding sequences of three sheep genes-RAB1A, RAB4A and RAB5A were amplified using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Sequence analysis revealed that the sheep RAB1A gene encodes a protein of 204 amino acids that shares high homology with the RAB1A, member RAS oncogene family (RAB1A) proteins of nine species-human (98%), mouse (98%), rat (98%), pig (98%), zebrafish (98%), Western clawed frog (97%), Atlantic salmon (96%), disc abalone (92%) and great pond snail (87%). The sheep RAB4A gene encodes a protein of 218 amino acids that shares high homology with the RAB4A, member RAS oncogene family (RAB4A) proteins of eight species-cattle (100%), dog (99%), human (99%), rhesus monkey (99%), horse (99%), chicken (99%), rat (98%) and mouse (98%). The sheep RAB5A gene encodes a protein of 215 amino acids that shares high homology with the RAB5A, member RAS oncogene family (RAB5A) proteins of fifteen species-cattle (98%), rabbit (98%), dog (98%), horse (98%), pig (98%), sumatran orangutan (98%), human (97%), mouse (97%), rat (97%), chimpanzee (97%), chicken (95%), rhesus monkey (93%), African clawed frog (93%), Western clawed frog (93%) and zebrafish (92%). Finally, these three novel sheep genes were assigned to GeneIDs: 100302065, 100302066 and 100302084. Phylogenetic analysis indicated that the sheep RAB1A gene has a closer genetic relationship with the RAB1A genes of human, mouse and rat. The sheep RAB4A and RAB5A genes both have closer genetic relationships with the RAB4A and RAB5A genes of cattle. Tissue expression profile analysis was also carried out and results demonstrated that sheep RAB1A, RAB4A and RAB5A genes were all generally but differentially expressed in detected tissues.

INTRODUCTION

RAB1A, member RAS oncogene family (RAB1A) is a member of Rab1 subfamily. Rab1 is found in eukaryote and is an important regulatory factor for the transport of vesicles from the ER to the Golgi apparatus. Latest researches suggested a novel function for Rab1a in the regulation of cell migration through controlling integrin beta1 recycling and localization to lipid rafts via a specific downstream effector pathway. Researches also revealed that the Rab1a plays a crucial role in mammalian autophagy (Huang et al., 2011; Wang et al., 2010; Diao et al., 2008).

RAB4A, member RAS oncogene family (RAB4A) is a member of Rab4 subfamily. Rab4 has been implicated in numerous functions within the cell. Experimental data revealed that overexpression of Rab4 regulates angiotensin II type I receptor phosphorylation and sensitization. Rab4A had also been identified to be a critical effector of VEGFR1 during branching morphogenesis of the vasculature (Esseltine et al., 2011; Schonhoff et al., 2009; Kachhap et al., 2007).

RAB5A, member RAS oncogene family (RAB5A) is a member of Rab5-related subfamily. This subfamily includes Rab5 and Rab22 of mammals, Ypt51/Ypt52/Ypt53 of yeast and RabF of plants. Recent researches showed that selective upregulation of Rab5 level is associated with mild cognitive impairment, Alzheimer's disease and sporadic motor neuron disease. Rab5a can promote proliferation of ovarian cancer cells (Ginsberg et al., 2010; Zhao et al., 2010; Matej et al., 2010).

As mentioned before, RAB1A, RAB4A and RAB5A genes are three genes which have important functions. Until today, RAB1A, RAB4A and RAB5A genes had been reported in human and other animals but the sheep RAB1A, RAB4A and RAB5A genes have not been reported yet.

In present experiment, the researchers will isolate the coding sequences of sheep RAB1A, RAB4A and RAB5A genes based on the coding sequence information of RAB1A, RAB4A and RAB5A genes from human or other mammals and their highly homologous sheep ESTs sequence information, subsequently perform some necessary sequence analysis and tissue expression profile analysis for these genes. These will establish the primary foundation of understanding these three sheep genes.

MATERIALS AND METHODS

Animals and sample preparation: Five adult Yunnan local sheep were slaughtered. Spleen, skin, lung, fat, muscle, heart, liver, kidney and ovary samples were collected, frozen in liquid nitrogen and then stored at -80°C. The total RNA was extracted using the total RNA extraction kit (Gibco, USA). First-strand cDNA synthesis was performed as that described by Liu et al. (2004). These first-strand cDNA samples were used to perform RT-PCR for the isolation of sheep RAB1A, RAB4A and RAB5A genes and for the tissue expression profile analysis.

Isolation of the sheep RAB1A, RAB4A and RAB5A genes: The primers for sheep RAB1A gene isolation were designed based on the coding sequence information of human RAB1A gene and its highly homologous sheep EST sequences: EE751788 and EE747095. Similarly, the primers for sheep RAB4A gene isolation were designed based on the coding sequence information from human RAB4A gene and its highly homologous sheep EST sequence: EE794089. The primers for sheep RAB5A gene isolation were designed based on the coding sequence information from human and mouse RAB5A genes and their highly homologous sheep EST sequences: EE806405 and EE791858. These primer sequences and their annealing temperature for RT-PCR reaction were shown in Table 1. The RT-PCR was performed to isolate these three sheep genes using the pooled cDNAs from different tissues above. The 25 μL reaction system was 2.0 μL cDNA, 2.5 μL 2 mM mixed dNTPs, 2.5 μL 10xTaq DNA polymerase buffer, 2.5 μL 25 mM MgCl₂, 2.0 μL 10 μM forward primer, 2.0 μL 10 μM reverse primer, 2.0 units of Taq DNA polymerase (1 U/1 μL) and 9.5 μL sterile water. The PCR program initially started with a 94°C denaturation for 4 min followed by 35 cycles of 94°C/50 sec, Ta°C/50 sec, 72°C/50 sec then 72°C extension for 10 min, finally 4°C to terminate the reaction.

Table 1:	Primers for sheep RAB1A, RAB4A, RAB5A and Beta-actin genes and their annealing temperatures

These PCR products for sheep RAB1A, RAB4A and RAB5A genes were then cloned into PMD18-T vector and sequenced bidirectionally with the commercial fluorometric method. At least five independent clones were sequenced for every gene.

RT-PCR for tissue expression profile analysis: RT-PCR for tissue expression profile analysis was performed as previously described elsewhere (Liu and Gao, 2009; Yonggang and Shizheng, 2009; Liu, 2009). We selected the housekeeping gene beta-actin (Accession No.: NM_001009784) as a positive control. The primers of sheep RAB1A, RAB4A and RAB5A genes which were used to perform the RT-PCR for tissue expression profile analysis were same as the primers for isolation RT-PCR above. The PCR reactions were optimized for a number of cycles to ensure product intensity within the linear phase of amplification. The 25 μL reaction system was 1 μL cDNA (100 ng μL^-1), 5 pmoles each oligonucleotide primer, 2.5 μL 2 mmol L^-1 mixed dNTPs, 2.5 μL 10xTaq DNA polymerase buffer, 2.5 μL 25 mmol L^-1 MgCl₂, 1.0 unit of Taq DNA polymerase and finally add sterile water to volume 25 μL. The PCR program initially started with a 94°C denaturation for 4 min followed by 25 cycles of 94°C/50 sec, Ta°C/50 sec, 72°C/50 sec then 72°C extension for 10 min, finally 4°C to terminate the reaction.

Sequence analysis: The cDNA sequence prediction was conducted using GenScan software (http://genes.mit.edu/GENSCAN.html). The protein prediction and analysis were performed using BLAST tool at the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST) and the ClustalW software (http://www.ebi.ac.uk/clustalw).

RESULTS AND DISCUSSION

RT-PCR results for sheep RAB1A, RAB4A and RAB5A genes: Through RT-PCR with pooled tissue cDNAs for sheep RAB1A, RAB4A and RAB5A genes, the resulting PCR products were 615, 657 and 648 bp (Fig. 1).

Sequence analysis: The cDNA nucleotide sequence analysis using the BLAST software at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST) revealed that these three genes were not homologous to any of the known sheep genes and they were then deposited into the GenBank database (Accession No.: FJ943970, FJ943971 and FJ943983). The gene prediction was carried out using the GenScan software and results showed that the 615, 657 and 648 bp cDNA sequences represent three single genes which encoded 204, 218 and 215 amino acids, respectively.

Fig. 1:	RT-PCR results for sheep RAB1A, RAB4A and RAB5A genes. M: DL2000 DNA markers; 1: PCR product for sheep RAB1A gene; 2: PCR product for sheep RAB4A gene and 3: PCR product for sheep RAB5A gene

Finally, these three novel sheep genes were assigned to GeneIDs: 100302065, 100302066 and 100302084.

Further BLAST analysis of these deduced proteins revealed that the sheep RAB1A protein has high homology with the RAB1A, member RAS oncogene family (RAB1A) proteins of nine species human (Accession No.: NP_004152; 98%), mouse (Accession No.: NP_033022; 98%), rat (Accession No.: NP_ 112352; 98%), pig (Accession No.: NP_0010269 57; 98%), zebrafish (Accession No.: NP_001007162; 98%), Western clawed frog (Accession No.: NP_001004787; 97%), Atlantic salmon (Accession No.: ACN11413; 96%), disc abalone (Accession No.: ABO26625; 92%) and great pond snail (Accession No.: Q05974; 87%) (Fig. 2).

The sheep RAB4A protein has high homology with the RAB4A, member RAS oncogene family (RAB4A) proteins of eight species cattle (Accession No.: DAA14394; 100%), dog (Accession No.: XP_536353; 99%), human (Accession No.: NP_004569; 99%), rhesus monkey (Accession No.: XP_001082985; 99%), horse (Accession No.: XP_001498005; 99%), chicken (Accession No.: XP_419573; 99%), rat (Accession No. NP_037151; 98%), mouse (accession number: NP_033029; 98%) (Fig. 3).

Fig. 2:	The alignment of the protein encoded by sheep RAB1A gene and nine other kinds RAB1A proteins

Fig. 3:	The alignment of the protein encoded by sheep RAB4A gene and eight other kinds of RAB4A proteins

The sheep RAB5A protein has high homology with the RAB5A, member RAS oncogene family (RAB5A) proteins of fifteen species-cattle (Accession No.: NP_001069654; 98%), rabbit (Accession No.: XP_002716250; 98%), dog (Accession No.: NP_001003317; 98%), pig (Accession No.: NP_001116652; 98%), horse (Accession No.: XP_001495368; 98%), sumatran orangutan (Accession No.: XP_002814060; 98%), human (Accession No.: NP_004153; 97%), mouse (Accession No.: NP_080163; 97%), rat (Accession No.: NP_073183; 97%), chimpanzee (Accession No.: XP_516319; 97%), chicken (Accession No.: NP_001006363; 95%), rhesus monkey (Accession No.: XP_001086669; 93%), African clawed frog (Accession No.: NP_001080535; 93%), Western clawed frog (Accession No.: NP_001008068; 93%) and zebrafish (Accession No.: NP_958893; 92%) (Fig. 4).

Based on the results of the alignment of RAB1A, RAB4A and RAB5A proteins, three phylogenetic trees were constructed using the Dendrogram procedure of ClustalW software (http://align.genome.jp/) as shown in Fig. 5-7.

The phylogenetic analysis revealed that the sheep RAB1A gene has a closer genetic relationship with the RAB1A genes of human, mouse and rat. The sheep RAB4A and RAB5A genes both have closer genetic relationships with the RAB4A and RAB5A genes of cattle.

Tissue expression profile: Tissue expression profile analysis was carried out and results revealed that the sheep RAB1A, RAB4A and RAB5A genes are all generally but differentially expressed in tissues including spleen, lung, muscle, kidney, ovary, skin, liver, heart and fat (Fig. 8).

In the current study, the researchers firstly get the coding sequences of sheep RAB1A, RAB4A and RAB5A genes by RT-PCR. With the development of modern bioinformatics and establishment of specific sheep NCBI EST database, researchers can easily find the useful ESTs which were highly homologous to the coding sequences of human genes. Based on these sheep EST sequences, the researchers can obtain the complete coding sequences of some novel sheep genes through the some experimental methods such as RT-PCR. From the clone and sequence analysis of sheep RAB1A, RAB4A and RAB5A genes, it could be seen that this is an effective method to isolate some novel sheep genes.

Through sequence analysis, the researchers found that the encoding protein of the sheep RAB1A, RAB4A and RAB5A genes are highly homologous with RAB1A, RAB4A and RAB5A proteins of human and some other animals.

Fig. 4:	The alignment of the protein encoded by sheep RAB5A gene and fifteen other kinds of RAB5A proteins

This implied that the RAB1A, RAB4A and RAB5A genes were highly conserved in some species and the sheep RAB1A, RAB4A and RAB5A genes will have similar functions as the RAB1A, RAB4A and RAB5A genes of human and other animals. The researchers also found that the sheep RAB1A, RAB4A and RAB5A proteins do not show complete identity to human or other animals. This implied that the sheep RAB1A, RAB4A and RAB5A genes will have some differences in functions to those of human or other mammals.

Fig. 5:	The phylogenetic analysis for ten kinds of RAB1A genes

Fig. 6:	The phylogenetic analysis for nine kinds of RAB4A genes

The phylogenetic analysis revealed that the sheep RAB1A gene has a closer genetic relationship with the RAB1A genes of human, mouse and rat. This implied that the researchers can use human, mouse and rat as model organisms to study the sheep RAB1A gene or use sheep as model organism to study the human, mouse and rat RAB1A genes.

The sheep RAB4A and RAB5A genes both have closer genetic relationships with the RAB4A and RAB5A genes of cattle. Similarly, we can use cattle as a model organism to study the sheep RAB4A and RAB5A genes or use sheep as a model organism to study the cattle RAB4A and RAB5A genes.

From the tissue distribution analysis in the experiment it can be seen that the sheep RAB1A, RAB4A and RAB5A genes were obviously differentially expressed in some tissues. As the researchers did not study functions at protein levels yet, there might be many possible reasons for differential expression of sheep RAB1A, RAB4A and RAB5A genes.

Fig. 7:	The phylogenetic analysis for sixteen kinds of RAB5A genes

Fig. 8:	Tissue expression distribution of sheep RAB1A, RAB4A and RAB5A genes. The beta-actin expression is the internal control. 1: Spleen; 2: Skin; 3: Lung; 4: Muscle; 5: Heart; 6: Fat; 7: Liver; 8: Kidney and 9: Ovary

The suitable explanation for this under current conditions is that at the same time those biological activities related to the mRNA expression of sheep RAB1A, RAB4A and RAB5A genes were presented diversely in different tissues.

CONCLUSION

In this study, the researchers first isolated the sheep RAB1A, RAB4A and RAB5A genes and performed necessary sequence analysis and tissue transcription profile analysis. This established the primary foundation for further insight into these novel sheep genes.

ACKNOWLEDGEMENTS

This study was supported by grants from the Natural Science Foundation Key Project of Yunnan Province (No. 2009CC015), National Nature Science Foundation of China (No. 30800810) and the Candidates of the Young and Middle Aged Academic and Technical Leaders of Yunnan province (2009CI055).