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Science in China Series C: Life Sciences 2006 Vol.49 No.4 342—348

DOI: 10.1007/s11427-006-2012-3

Cloning and analysis of differentially expressed ESTs in swine muscle tissue LI Chongsheng, CHEN Yaosheng, WANG Chong & LI Jiaqi College of Animal Science, South China Agricultural University, Guangzhou 510642, China Correspondence should be addressed to Wang Chong (email: [email protected])

Received March 29, 2005; accepted November 16, 2006

Abstract The obvious difference in muscle growth and meat quality traits exists between Chinese indigenous pig and exotic pigs. In order to study the reason of these phenotypic differences and search the potential gene related to growth and meat quality traits, silver-stained mRNA differential display technique was used to detect the difference with mRNA of loin-eye muscle tissue from maturity pigs of Lantang in Guangdong Province and Large Yorkshire. One of the newly discovered expressed sequence tag (ESTsp3) was analyzed by using bioinformatic technique. The results showed: (1) nearly 2000 cDNA fragments were detected with 30 primer pairs, and 6 differentially expressed ESTs in the loin-eye muscle tissues from the two breeds were isolated and obtained. The differential fragments were cloned and sequenced. The all sequences were recorded in the GenBank. (2) The 786 bp fragment of ESTsp3 was obtained with in silico elongation system, the ORF analysis revealed that it existed as an 83 aa complete open reading frame, and the elongation sequences were verified by RT-PCR. The analysis of in silico expression profile showed that ESTsp3 is expressed in various growth stages and in most tissues and organs, such as soft tissue, skin, skeletal muscle and kidney, but with variant expression quantity. Keywords: pig, muscle, mRNA differential display, in silico expression profile.

Pig growth and meat quality traits are widely studied, since pork is the main source of animal protein. In the past 20 years, alone with the development of animal genome project and molecular marker techniques, much progress was achieved on QTLs[1,2] and candidate gene analysis[3,4] for pig growth and meat quality traits (such as muscle pH, color, water holding capacity, fat content etc.), but it is far away from accurately cloning the QTL. The efficiency of traditional candidate gene technique was very low, since it was based on the identification of genetic effects of functional gene on quantitative trait with physiological and biochemical methods. It is necessary to exploit new techwww.scichina.com

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niques to isolate candidate genes. There are obvious phenotypic differences in muscle growth and meat quality traits between Chinese indigenous pigs and exotic pigs. Many studies have proved that the improved exotic pigs have some characteristics such as faster growth rate, higher feed efficiency and more lean meat content. The Chinese indigenous pigs are just the opposite[5,6]. One of the causes of the genetic differences is that the high intensive selection of breeding[7] and it results in much phenotypic differences, which has been demonstrated by the studies on cytogenetics and molecular genetics ― in recent years[8 10]. In order to explore the causes of

Cloning & analysis of differentially expressed ESTs in swine muscle tissue

the phenotypic differences on the level of RNA, Pan et al. obtained 10 differentially expressed cDNAs from Duroc and Erhualian pigs by differential display techniques[11]. In order to get some molecular genetic information about the difference on muscle growth and meat quality between Lantang pig and Large Yorkshire pig, silver-stained mRNA differential display technique was used to compare and analyze mRNA of loin-eye muscle tissue from the two pig breeds, also the bioinformatics technique was used to analyze the newly discovered EST sequence. 1 1.1

Materials and methods Loin-eye muscle samples

Two purebred Lantang pigs and two Large Yorkshire pigs (one boar and one sow for each breed) were randomly sampled in the research, and all of them were healthy adult pigs. Samples of the loin-eye muscle of pigs were rapidly taken in 5 min after slaughter and were preserved in liquid nitrogen. 1.2 Extraction, processing, detection of the total RNA The total RNA of the loin-eye muscle tissue in the two breeds were extracted according to the protocol in the manual of TRIZOL Reagent of Invitrogen Co. Ltd. Non-RNase DNase-I was used to process RNA, which eliminated the trace quantity of contamination in DNA. The integrality of RNA was detected by the ordinary agarose gel electrophoresis method. The concentration was measured by DNA analyzer. RNA pool of boar and sow for each breeds was isometrically mixed. 1.3

Reverse transcription and PCR amplification

2 μg total RNA, 5 μL 5×RT buffer, 2 μL 2.5 mmol/L dNTPs, 2 μL 100 mmol/L DTT, 4 μL 8 U/μL RNasin and 2 μL 50 pmol/L anchored primer were added in a 0.5 mL sterile centrifuge tube, and DEPC disposal water was added to 24 μL finally. Then they were mixed and incubated at 65℃ for 5 min, incubated at 37℃ for 60 min (added 1 μL 200 U/μL M-MLV reverse transcriptase at 10 min) and incubated at 95℃ for 5 min to inactivate reverse transcriptase. cDNA after reverse transcription was preserved at

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−20℃. PCR reaction system: 2 μL 10× PCR buffer, 1 μL 50 pmol/L anchored primer and random primer, 2 μL 25 mmol/L MgCl2, 2 μL 2.5mmol/L dNTPs, 1 μL reverse transcription mixture, 5 U Taq enzyme, and double distilled water was added to 20 μL finally. PCR cycle parameters: denaturing at 94℃ for 2.5 min→ (denaturing at 94℃ for 30 s→annealing at 40℃ for 2 min → extending at 72℃ for 30 s) 40 cycles → extending at 72℃ for 10 min→preserved at 4℃. 1.4 Non-denatured polyacrylamide gel electrophoresis and silver-stained display 6.5% polyacrylamide gel was made. The mixture of 10 μL PCR product and 4 μL 1× loading buffer was loaded and electrophoresed at 300 V stable voltage for 2.5 h. The gel was stained with silver and gel imaging system was used to photograph. 1.5 Reamplification, collection and purification of the differential display ESTs The display bands were cut from the gel and put in a 0.5 mL centrifuge tube. 50 μL double distilled water was added and boiled away (or 95―100℃) for 10 min. 2 μL of the upper clear fluid was taken for reamplification. All conditions of the reamplification were the same as the first amplification (note: the primer pair of the two amplifications must be corresponding to each other). The reamplification products were purified and collected with TaKaRa Agarose Gel DNA Purification Kit. 1.6 Cloning, sequencing and analyzing of the differential display ESTs The purified product was connected with the pUCm-T vector. Connecting system: 1 μL pUCm-T vector (50 ng/μL), 1 μL insert DNA (50 ng/μL), 3 μL ddH2O, 5 μL solution I. 5 μL connection product was transformed to DH5α competent bacteria. 200 μL revival bacterial fluid was spread on the gel plate containing ampicillin. After being cultured by gently shaking for 12―16 h at 37℃, single positive colony was selected and inoculated in 3 mL LA culture fluid. Plasmids were extracted after culturing at 37℃ with gently shaking and digested with two enzymes for identification. The positive plasmids were sent to

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Boya Shanghai Co. Ltd. for DNA sequencing. The attained sequences were recoded by accessing to the National Center for Biotechnology Information (NCBI) at the website (http://www.ncbi.nih.nlm.gov), and the homology of the sequences from diffent species was compared in GenBank by BLAST software. 1.7 In silico elongation of the differentially expressed ESTsp3 The homological EST sequence compared with the seed sequence was searched in GenBank database by BLAST software. The searched sequences were assembled into a new sequence, and the new sequence was regarded as the new seed sequence for the second search. The iteration progress was repeated till there were no new sequences that could be selected. The last new sequence was in silico elongation production. The open reading frame (ORF) was analyzed for the in silico elongation gene sequence at the website of NCBI with the standard genetic code. 1.8

nm was between 1.8 and 2.0, indicating that it had not been contaminated by proteins and other impurities. So that the quality of the total extracted RNA was perfect and could be used for further studies. 2.2

Results of the differential display

About 2000 cDNA fragments were detected from differential display of 30 primer combinations (cross combinations of 3 anchored primers and 10 random primers) in the research, of which 6 could be repeatable and the differential display ESTs could be collected and amplified. They were named ESTsp1, ESTsp2, … , ESTsp6, respectively. The expression quantities of 2 fragments (ESTsp1, ESTsp5) in Large Yorkshire pigs were more than in Lantang pigs, whereas that of the other 4 fragments in Lantang pigs were more than in Large Yorkshire pigs. Fig. 1 shows the typical results of differential display.

Verification of the in silico elongation sequence

Special gene amplification primers MP1 and MP2 were designed according to the cDNA sequence obtained from the in silico elongation. The aim gene was searched with reverse transcription PCR (RT-PCR) techniques. PCR products were cloned and sequenced by the method described in subsec. 1.7. 1.9

Analysis of ESTsp3 in silico expression profiles

The in silico expression profiles were analyzed with UniGene database by comparing the sequences of ESTsp3 and searched sequences in the database. The tissue expression profile of the gene was deduced from the source of the tissue corresponding to EST sequence which was highly homologized to the analyzed nucleic acid sequence. 2 2.1

Results and analysis Extraction and detection of the total RNA

The agarose gel electrophoresis showed that there were 3 clear bands: 28, 18 and 5 s, but no dragged bands to show non-degradation of the total RNA. Measurement with the DNA analyzer showed that the ratio of absorption value at 260 nm to the value at 280

Fig. 1. Typical results of differential display. L, Lantang pigs; Y, large Yorkshire pigs; M, 2000 bp DNA marker. The arrow indicates the differential display band.

2.3

Repeatability of differential display ESTs

To reduce the influence of individual genetic differences on the results and decrease of the rate of false

Cloning & analysis of differentially expressed ESTs in swine muscle tissue

positive, the total RNA pool was used to eliminate the differences of individual and sex in the research, and repeative experiments were utilized to determine the differential display bands that could be used in the further study. The repeative processes were from reverse transcription to gel separating differential display product. The 6 repeatable differential display bands were cloned and sequenced. 2.4 Repeating amplification and sequencing results of differential display ESTs

Fig. 2 shows the results of differential display ESTs. The positive clone plasmids identified by PCR were sequenced by Boya, Shanghai Co. Ltd. The length of the 6 EST fragments was between 111 and 372 bp, and the average length was 198 bp. The EST sequences were recorded in the GenBank, with the accession numbers from CB274875 to CB274880.

Fig. 2. Repeating amplification results of differential display bands. M, 2000 bp DNA marker; 1―6, repeating amplification products.

2.5

Sequence analysis of differential display ESTs

Through comparison of the homology, among the 6 cloned differential display ESTs, three ESTs (ESTsp1, ESTsp2, ESTsp6) could not be found to be homologous to the known sequences, but there was high homology between ESTsp 5 and IGF2 (insulin-likegrowth factor 2 preprotein genes) of pig; ESTsp4 and methionine aminopeptidase-like 1 gene of mouse; ESTsp3 and CAV1 (caveolin 1) gene of pig, human, mouse and rat. The homologous degree was 93%, 96.07%, 96.07% and 93.28%, respectively. 2.6

Results of ESTsp3 in silico elongation sequence

The sequence length after in silico elongation of ESTsp3 was 786 bp. According to ORF analysis, a

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complete open reading frame might exist in −2 reading frame, with the initiation codon ATG located in 269― 267 nt and the −3 region was A which according to Kozak rule[12]. The upstream 18 bp region had a stop codon (in the same reading frame) containing 83 aa. Fig. 3 shows the detailed sequence information. The black and italic letters indicated the primary ESTsp3 sequence, while the others were the in silico elongation sequences. Through homologous analysis, the predicted open reading frame was shown to be located in different regions compared with the known open reading frame of CAV1 in pigs and humans. However, this result needs to be further verified. 2.7

The verification of in silico elongation sequence

Two primers MP1 and MP2 were designed according to the sequences obtained from in silico elongation sequences. A very bright aim band was amplified by RT-PCR from the total RNA in loin-eye muscle of adult pig. The size of the band was equal to the predicted band. The aim band was cut and collected from the low melting point agarose gel electrophoresis and used for TA cloning, then a positive clone was randomly selected. The plasmids were extracted and identified by PCR method through enzyme digestion. The positive clone plasmids were sequenced by Boya, Shanghai Co., Ltd. Homology between the sequencing results and in silico elongation sequences was analyzed with BLAST2 software. The homology was up to 98%, which further demonstrated that the in silico elongation results were right. The sequencing result was recorded in GenBank, with the recorded number of CB333831. 2.8

Analysis of ESTsp3 gene expression profile

Analysis of in silico expression profile showed that the transformation quantities of CAV1(ESTsp3) in every 1000000 transformation products were as follows: 178 in bladder, 57 in blood, 454 in bone, 157 in bone marrow, 134 in brain, 0 in cervix, 59 in colon, 124 in eye, 768 in heart, 207 in kidney, 105 in larynx, 311 in liver, 483 in lung, 0 in lymph node, 132 in mammary gland, 116 in muscle, 40 in ovary, 176 in pancreas, 193 in peripheral, 293 in placenta, 130 in prostate, 307 in skin, 0 in small intestine, 1099 in soft tissue, 50 in spleen, 134 in stomach, 66 in tongue, 57

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Fig. 3. ESTsp3 in silico elongation and analysis results of open reading frame. Y, T or C; R, A or G; K, G or T; M, A or C; W, A or T. The italics are the sequences of ESTsp3, the others are elongation sequences.

in testis, 142 in thymus, 277 in uterus, 596 in vascular, 224 in embryo, 242 in juvenile, 176 in adult. It indicated that ESTsp3 was expressed in most tissues and organs in different growth stages, the expression distribution range was very extensive, the expression quantity was highest in soft tissues and higher in heart and vascular, whereas the expression quantity was lower in blood, colon, ovary, spleen, tongue, testis, thymus and was none in cervix, lymph node, small intestine. For growth stages: the highest expression quantity was in juvenile, whereas the lower in adult relatively. The above results showed that the expression distribution of EST was very extensive but the expression quantities were different in tissues and organs and in different growth stages. 3 3.1

Discussion Differential display techniques

The quality of total RNA was most important for successful differential display[13]. It was essential to eliminate the contamination of trace quantity DNA with non-RNase DNase-I processing the total RNA template and to avoid the contamination of RNase. Silver-stained differential display techniques were used to separate the aim bands in the research, 6 dif-

ferential display fragments were successfully obtained and could be used for repeating amplification. Differential display bands were all repeated twice to avoid false positive results. The bands without repeatability would not be used in the late research. 3.2 Screening differential display gene of muscle tissue in the two pig breeds by differential display technique It was very complicated for growth regulation of muscle tissue in pig, since it involved the interaction of many biochemical processes and environments. Identifying gene expression in the processes was very important for understanding growth regulation mechanism. It was important to analyze differential display genes in muscle tissues for different pig breeds, which would be valuable for understanding the molecular mechanism of variation in growth and meat quality traits. In this research, two pig breeds, purebred Lantang pig and Large Yorkshire pig, which were significantly different from the phenotypic performance, were used to analyze gene expression difference. The results showed that most gene expression of loin-eye muscle tissue in the adult pig of the two breeds was similar, since the two breeds had similar genetic basis. However, there were also some differential expression

Cloning & analysis of differentially expressed ESTs in swine muscle tissue

genes in their genome, because their breeding histories were completely different and they had long time geographical isolation. The high homology between ESTsp3 and CAV1 of pig and human was found (the homology was 93% and 96.07%, respectively) through analyzing the homologies among 6 differential expression ESTs. It could be deduced that ESTsp3 was one part of cDNA in pig CAV1 gene. Human CAV1 was a member of human caveolin family. Its molecular weight was 21―24 kD. As an integrity membrane protein, it is the main structural component of caveolin, and the content is abundant in endothelial cell, smooth muscle cell, skeletal muscle parent cell, fibrogenous cell and adipose cell[14]. The action of CAV1 related to muscle dystrophia was to restrain the activity of GTPase and concentrate special lipid[15]. And perhaps the implementation of ESTsp3 was similar to the process of CAV1. Now it has been found that many proteins have interaction with CAV1 or phosphotyrosine CAV1(pY14), indicating that CAV1 plays an important role in lipid communication, membrane communication and signal conduction[16]. This implies that the differential expression of ESTsp3 in Lantang pig and Large Yorkshire pig may be one of the genetic factors of phenotypic difference in the two breeds, but verified by late studies. 3.3 In silico cloning and analyzing of expression profile The length of the different fragments found in this research was all about 200 bp. These cDNA did not really represent the different expression genes. In recent years, RACE technique played an important role in cDNA cloning for the whole length of new genes. But RACE had much limitation since it would generate many unspecific products that would make it difficult to select the aim band[17]. With the rapid development of bioinformatics, the technique of cloning whole length cDNA by using computer was a rapid, simple and economical method, which reduced much repeating and exploring work[18]. The technique of cloning with computer was successfully used in this research to elongate the ESTsp3 in silico and obtain 786 bp sequence. The reliability of the cloning results with computer has been verified by RT-PCR. Studies on tissue expression profile of gene could

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provide important information for gene functional research[19]. Analysis of in silico expression profile based on the bioinformatical principle could assay the different gene expression from various tissues and in different growth stages, which had much important reference to research. Analysis of in silico expression profile indicated that ESTsp3 was expressed in many tissues and in different growth stages, but the expression quantity was obviously different, which was valuable for future study on its molecular genetics basis. Acknowledgements This work was supported by Key Project of Chinese National Programs for Fundamental Research and Development (973 Programe) (Grant No. 2004CB117506), Natural Science Foundation of Guangdong Province (Grant No. 010277), and Group Program of Natural Science Foundation of Guangdong Province (Grant No. 04205804)

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