Microsoft Word - 5-Agra_26138 - 1024 Original Article Biosci. J., Uberlândia, v. 31, n. 4, p. 1024-1029 , July/Aug. 2015 MOLECULAR CHARACTERIZATION AND EXPRESSION PATTERN OF A NOVEL CADMIUM RESISTANCE GENE OF TOBACCO CARACTERIZAÇÃO E PADRÃO DE EXPRESSÃO MOLECULAR DE UM NOVO GENE DE FUMO PARA RESISTÊNCIA AO CÁDMIO Nian FUZHAO 1 ; Zhao LEIFENG 1 1. College of Tobacco Science, Yunnan Agricultural University, Kunming 650201, China. zhleifeng@163.com ABSTRACT: Cadmium (Cd) of tobacco is a pollutant that is extremely toxic to the health of humans. Protein plant cadmium resistance 8 gene has been characterized to increase the plant Cd resistance. In present experiment, the complete mRNA sequence of tobacco protein plant cadmium resistance 8 gene was amplified using the rapid amplification of cDNA ends methods. The full-length tobacco protein plant cadmium resistance 8 gene mRNA was 887bp containing an 555 bp open reading frame, which encodes a protein of 184 amino acids. BLAST analysis revealed that tobacco protein plant cadmium resistance 8 protein shares high homology with the protein plant cadmium resistance 8 of potato (81%), Lycopersicon esculentum (80%), Eutrema salsugineum (60%), Capsella rubella (58%) and thale cress (57%). Results also showed that tobacco protein plant cadmium resistance 8 gene has a closer genetic relationship with the protein plant cadmium resistance 8 gene of Lycopersicon esculentum. The expression profile was studied and the results indicated that tobacco protein plant cadmium resistance 8 gene was highly expressed in root, moderately expressed in stem, and hardly expressed in flower and leaf. These results established the primary foundation of utilizing tobacco protein plant cadmium resistance 8 gene to decrease the cadmium content of tobacco and benefit the health of humans in the future. KEYWORDS: Tobacco. Gene. Protein plant cadmium resistance 8. Expression pattern. INTRODUCTION Cadmium (Cd) of tobacco is a pollutant that is extremely toxic to the health of humans. Cd has caused neurotoxicologic and behavioral changes in both humans and experimental animal studies (Liu et al., 2013; Counter et al., 2009). Cd exposure may be implicated in some neurological disorders including hyperactivity and increased aggressiveness in humans (Liu et al., 2013; Matés et al., 2010). In the case of coronary risk with metal levels, Cd may be more important for females (Liu et al., 2013; Olsen et al., 2012). Cd was reported to damage bone microstructures (Liu et al., 2013; Chen et al., 2011) and can negatively influence growth in newborns. Several studies have reported an inverse relationship between anthropometric measurements of the newborn and the placental or umbilical cord Cd levels (Liu et al., 2013; Llanos et al., 2009; Ronco et al., 2009). Cd exposure exerts inhibitory effects on testicular steroidogenesis(Liu et al., 2013; Pillai et al., 2012). Recent researches have proved that a lot of genes, including protein plant cadmium resistance 8, can increase the plant Cd resistance (Thomine et al., 2000; Song et al., 2004; Ishikawa et al., 2012). Protein plant cadmium resistance 8 gene has been identified from many plants such as thale cress, tomato and potato. Until today, the tobacco protein plant cadmium resistance 8 gene has not been reported yet. In the present work, we isolated the full- length mRNA sequence of this tobacco gene, subsequently performed some necessary sequence analysis and tissue expression analysis for this gene. These will establish the primary foundation of utilizing tobacco protein plant cadmium resistance 8 gene to decrease the cadmium content of tobacco and benefit the health of humans in the future. MATERIAL AND METHODS Samples collection, RNA extraction and first- strand cDNA synthesis Tobacco plants (Chinese local variety Yunyan 87) were grown in a naturally lit glasshouse with normal irrigation and fertilization. The tissues including leave, stem, root and flower were harvested, immediately frozen in liquid nitrogen and stored at -80℃. Total RNA extraction and first- strand cDNA synthesis for these tissue samples were performed as the methods describe by Liu (2009). 5’and 3’-RACE 5’- and 3’-RACE were performed as the instructions of SMARTTM RACE cDNA Amplification Kit. For the tobacco protein plant cadmium resistance 8 gene, the gene specific primers (GSPs) were designed based on one tobacco EST sequence: EH623456. 5’-RACE GSP: 5’- GCCCATTATCCATTGAGAGCAAACA-3’ 3’- Received: 05/04/14 Accepted: 15/10/14 1025 Molecular characterization and expression.... FUZHAO, N.; LEIFENG, Z. Biosci. J., Uberlândia, v. 31, n. 4, p. 1024-1029 , July/Aug. 2015 RACE GSP: 5’- GCCTGCTGTTTGCTCTCAATGGATA-3’. RACE touchdown PCRs were carried out with 5 cycles of 94°C 30 sec and 72 °C 3 min, followed by 5 cycles of 94°C 30 sec, 70°C 30 sec and 72 °C 3 min, finally with 25 cycles of 94°C 30 sec, 66°C 30 sec , 72°C 3 min to terminate reaction. These RACE PCR products were then cloned into PMD18-T vector (TaKaRa, China) and sequenced bidirectionally with the commercial fluorometric method. Quantitative real time PCR (qRT-PCR) for tissue expression profile analysis qRT-PCR for evaluating the level of mRNA for protein plant cadmium resistance 8gene was performed by the ABI Prism 7300 Sequence Detection Systems (Applied Biosystems, Foster City, CA, USA). 25µ l reaction volume of PCR reaction contained 1µl SYBR Green real-time PCR Master Mix, 100 ng cDNA template and 200 nM each primer. Conditions for real-time PCR were: an initial denaturation at 95 °C for 3 min, 40 cycles of 95 °C for 15 s, optimal annealing temperature for each specific primer for 15 s (Table 1), 72°C for 20 s. The gene relative expression levels were quantified relative to the expression of the reference gene, actin (GenBank Accession No. GQ339768), by employing the 2 -∆∆Ct value model (Livak et al., 2001). Table 1. qRT-PCR primers for tobacco protein plant cadmium resistance 8 and actin genes Gene Primer sequence Ta/ °C Length/(bp) protein plant cadmium resistance 8 Forward : 5’- TGGATACAATGGGAAGAG -3’ Reverse: 5’- ATGGACAGAATATGTGGG -3’ 52 420 Actin Forward :5’- CCATTCTTCGTTTGGACCTT -3’ Reverse: 5’- TTCTGGGCAACGGAACCT- 3’ 56 257 Sequence analysis Gene prediction of cDNA sequence was performed by GenScan software (http://genes.mit.edu/GENSCAN.html). Theoretical isoelectric point (pI) and molecular weight (Mw) of the deduced protein was computed using the Compute pI/Mw Tool (http://www.expasy.org/tools/pi_tool.html). Protein analysis were carried out using the 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 RACE results for tobacco protein plant cadmium resistance 8 gene For tobacco protein plant cadmium resistance 8 gene, through 5’-RACE, one PCR product of 380-bp was obtained. The 3’-RACE product was 538-bp in length. These products were then cloned to T-vector and sequenced. Taken together, a 887-bp cDNA sequence was finally obtained (Figure 1). Figure1. RACE results for tobacco protein plant cadmium resistance 8 gene. M DL2000 DNA markers; 1, 3’- RACE product for tobacco protein plant cadmium resistance 8 gene; 2 , 5’-RACE product for tobacco protein plant cadmium resistance 8 gene 1026 Molecular characterization and expression.... FUZHAO, N.; LEIFENG, Z. Biosci. J., Uberlândia, v. 31, n. 4, p. 1024-1029 , July/Aug. 2015 Sequence analysis BLAST analysis of this cDNA sequence revealed that this sequence was not homologous to any of the known tobacco gene and it was then deposited into the Genbank database (Accession number: KJ159912). Sequence prediction was carried out and results showed that this 887-bp cDNA sequence represents one single gene which encodes 184 amino acids(Figure 2). The theoretical isoelectric point (pI) and molecular weight (Mw) of this deduced protein was also computed. The pI of tobacco protein plant cadmium resistance 8 is 5.29. The molecular weight is 20371.27. AATTTTCTTCATCCACTTGAGGTGTATACGTAGGTGTAAAATTTTTGGATACAATGGGAAGAGTTG A A G A A A A T A A T G A A A T A G A A A C T C C T M G R V E E N N E I E T P AAACCAGGTGAGAGTGGTGAACCAGTTGCCTCACAGCCTCCTCCACAGTACCAAGGAGTAAAGG A T G T A C A G C C G C C A T C G C C A T C A C A A K P G E S G E P V A S Q P P P Q Y Q G V K D V Q P P S P S Q CCAATTGGAGCTCCCTGGAGCACTGGCTTATTTGATTGTCATTTGAACCAGATTAATGCTGTTATG A C A T C A T T C T T A C C T T G T G T A A C A P I G A P W S T G L F D C H L N Q I N A V M T S F L P C V T TTCGGACAGATAGCAGAAGTCCTCGATGCAGGAGAAATGACATGTCCTTTGGGGACTTTCATATA C T T G C T G A T G A T G C C T G C T G T T T G C F G Q I A E V L D A G E M T C P L G T F I Y L L M M P A V C TCTCAATGGATAATGGGCTCTAAGTATAGAACTAAGCTGAGACAGAAATATAATCTTGTCGAAGC T C C T T A T T C A G A C A T A G T T T C C C A C S Q W I M G S K Y R T K L R Q K Y N L V E A P Y S D I V S H ATATTCTGTCCATGTTGCTCTCTTTGTCAAGAGTTCAGAGAACTTCAACACAGGGGACTTGATCCT G C T C T A G G A T G G A A T G G T A T A G T T I F C P C C S L C Q E F R E L Q H R G L D P A L G W N G I V GCTCAGCAGCATTATGGGAACCAACAAGTGAATCAAGCTCCCCAAGTACAATCCATGTCTAAGTA A A A T T C A A G A T T C T G A T T C T T T A T T T G A Q Q H Y G N Q Q V N Q A P Q V Q S M S K * TTTGAGTTAAGGTTGTAATATGTTACATGTGTTTTGACACACCCAGATTCTGTTCATGTTGTAGCCT TTATTATTTATTCCAACAATCTTGTTGGCAGTGTTGATTGTTGTACTATGTTTCTAGAACTCTATTTT TGGGAGTTACATACTTCCGCGTATATGAAAATTGAGTTTGCAATGATCTAATGAAGTCTTGGCATC AATTATTAATTGGGCAGTTCGATAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Figure 2. The complete mRNA of tobacco protein plant cadmium resistance 8 gene and its encoding amino acids. *indicates the stop codon. Further BLAST analysis of this protein revealed that tobacco protein plant cadmium resistance 8 shares high homology with the protein plant cadmium resistance 8 of potato(Accession number: XP_006356149, 81%), Lycopersicon esculentum (Accession number: XP_004241740, 80%), Eutrema salsugineum (Accession number: XP_006392946, 60%), Capsella rubella (Accession number: XP_006305669, 58%) and thale cress (Accession number: AAM62872, 57%) (Figure 3). Based on the results of the alignment of different protein plant cadmium resistance 8 proteins, a phylogenetic tree was constructed using the ClustalW software, as shown in Figure 4. Phylogenetic analysis revealed that the tobacco protein plant cadmium resistance 8 gene has a closer genetic relationship with that of Lycopersicon esculentum Tissue expression profile Tissue expression profile analysis was carried out and results revealed that the tobacco protein plant cadmium resistance 8 gene was highly expressed in root, moderately expressed in stem, and hardly expressed in flower and leaf (Figure 5). 1027 Molecular characterization and expression.... FUZHAO, N.; LEIFENG, Z. Biosci. J., Uberlândia, v. 31, n. 4, p. 1024-1029 , July/Aug. 2015 Figure 3. The alignment of the proteins encoded by tobacco and other protein plant cadmium resistance 8 genes Figure4. The phylogenetic tree for six kinds of protein plant cadmium resistance 8 genes Figure 5. Expression analysis of protein plant cadmium resistance 8 gene in various tobacco tissues 1028 Molecular characterization and expression.... FUZHAO, N.; LEIFENG, Z. Biosci. J., Uberlândia, v. 31, n. 4, p. 1024-1029 , July/Aug. 2015 DISCUSSION Comparative genomics research has revealed that virtually all (99%) of the protein- coding genes in humans align with homologs in mouse, and over 80% are clear 1:1 orthologs for human and mouse both belong to mammalian (Hardison, 2003; Liu,2009). This extensive conservation in protein-coding regions implied that this conservation of protein-coding sequences may be expected in tobacco and other plants of solanaceae. From the sequence analysis of protein plant cadmium resistance 8 genes, it can be seen that the coding sequences of protein plant cadmium resistance 8 genes were highly conserved in three solanaceae plants-tobacco, potato and Lycopersicon esculentum. The phylogenetic tree analysis revealed that the tobacco protein plant cadmium resistance 8 gene has a closer genetic relationship with that of Lycopersicon esculentum. This implied that we can use Lycopersicon esculentum as model organism to study the tobacco protein plant cadmium resistance 8 gene or use tobacco as model organism to study the Lycopersicon esculentum protein plant cadmium resistance 8 gene. From the tissue distribution analysis in our experiment it can be seen that protein plant cadmium resistance 8 gene was highly expressed in root. For protein plant cadmium resistance 8 can increase the plant Cd resistance (Thomine et al., 2000; Song et al., 2004; Ishikawa et al., 2012), the suitable explanation for this is that tobacco cadmium resistance is mainly existed in root. In conclusion, we first isolated the tobacco protein plant cadmium resistance 8 gene and performed necessary sequence analysis and tissue expression profile analysis. These will establish the primary foundation of utilizing tobacco protein plant cadmium resistance 8 gene to decrease the cadmium content of tobacco and benefit the health of humans in the future. ACKNOWLEDGMENTS This work was supported by grants from the Science and Technology Development Plan of Yunnan Provincial Tobacco Monopoly Administration (Corporation) (2013YN05). RESUMO: Cádmio (Cd) oriundo do fumo é um poluente que é extremamente tóxico a saúde humana. Um gene encontrado na planta de fumo que codifica uma proteína para resistência (gene 8) tem sido caracterizado para aumentar a resistência do fumo na absorção do elemento Cádmio (Cd).No experimento realizado a seqüência do mRNA de fumo que codifica uma proteína foi amplificada usando métodos de amplificação de CDNAs. A proteína completa codificada pelo gene 8 apresenta um mRNA com 887 pb com uma fita de leitura de 555 pb, a qual codifica 184 aminoácidos. A análise de BLAST demonstrou uma homologia de 81 % com o gene 8 da batateira, de 80 % com o tomateiro (Lycopersicon esculentum), 60 % para Eutrema salsugineum , 58 % para Capsella rubella e 57 % para o agrião. 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