Identification and expression profiling of a novel Kunitz trypsin inhibitor (KTI) gene from turmeric, Curcuma longa, by real-time quantitative PCR (RT-qPCR)

Acta Physiologiae Plantarum, Dec 2016

Kunitz trypsin inhibitor (KTI) is one of the widely studied protease inhibitors (PIs) and has been reported to take part in plant defense mechanism during pathogenesis. Numerous plant-origin recombinant KTIs have been described to exhibit anti-pathogenic properties and were used to fight pathogens in the field of pharmacology and agriculture. In this study, a novel Kunitz trypsin inhibitor gene, ClKTI, was isolated from a medicinal herb plant, turmeric, Curcuma longa. The full-length ClKTI gene is 754 bp long (Accession No. KF889322.1 in NCBI database) and it was obtained using 5′/3′ rapid amplification of cDNA ends (RACE) technique. ClKTI has an open reading frame of 639 bp length which encodes for 213 amino acids and contains the Kunitz-family motif, (V-X-D-X2-G-X2-L-X5-Y-X-I) and an altered reactive site motif, (G/E-I-S). Sequence similarity search using BLASTX showed that ClKTI shared the highest similarity to KTI from Theobroma cacao with 58% max identity while conserved domains search resulted in ClKTI having specific hits with Kunitz-family soybean trypsin inhibitor (STI). Phylogenetic studies suggested that ClKTI is related to T. cacao while protein homology modeling analysis indicated that it has 12 β-sheets with three disulfide bridges. Using real-time quantitative PCR, the ClKTI gene expression pattern in five different tissues (flower, basal stem, stem, rhizome and root) treated with methyl-jasmonate (MeJA) was studied where MeJA was suggested to regulate expression of PI genes in plants. The results indicated that the expression of ClKTI generally increased in the MeJA-treated tissues with the root tissues possessing the highest expression and stem tissues showed the highest expression fold-change.

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Identification and expression profiling of a novel Kunitz trypsin inhibitor (KTI) gene from turmeric, Curcuma longa, by real-time quantitative PCR (RT-qPCR)

Acta Physiol Plant Identification and expression profiling of a novel Kunitz trypsin inhibitor (KTI) gene from turmeric, Curcuma longa, by real-time quantitative PCR (RT-qPCR) Seow-Neng Chan 0 1 2 3 Norliza Abu Bakar 0 1 2 3 Maziah Mahmood 0 1 2 3 Chai-Ling Ho 0 1 2 3 Noraini Mohamad Dzaki 0 1 2 3 Noor Azmi Shaharuddin 0 1 2 3 0 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences , Universiti Putra Malaysia (UPM), 43400 Serdang , Malaysia 1 Biotechnology and Nanotechnology Research Center, Malaysian Agricultural Research and Development Institute (MARDI) , 43400 Serdang , Malaysia 2 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences , Universiti Putra Malaysia (UPM), 43400 Serdang , Malaysia 3 Laboratory of Plantation Crops, Institute of Tropical Agriculture , Universiti Putra Malaysia (UPM), 43400 Serdang , Malaysia Kunitz trypsin inhibitor (KTI) is one of the widely studied protease inhibitors (PIs) and has been reported to take part in plant defense mechanism during pathogenesis. Numerous plant-origin recombinant KTIs have been described to exhibit anti-pathogenic properties and were used to fight pathogens in the field of pharmacology and agriculture. In this study, a novel Kunitz trypsin inhibitor gene, ClKTI, was isolated from a medicinal herb plant, turmeric, Curcuma longa. The full-length ClKTI gene is 754 bp long (Accession No. KF889322.1 in NCBI database) and it was obtained using 50/30 rapid amplification of cDNA ends (RACE) technique. ClKTI has an open reading frame of 639 bp length which encodes for 213 amino acids and contains the Kunitz-family motif, (V-X-DX2-G-X2-L-X5-Y-X-I) and an altered reactive site motif, (G/E-I-S). Sequence similarity search using BLASTX showed that ClKTI shared the highest similarity to KTI from Theobroma cacao with 58% max identity while conserved domains search resulted in ClKTI having specific hits with Kunitz-family soybean trypsin inhibitor (STI). Phylogenetic studies suggested that ClKTI is related to T. cacao while protein homology modeling analysis indicated that it has 12 b-sheets with three disulfide bridges. Using real-time quantitative PCR, the ClKTI gene expression pattern in five different tissues (flower, basal stem, stem, rhizome and root) treated with methyl-jasmonate (MeJA) was studied where MeJA was suggested to regulate expression of PI genes in plants. The results indicated that the expression of ClKTI generally increased in the MeJA-treated tissues with the root tissues possessing the highest expression and stem tissues showed the highest expression fold-change. Protease inhibitors; Real-time qPCR; Turmeric; Kunitz trypsin inhibitor; Anti-pathogenic - Protease inhibitors (PI) are proteins that are capable to inhibit proteases by regulating proteolytic activities inside cells. They form stable complex with target proteases resulting in either blockage or alteration of active structures of the proteases (Habib and Fazili 2007; Oddepally et al. 2013). PIs are commonly found in a diverse group of microorganisms, animals and also plants (Lawrence and Koundal 2005; Haq et al. 2004; Habib and Fazili 2007). In fact, a large amount of plant-origin PIs has been identified and characterized (Bijina et al. 2011; de Oliveira et al. 2012) and most of them were isolated from storage organs such as seeds and tubers. In plant system, PIs involve in numerous physiological roles ranging from plant development to stress-related responses. One of the interesting roles of PIs is their involvement in plant defense mechanism. Their concentration were significantly increased at the sites of pathogen attack, followed by subsequent accumulation at all other parts of the plant (Lawrence and Koundal 2005; Haq et al. 2004; Fan and Wu 2005; Habib and Fazili 2007). In another case, PIs that were fed to larvae of flour beetle were shown to be toxic and retarded the larvae growth (Habib and Fazili 2007; Koiwa et al. 1997). Therefore, it was concluded that proteases and other proteolytic enzymes secreted by the pathogens during pathogenesis had been deactivated by the PIs. The PI characteristic of being able to deactivate pathogenic proteases has since attracted many researches to develop recombinant PIs (Lawrence and Koundal 2005). PI genes could be incorporated into crop plants and expressed as a defensive mechanism tool against insects and pathogens. Recombinant PIs could also be synthesized as medicines to treat viral and bacterial infections (Haq et al. 2004; Oddepally et al. 2013). PIs are classified based on the DNA sequence homology of their inhibitory domains. Common PI families that gain the interest of researchers include Bowman–Birk, serine protease inhibitors (Serpin), cysteine protease inhibitors (cystatins) and Kunitz type. These PIs families have been reported to display anti-pathogenic, anti-fungal, anti-viral and anti-feedant properties (Habib and Fazili 2007; Haq et al. 2004). PIs in Kuni (...truncated)


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Seow-Neng Chan, Norliza Abu Bakar, Maziah Mahmood, Chai-Ling Ho, Noraini Mohamad Dzaki, Noor Azmi Shaharuddin. Identification and expression profiling of a novel Kunitz trypsin inhibitor (KTI) gene from turmeric, Curcuma longa, by real-time quantitative PCR (RT-qPCR), Acta Physiologiae Plantarum, 2017, pp. 12, Volume 39, Issue 1, DOI: 10.1007/s11738-016-2311-7