To investigate the percentage homology of NpNBS, a BLAST search was performed with predicted amino acid NCS sequence form various plant species, which shares a significant homology with NpNBS (Figure 2). Notably, NpNBS shares 30-35% similarity with TfNCS, which has been reported to shares 28-38% with PR-10 member protein , e.g. rice (Oryza sativa), mung bean (Vigna radiata) and pollen allergens such as birch (Betula pendula) [14, 21, 22]. NpNBS displays comparatively low homology (22-25%) with Stylophorum diphyllum (SDINCS1), Argenome mexicana (AMENCS1) and Eschscholzia californica (ECANCS1). The length of NpNBS was consistent with other NCS members of this group.
A motif scan was performed using myhits (http://myhits.isb-sib.ch/cgi-bin/motif_scan) revealed a presence of Bet v1 domain in CjPR10, Bet v1_PR10, TfNCS and NpNBS. This result further supports a relation between NpNBS, NCS and PR-10. Interestingly, these results also suggest absence of signal peptides in CjPR10, Bet v1_PR10 and NpNBS except TfNCS (Figure 3). TfNCS was previously reported with a signal peptide and has no role in role in enzyme activity [14]. Moreover, truncation of first 19 amino acids of TfNCS has shown a better activity compared to non-truncated protein [23].
Therefore, to investigate the evolutionary history of NpNBS and its relationship with PR-10 gene, a phylogeny tree was constructed with predicted amino acid sequence of NpNBS suggesting that NCS and PR-10 evolved as individual gene over the period of time. The phylogenetic tree was divided into two main clusters: NCS and PR-10. NpNBS sequence was placed in the NCS group and shows close homology with TFLNCS5 (Figure 4).
RT-PCR analysis
The study of NpNBS expression profile in different tissues of N. pseudonarcissus ‘King Alfred’ by quantitative Real-time PCR revealed high expression of NpNBS in bulbs compared to roots, stems, leaves and flowers (Figure 5). The expression of NpNBS was approximately 1500 folds higher in bulbs than in other tissues. This data is also supported by its high FPKM value (31543) reported in the transcriptome. It was reported among the top expressed genes, which altogether comprises 16.12% of the N. pseudonarcissus ‘King Alfred’ transcriptome. Therefore, based on the expression data we conclude that NpNBS has a crucial role in AAs metabolism.
NpNBS gene cloning and heterologous expression
PCR amplified product of NpNBS shows an attB flanked sequence of 566 bp including the length of gateway primers (74 bp-without gene specific region) on gel electrophoresis image (Figure 6A). This amplified PCR product was used to perform a gateway cloning. The gateway-adapted attP-flanked pDONR 221 vector with kanamycin resistance gene was used for BP recombinase reaction to generate an attL-flanked entry clones with attB-flanked NpNBS DNA fragment. This reaction was catalyzed by BP clonase. These entry clones were transformed into E. coli DH10” competent cells and positive clones were obtained on kanamycin selection plate. These clones were used to perform a LR recombination reaction between an attL-containing entry clone and attR-containing pET301/CT-DEST destination vector with a histidine tags and T7 promoter and terminator. Positively transformed E. coli DH10” competent cells were selected from ampicillin-Luria-Bertani (LB) media plates. A gel electrophoresis image of five positive clones of LR reaction shows band of 566 bp (Figure 6B). The resulting clones were cell cultured overnight at 37”C and 25”C in 10 ml LB broth containing ampicillin for expression vector isolation, which were transformed into E. coli Rosetta (DE3)pLys strain via heat shock method for and grown on LB plates with ampicillin.
To produce recombinant NpNBS with 6x-His tags, a positive colony from above plate was inoculated in LB broth with ampicillin and chloramphenicol and incubated overnight for starting a pre-culture. Eventually, culture media with ampicillin and chloramphenicol was inoculated with a pre-culture in a ratio of 1:100. Culture was mixed vigorously to measure an initial optical density (O.D) and incubated until it reaches a final O.D. Once the right O.D is attained, the transformed E. coli Rosetta (DE3)pLys cell cultures were induced with isopropyl-”-D-thiogalactopyranoside (IPTG). Supernatant and pellet of IPTG induced bacterial culture were separated by centrifugation. E. coli Rosetta (DE3)pLys crude cell pellet were purified to elute NpNBS protein. Non-induce protein were also purified by adding lysis buffer and sonication, followed by centrifugation to separate supernatant and pellet. Later, the pellet was resuspended in elution buffer (without IPTG) for SDS-PAGE and western blot analysis.
In order to reveal NpNBS expression and its molecular weight from bacterial cell cultures incubated at 37”C and 25”C, SDS-PAGE analysis was performed with different fractions obtained during protein purification process which shows few unspecific bands of different sizes, suggesting a presence of bacterial cell proteins in crude (Cr), lysate (L1 and L2) and wash buffer (W1 and W2). An apparent molecular mass of 19 kDa in elution buffer 1 (E1) was observed, which was absent in non-induce protein (NI) in protein samples incubated at 37”C (Figure 7A). Interestingly, no such protein band was visible in SDS-PAGE analysis of 25”C incubation temperature (Figure 7B). Western blot was performed using 6x HIS-tag monoclonal antibody shows a 6His-tag NpNBS protein expression band of 19 kDa in crude (Cr) and purified extracts (E1) only in purified protein isolated from transformed bacterial cell culture grown at 37”C. No protein expression was observed in cultures without IPTG (Figure 8). The above analysis suggests that optimum temperature for NpNBS expression is 37”C and expressed only in IPTG induced cell culture.