Cloning, expression, and enzymatic activity analysis of strawberry S-adenosyl-L-metnionine synthetase gene FaSAMS1

The S-adenosyl methionine synthetase gene FaSAMS1 is involved in the regulation of strawberry fruit ripening, however, the biochemical properties of FaSAMS1 protein remain unclear. Here, a coding cDNA sequence of FaSAMS1 was cloned by RT-PCR and inserted into a recombinant yeast expression vector pPICZA, then transformed into the yeast expression strain X-33. The fusion protein FaSAMS1 was induced, expressed, and purified. The enzymatic activity analysis of FaSAMS1 showed that the reaction system contains 0.09 mg of FaSAMS1, protein concentration reached at 0.454 mg·mL -1 , the activity of the enzyme was 0.32 U, specific activity was 3.56 U·mg -1 . This study has provided biochemical evidence for the involvement of ethylene in the regulation of strawberry fruit ripening.


Introduction
The gaseous plant hormone ethylene plays an important role in various aspects of plant growth and development, as well as environmental stress (Fluhr and Mattoo, 1996;Wang et al., 2002). Especially, much progress has been made in understanding the molecular mechanisms of ethylene in the regulation of climacteric fruit ripening using the model plant tomato (Klee and Giovannoni, 2011). Notably, in recent years, ethylene is also involved in the regulation of non-climacteric fruit ripening using the model plant strawberry (Sun et al., 2013), demonstrating that downregulation of the ethylene biosynthesis-related gene FaSAMS1inhibits ripening. However, the biochemical nature of the FaSAMS1 protein remains yet unknown.
The ethylene biosynthesis in model plant Arabidopsis are well defined (Thomas and Surdin-Kerjan, l99l; Kende, 1993;Bleecker and Kende, 2000): (1) conversion of methionine to S-adenosyl-L-methionine (SAM) catalyzed by SAM synthetase (SAMS), (2) formation of 1aminocyclopropane-1-carboxylic acid (ACC) from SAM via ACC synthase (ACS) activity, and (3) conversion of ACC to ethylene through ACC oxidase (ACO). Owing to a critical role of ethylene in triggering tomato fruit ripening, to a large extent, this model has also been further developed in the fruit (Barry and Giovannoni, 2007), finding that ACC synthase LeACS2 and ACC oxidase LeACO1 control tomato fruit ethylene production and tomato ethylene receptor tCTR1 is induced by both I S S N 2 3 4 9 -0 8 3 7 V o l u m e 6 N u m b e r 1 J o u r n a l o f A d v a n c e s i n A g r i c u l t u r e 842 | P a g e c o u n c i l f o r I n n o v a t i v e R e s e a r c h A p r i l 2016 w w w . c i r w o r l d . c o m ripening and exogenous ethylene (Klee, 2002). Downregulation of FaSAMS1 or FaCTR1 transcripts not only inhibits strawberry fruit red-coloring, but also applied ethephon could promote natural strawberry fruit red-coloring and softening, as well as rescues partially anthocyanin biosynthesis in the RNAi fruit, demonstrating that ethylene is required for strawberry fruit ripening (Sun et al., 2013). Although SAM synthetase committed the first step in ethylene biosynthesis is proved to plays a role in strawberry fruit ripening (Sun et al., 2013), its biochemical properties of FaSAMS1 protein remain unclear.
In the present study, fusion protein FaSAMS1 was induced, expressed, and purified using prokaryotic expression system. Enzymatic activity analysis of FaSAMS1 showed that the specific activity of FaSAMS1 reached to 3.56 U·mg -1 , demonstrating that strawberry SAM synthetase FaSAMS1 has high Enzymatic activity. This study has provided biochemical evidence for the involvement of ethylene in the regulation of strawberry fruit ripening.

Cloning of the FaSAMS1 gene
The cDNA obtained above was used as a template for amplifying the FaSAMS1 gene with primers (forward, 5′-ATGGAGACTTTCCTATTCACATCTG-3′; reverse, 5′-TTAAGACTGAGGCTTCTCCCAC-3′). PCR was performed under the following conditions: 94℃for 5 min, followed by 32 cycles at 94℃ for 30 s, 54℃ for 30 s, and 72℃ for 1.5 min, with a final extension at 72℃ for an additional 5 min. The PCR products were ligated into a T1 simple vector and subsequently transformed into Trans1-T1. Positive colonies were selected, amplified, and sequenced by Invitrogen China (Shanghai, China).

Expression and purification of FaSAMS1 recombinant protein
The expression and purification of the recombinant protein FaSAMS1 was used EasySelect™ Pichia Expression Kit and ProBond™ Purification System (Invitrogen, USA), respectively. The coding sequence of FaSAMS1 was amplified by PCR from a cDNA synthesized above using primers [forward, GGAATTCAAAAATGGAGACT TTCCTATTCACATCTG (EcoRI site inderlined); and reverse, GGGGTACCAGACTGAG GC TTCTCCCAC (KpnI site inderlined)] and cloned into the expression vector pPICZ A in frame with the C-terminal tag fusion, which was transformed into E. coli to select transformants on Low Salt LB plates containing 25 μg/mL Zeocin™. Twenty transformants were selected to confirm the frame fusion with the C-terminal tag by sequencing. The purified and linearized recombinant plasmids were transformed into Pichia pastoris X-33 to select the Zeocin™-resistant yeast transformants by YPDS plates containing the appropriate concentration of Zeocin™. The FaSAMS1 fusion protein was expressed and purified by Ni-NTA His Bind Resins according to manufacturer's protocols. The eluted fusion protein was stored at -80℃ until use. The analysis of enzyme activity The SAM enzyme activity assay uses enzyme catalysis. Enzyme activity standard curve was made. S-adenosyl methionine (5.5 mg) was formulated as the concentration of mother liquid 1.1 mg / ml. Taking 800 µl of mother liquor diluted to 5 ml as standard 1, then we diluted 2 times of standard 1 as standard 2, standard 3, 4, and 5 were made by using twice gradient dilution method above. Take these five samples draw the S-adenosyl methionine standard curve. To determine the enzyme activity, enzyme catalyzed reaction of substrate ATP and L-methionine carried out in the buffer, the amount of enzyme reaction product of SAM was detected. Enzyme reaction system included ATP (final concentration of 1.5 mM) 2 μl, Met (final concentration of 150 μM) 5 μl, buffer solution 93 μl, 100 μl recombinant strain. To each tube, we added 500 ml of the substrate mixture, which contained 100 mM Tris (pH 9.0), 20 mM MgCl 2 , 150 mM KCl, and 5 mM of DTT. The tubes were then incubated at 37 ℃ for 10 min. After the end of enzyme reaction, reaction liquid was centrifuged in 10,000 rpm for 20 min. The supernatant (1 ml) was used for measuring the amount of product S-adenosylmethionine (S-adenosylmethionine in 254 nm has the maximal light absorption).

Cloning of FaSAMS1 gene from strawberry fruit
To clone the FaSAMS1 gene, an Arabidopsis SAMS1 protein was used to BLAST in a strawberry gene library (https://strawberry.plantandfood.co.nz/index.html), and a high homology protein with the gene was found in the 24,556 position of the gene locus. Based on the nucleotide sequence, specific primers (forward, 5′-ATGGAGACT TTCCTATTCACATCTG-3′; reverse, 5′-TTAAGACTGAGGCTTCTCCCA C-3′) were designed to amplify a coding sequence of the FaSAMS1 gene from strawberry fruit. The results showed that an 1,182 bp cDNA homologous to the Arabidopsis gene SAMS1 (named FaSAMS1) was isolated from strawberry fruit through RT-PCR. The cDNA included an open reading frame that encodes a deduced protein of 393 amino acids, in which the putative conserved domains were detected by homology analysis using BLAST for the FaSAMS1 protein on the NCBI website, http://blast.ncbi.nlm.nih.gov/Blast.cgi (Fig. 1), suggesting that the putative strawberry potassium channel gene FaSAMS1, was cloned successfully.

Construction and expression of FaSAMS1 fusion protein
The FaSAMS1 cDNA sequence (1,182 bp) was inserted into the yeast expression vector PpiczA by EcoR1 and Kpn1. The recombinant plasmid was extracted and identified by double enzyme digestion witn EcoR1 and Kpn1. The FaSAMS1 gene was successfully constructed and expressed in a yeast expression vector ( Fig. 2A, arrow). Expression vector containing FaSAMS1 gene was electroporated into yeast strain to express FaSAMS1 fusion protein. The results of electrophoresis for purified protein demonstrated that 55 kD fusion protein was collected by affinity chromatography with His tag (Fig 2B).

Enzymic activity analysis of FaSAMS1
In order to determine the activity of FaSAMS1, the chemical reaction and spectrophotometry were used in this study. L-methionine generated S-adenosylmethionone under the reaction of S-adenosylmethionine synthetase. S-adenosylmethionine in 254 nm has the maximal light absorption, depending on the amount of S-adenosylmethionine after enzymatic reactions to determine of SAMS activity by standard curve (Fig. 3). The results demonstrated that the reaction in 37℃ for 10 min generated S-adenosylmethionine with the concentration of 0.316 nmol·ml -1 , FaSAMS1 protein concentration was 0.454 mg·ml -1 , the measured enzyme activity was 0.32 U and the specific activity was 3.56 U· mg -1 . Fig. 3 The criterion curve of standard SAM.

Discussion
The expression of foreign proteins in yeast expression system has post translational modification, which makes the expressed protein with native activity. Thus, yeast expression provides a powerful tool for the identification of protein biochemical functions. In the present study, FaSAMS1 gene was integrated into yeast expression vector pPICZA and then the recombinant plasmid was transformed into X-33 yeast strain. The fusion protein was induced by methanol and the activity of this fusion protein was analyzed by enzymatic reaction and spectrophotometry. Our results demonstrated that the expressed protein had high enzymatic activity, reaching to specific activity at 3.56 U·mg -1 .
It is previously reported that downregulation of FaSAMS1 transcripts inhibits fruit red-coloring, and furthermore, exogenous ethephon could rescue anthocyanin biosynthesis in the RNAi fruit, demonstrating that FaSAMS1plays a role in the regulation of strawberry fruit ripening (Sun et al., 2013). Although SAM synthetase committed the first step in ethylene biosynthesis is proved to plays a role in strawberry fruit ripening (Sun et al., 2013), the biochemical properties of FaSAMS1 protein remain unclear. In the present study, it is confirmed that FaSAMS1had high enzymatic activity. This study has provided biochemical evidence for an important role of ethylene in the regulation of strawberry fruit ripening.