{'fraction_non_alphanumeric': 0.043622641509433964, 'fraction_numerical': 0.026867924528301886, 'mean_word_length': 5.098021168890934, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 2, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Cloning and Identification of an ACO Gene Involved in Ethylene Production Pathway from Ginkgo biloba

Ginkgo biloba is a living fossil from the Cenozoic Quaternary glacial period. Itis a precious tree species in China and has been extensively studied given its important medical and ornamental value. It has a long juvenile phase and spends 15 to 20 years in the flowering transition, and ethylene has played a very important role in its life cycle.As we all know, ethylene, one of the most important plant hormones, involves in the various aspects of plant growth and development, such as seed dormancy and germination, cell elongation, root growth and nodulation, shoot and leaf formation, flower and fruit development, sexual development, plant defense mechanism, and so on (Abeles et al., 1992;Wang et al., 2002; Chaves and Mello-Farias, 2006; Argueso et al., 2007; Lin et al., 2009). Although ethylene is plays an essential role for proper plant growth, development, and survival, it may also be deleterious to plants in some instances. Ethylene is biologically active in trace amounts, and the effects of this hormone are commercially important (Yang and Hoffman, 1984). Lieberman and Mapson (1964), Murr and Yang (1975), Adams and Yang (1979) made major contributions to our International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 5 (2017) pp. 110-119 Journal homepage: http://www.ijcmas.com

understanding of ethylene biosynthesis. A simplified overview of the pathway is presented in figure 1. The number of sequenced lower plant genomes has increased extensively in the past few years, providing opportunities to trace the evolutionary origins of biosynthetic and signaling pathways of plant hormones (Ross and Reid, 2010;Ju et al., 2015). The ethylene biosynthetic pathway, which includes three key enzymes reactions, has been well-documented in higher plants. First, the biosynthesis of ethylene begins with the conversion of the amino acid methionine to S-adenosyl-methionine (SAM) via enzyme SAM synthase (Yang and Hoffman, 1984;Zarembinski and Theologis, 1994). Second, the rate-limiting step catalyzed by 1aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) involves the cyclization of SAM to ACC, and 5'-methylthioadenosine (MTA) is produced by the ACS to utilize the synthesis of new methionine by the activated methyl cycle. Finally, ACC oxidase (ACO) catalyzes the oxygen-dependent conversion of ACC to ethylene (Yang and Hoffman, 1984;Liuet al., 1999;Choudhury et al., 2008).
Increased ethylene levels in plants when exposed to various types of stress including chilling, heat, nutrient deprivation, anaerobic life, wounding, and pathogen infection with increased damage to plant growth and health has been reported (Stearms and Glick, 2003). There is considerable commercial interest in modifying the amount of ethylene produced under ripening, senescent or stress conditions and thereby creating plants with more robust and/or desirable traits (Handa et al., 2010). The level of ethylene in plants is determined by the activity of two enzymes that catalyze the two final reaction steps in the biosynthetic pathway: ACS and ACO gene. Both ACS and ACO are encoded by multigene families that are differentially regulated by diverse developmental and environmental inputs (Barry et al., 1996;Chen and McManus, 2006), the majority of the regulatory mechanisms of ethylene biosynthesis at the level of ACC production by ACC. However, there are additional regulatory mechanisms. Under conditions of high ethylene production, the pathway can also be regulated at the level of conversion of ACC into ethylene by ACO (Vanderstraeten and Van Der Straeten, 2017). ACO enzyme belongs to the family of oxidoreductases which utilize Fe (II) as a cofactor and 2-oxoglutarases (2OG) as cosubstrate, although ACO uses ascorbate as a co-substrate (Prescott, 1993;Ryle and Hanusinger, 2002;Zhang et al., 2004;Mirica and Klinman, 2008). ACO needs bicarbonate as an activator and catalyzes the oxidation of ACC to give ethylene, CO 2 , and HCN. It has been revealed that ACO forms a complex with Fe (II) and its active site contains a single Fe (II) matched with three residues (Zhang et al., 2004;Jafari et al., 2013). In its sequence, two distinct domains can be distinguished: an N-terminal located highly conservative non-heme dioxygenase DIOXˍN region and a C-terminal located 2OG-FeⅡˍOxy region.
The practical implication of ethylene biosynthesis regulation for plant improvement has supported the continuous basic research on dissecting the structure of genes encoding ethylene biosynthetic enzymes, their differential expression patterns, and mechanisms underlying their transcriptional activity (Izabela et al., 2013). ACO has been cloned and characterized in many plant species such as peach, potato, strawberry, white clover, plum, Arabidopsis, rice, apple, pine, pear (Ruperti et al., 2001;Nie et al., 2002;Trainotti et al., 2005;Chen and McManus, 2006;Fernandez-Otero et al., 2006;Babula et al., 2006;Quyang et al., 2007;Binnie and McManus, 2009;Yuan et al., 2010;Qi et al., 2015). Here, we identified the GbACO gene originated from the G. bilobaleaves that promoted the growth of ginkgo, and analyzed the sequence structure and function.We constructed a phylogenetic tree for the comparison of GbACO with other members of the plant dioxygenase family through the neighbor-joining method. These results revealed that GbACO is a key enzyme gene in the ethylene biosynthesis pathway, and also laid a foundation for the analysis of gene regulatory network of ethylene synthesis in the G. biloba.

Plant materials
The 15-years-old G. biloba plants were grown in the Ginkgo Garden of Yangtze University (Jingzhou, China). Ginkgo leaves were immediately frozen in liquid nitrogen at the time of collection and then stored at -80℃for further use. Both primers synthesis and DNA sequencing were performed by Shanghai Sangon Biotechnology Company, China.

Cloning of ACO homologous from G. biloba
Total RNA was extracted from frozen ginkgo leaves using the TaKaRa MiniBEST Plant RNA Extraction kit (Dalian, China). First strand cDNA was synthesized using TaKaRa PrimeScript TM 1st Strand cDNA Synthesis Kit according to the manufacturer's instruction. A set of primers GbACO-up (5'-CTCATAACAATAATAGAGATTACTTTC TCA-3') and GbACO-dn (5'-TTAAGATTTGAACTAAAGTAAGATTGT -3') were designed based on the previous ginkgo transcriptome data (CL1905Contig1). Each 25 µl reaction volume contained 0.5 µg cDNA as the template; 1.5 mM Macl 2 ; 10×Taq buffer; 0.4 µM of each primer; 0.25 mM dNTPs; and 0.25U Taq polymerase. The reactions were initiated at 94℃ (4 min), followed by 35 cycles of denaturing at 94℃ (1 min), annealing at 53.5℃(50 s), and elongation at 72℃ (1 min), and ended with incubation at 72℃ for 10 min. The PCR products were separated on a 1% (w/v) agarose gel, then the target DNA (with the expected size) was excised from the gel and purified using TaKaRa Agarose Gel DNA purification Kit Ver.4.0. The purified products were ligated into the pMD19-T plasmid vector (TaKaRa), and then cloned into the Escherichia coli DH5α followed by sequencing.

Bioinformatics and molecular evolution analyses
The obtained nucleotide sequence from sequencing was analyzed and translated using Vector NTI 11.5 program, and the predictions for pI and molecular mass were using the pI/MW tool at Expasy (http://web.expasy.org/compute_pi/). Secondary structure was predicted by SOPMA (https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sop ma.html).The deduced protein sequence was searched for homologous proteins using the BLAST-Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences with the highest similarities were downloaded and multiple alignments were performed using Vector NTI 11.5 program, and the phylogenetic tree was constructed using Clustal X2 and MEGA 6.0 software.

Cloning and characterization of GbACO
In order to characterize the full-length GbACOc DNA, total RNA was isolated from ginkgo leaves. cDNA was reverse transcribed from RNA, and then, the corresponding GbACOcDNA amplification was performed using the primers (GbACO-up and GbACOdn), designed based on previous transcriptome data (CL1905Contig1). A single PCR fragment of the expected size was obtained, purified, ligated into the pMD19-T vector, and then cloned into the E. coli DH5α, finally sequenced. Sequencing results showed that the full-length cDNA of GbACO was 1288 bp and contained a 990bp ORF, which encoding a 329-amino-acid protein (Figure 2). The molecular weight and isoelectric point of the deduced polypeptide were 37.2 kDa and 5.20, respectively.

Characterization of the deduced GbACO protein
The comparison of the amino acid sequence of GbACO with other plant ACO proteins showed high sequence similarity. The GbACO protein showed 64%, 62%, 61%, 60%, 60%, 59%, 60%, 63%, and 61% similarity to the counterparts of Picea sitchensis Pinus pinaster (CBL95267.1), and Ricinus communis (XP_002520313.1), respectively. Multiple sequence alignments showed that the peptide is a member of ACO proteins, with DIOXˍN (non-haem dioxygenase in morphine synthesis N-terminal), and 2OG-FeⅡˍOxy (2oxoglutarate (2OG) and Fe (II)-dependent oxygenase) domains ( Figure 3). As we all know, ACO is a member of Fe (II)-dependent family of oxidases, which requires ascorbate as a co-substrate and Fe (II) as a cofactor for its enzymatic activity (McGarvey and Christoffersen, 1992). All motifs for binding of the cofactor (His-Xaa-Asp-Xaa-His) and the co-substrate (Arg-Xaa-Ser) that are highly conserved among all members of Fe (II) ascorbate family of dioxygenases were also well conserved in GbACO and other plant ACO proteins (Figure. 3). The Fe2OG dioxygenases catalyze a variety of twoelectron oxidations, including hydroxylations, desaturations, and oxidative ring closures (Costas et al., 2004). Although the sequence homology among these enzymes is not high, all their active sites contain a single ferrous ion bound in a tridentate ligand arrangement referred to as a "2-His-1-carboxylate facial triad" (Mirica and Klinman, 2008). Secondary structure analysis of GbACO protein by SOMPA revealed that GbACO consists of αhelix (40.43%), β-turn (10.33%), extended strand (13.07%), and random coil (36.17%). Figure. 1 Ethylene biosynthesis pathway. The enzymes catalyzing each step are shown above the arrows. The methionine is converted to S-adenosyl-methionine (SAM) via enzyme SAM synthase (SAMS). SAM is subsequently converted to ACC by ACC synthase (ACS). MTA finally, ACC is converted to ethylene by ACC oxidase (ACO). (Yang and Hoffman, 1984;Vanderstraeten and Van Der Straeten, 2017) Methionine

ACC Ethylene
Yang cycle MTA Figure.2 cDNA sequenceof GbACO and the translated amino acid sequence. The red box is the start codon, and the asterisk represents the termination codon Figure.3 Alignment of the deduced GbACO amino acid sequence with other ACO proteins., The species names are shown as following:    Moraceae, Solanaceae, Cruciferae, Palmaceae and Monocotyledonae). Taken together, phylogenetic analysis suggests that GbACO has a common evolutionary original with other plant ACO proteins based on the conserved sequence and sequence characteristics.

Conclusion and perspectives
ACO catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene that is involved in the regulation of array of biological processes in plants, including seed dormancy and germination, root growth, shoot and leaf formation, flower and fruit development, different organs senescence, plant defense mechanisms, and a number of interactions with other plant hormones. Here, an ACO homologue, GbACO was successfully isolated and characterized for the first time from ginkgo. Multiple sequence alignments indicated that GbACO had a high similarity to other plant ACO proteins. Phylogenetic tree analysis showed that GbACO keeps a strong conservation during the molecular evolution. All of these results not only contribute to understand the ethylene biosynthesis in Gymnosperms but also promote researches of the role of ethylene in the regulation of ginkgo growth and development.
Nowadays, the number of sequenced plant genomes has increased extensively in the past few years, providing opportunities to probe the evolutionary origins of biosynthetic and signaling pathways of plant hormones. There is a commercial interest in increasing the final products through increasing the expression level of key genes in the biosynthesis pathway. Even though ACO genes have been isolated from a lot of species, the biochemical properties, in vivo stability, and/or physiological functions of the gene products are in many cases not fully understood. Thus, further study is needed to verify the role played by different ACO genes in the regulation of ethylene production. A better understanding of both structure and function of genes involved in the ethylene biosynthesis, and also the mechanisms responsible for their activity during different developmental processes of plants. All this has a profound practical importance as it develops the tools for ginkgo improvement.