Isolation and partial characterization of the lipoxygenase gene in black pepper ( Piper nigrum L)

Black pepper is a spice that have an economic importance in the world by its wide application in industry and by medicinal properties. State of Pará is one of the largest producers of these spices, but the production of this species is committed by fusariosis, that causes root rot in plant. Study of molecular biology is important by support breeding improvement of black pepper. In this work we draw the primers Lox3R and Lox3F to amplify a larger fragment of the gene lipoxygenase (Lox) in black pepper. The amplified fragment using primers drawn was sequenced. The sequence isolated has 770 nucleotides that encode 258 amino acids. This sequence was characterized by comparison in biological databases and using computational programs. The analysis with BlastX showed that sequence isolated has high similarity with lipoxygenase proteins of Persea americana , Parasponia andersonii e Vitis vinífera . We verified that Lox of black pepper has the PLAT/ LH2 domain of plant lipoxygenase related proteins. Its description in black pepper is essential to clarify the molecular mechanisms of response of the plant to the Fungus and understand its paper in activation of the defense response, once the gene Lox is activated in plant to signal its defense in a possible attack against pathogen and may be precursors of metabolic regulators. . Se observó que Lox de pimienta negra tiene el dominio PLAT/LH2 de proteínas relacionadas con la lipoxigenasa vegetal. Su descripción en pimienta negra, es esencial para aclarar los mecanismos moleculares de respuesta de la planta al hongo, y entender su papel en la activación de la respuesta de defensa, ya que el gen de Lox se activa en la planta, para señalar su defensa, en un posible ataque contra un patógeno y pueden ser precursores de reguladores metabólicos.


Introduction
The black pepper (Piper nigrum L) is a spice consumed world widely, that has a great economic importance. This species is widely used in the food industry, cosmetics industry (Liu et al., 2010) and in the traditional medicine due to its antioxidant, anti-inflammatory and anticancer properties (Nishimura et al., 2011).
In Brazil, the state of Pára is one of the main productor of black pepper (EMBRAPA, 2010), Nonetheless the production is affected, since that this species has great susceptibility to a disease called fusarium (EMBRAPA, 2010). This disease affects the root system and the aerial part of the plant, resulting in the yellowing and fall of the leaves and root rot and consequently reducing the lifespan of peppers (Chu et al., 1997). Therefore, up to the present time, there are only palliative measures against fusarium such as; the use of fungicide, use of disinfected tutors, eradication and burning of the diseased plants (Serrano et al., 2006).
In general, the works that contribute to understand the plants' molecular answers are scarce and considered of great relevance specially to support programs for the genetic improvement of the species. From the molecular perspective, the lipoxygenases are protein found in most plants associated with important physiological processes as a defense against pathogenic attack (Song et al., 2016). These enzymes catalyze the addition of molecular oxygen, forming hydroperoxides (Axelrod et al., 1981;Siedow, 1991).
The hydroperoxides derived from lipoxygenase may be metabolized giving rise to volatile aldehyde molecules and jasmonates in vegetables (Mosblech et al., 2009). These molecules have an important role in the process of signaling and healing plants' injuries (Andreou & Feussner, 2009) and are associated to important physiologic processes in living beings such as: biosynthesis of regulatory compounds, growth and development (Siedow, 1991), senescence (Rouet Mayer et al., 1992), seed germination (PARK et al., 1994), wound response (Vieira et al., 2001), protein of vegetative reserve (Stephenson et al., 1998) and in resistance to biotic and abiotic stress (Song et al., 2016).
The transcriptome studies of Black Pepper showed the altered expression pattern of several genes in response to the pathogen Fusarium solani, including a lipoxygenase gene (LOX) (Moreira et al., 2017). Therefore, the objective of this work was to isolate and characterize partially the Lipoxygenase gene in Piper nigrum, providing the base to understand the functioning of the plant's molecular response mechanisms.

Biological material
Were used black pepper leaves (Piper nigrum cv. Bragantina), provided by the Brazilian Agricultural Research Corporation (EMBRAPA) Eastern Amazon (Belém -PA) around two months old. The leaves were disinfected using distilled water and then proceeded to extract the genomic DNA following the protocol of the Kit "PureLink Plant Total DNA Purification Kit", commercially available.

Design of the initiators
For the construction of the initiators were used sequences of nucleotides from different plant species available at NCBI (National Center for Biotechnology Information) (https://www.ncbi.nlm.nih.gov/). After obtaining the sequences, they were placed in fasta format and then aligned using the Multalin program (Corpet, 1988). From the alignment the Forward initiators were built (in the chemical sense 5'-3') and reverse (in the chemical sense 3´-5') using the program Primer3plus (Untergasser et al., 2007).

Amplification and sequencing
The gene was amplified by PCR with a total volume of 25 μl, being 2.5 μl of 10x Buffer, 0.5 μl of MgCl2

Lox gene characterization using biological databases
The nucleotides sequence of Lox of Piper nigrum L was analyzed by comparison in a biological database using the program Blast-X (Altschul et al., 1990). The program Expasy (Artimo et al., 2012) was used to identify possible reading frames and identify amino acid sequences. The evaluation of possible domains in the isolated sequence was performed using the Blast-X and pfam databases (Finn, 2016). The Phyre2 server (Kelly et al., 2015) was used to model and predict the secondary structure of the obtained protein. The PDB program (Berman et al., 2000) was consulted to visualize the threedimensional structure of the lipoxygenase protein with greater similarity to that of Piper nigrum. Then the Lox sequence was predicted through comparative modeling using the Swiss Model server (Guex et al., 2009). The evaluation of the model's quality was observed from the GMQE (Global model Quality estimation) and Qmean scores.

Design of primers for partial isolation of the Lox in P. nigrum
After the isolation of the DNA, After DNA isolation, primers were designed by aligning lipoxygenase gene sequences from different plant species available in the database. NCBI. The species used, as well as their accession number in the database are shown in Table 1. Research, Society and Development, v. 11, n. 9, e57411932254, 2022 (CC BY 4. The alignment shown in table 1indicates a high degree of conversation between the sequences. The regions selected for the design of the primers Lox3R (in the 5'-3' chemical sense) and Lox3F (in the 3'-5' chemical sense) are highlighted ( Figure 1).

Figure 1 -Alignment of Lipoxygenase gene sequences in different plant species, the synthesized primers (F) Forward and (R)
Reverse are indicated in green.

Comparative analyzes of the isolated partial nucleotide sequence
The isolated sequence was obtained from the sequencing of Moreira et al. (2017) and was characterized through computer programs described below. The analysis of the nucleotide sequence was performed with the BlastX program, and Research, Society andDevelopment, v. 11, n. 9, e57411932254, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i9.32254 5 showed that it has identity with other lipoxygenase-type proteins from different plant species. Among the species that showed greater identity with the isolated partial protein, Persea americana, Parasponia andersonii, Vitis vinífera, Prunus dulcis, Trema orientalis, Cephalotus follicularis e Ziziphus jujuba (Figure 2). The analysis with the program Expasy showed that the isolated protein has 777 nucleotides, which encode 258 amino acids (Figure 3).    (Song et al., 2016). The Plat domain is found in a large number of membrane-associated proteins or lipids and may present a single domain or be repeated (Bateman and Sandford, 1999;Hong et al., 2000;Minor et al., 2006;Shin et al., 2004). It is reported in the literature that this domain is always present in monocot and dicot plant species, in addition, a study with Arabidopsis suggests that the promotion of stress tolerance may be the general function of PLAT (Hyun et al., 2015), however, studies on the Plat domain for vegetables are still scarce or non-existent (Shin et al. 2004).
According to the analysis of the Lox domain of the isolated partial protein, we can say that it is a 9-lipoxygenase (9-LOXS) type protein. These 9-Loxs proteins catalyze the oxygenate at the C-9 position of fatty acids and have been reported to activate plant defense (Walper et al., 2016). Genetic studies show that the 9-Loxs pathway plays an important role in activating the local defense against pathogens (Hwang & Hwang, 2010;López et al., 2011;Montillet et al., 2013), in addition, this pathway helps in the triggering of systemic resistance (Vicente et al., 2012). Studies by Marcos et al., (2015) also show that derivatives of the 9-Loxs pathway activate cell wall-based defense responses, participating in the signaling of cell wall damage. Furthermore, the analysis of the secondary structure of the Lox domain of the partial protein performed in the Phyre2 program showed that it has great similarity to the soy protein Lipoxygenase (Data not shown).
Regarding the tertiary structure, the isolated protein is similar to the structure of the soy lipoxygenase protein (1rrl2.A). The tertiary structure model built for black pepper using the Swiss model server is shown in the figure below ( Figure   5). The regions shown in blue are polar regions and in red are highlighted hydrophilic regions. The model built is a reliable model according to the QMEAN and GMQE measure around 0.77 observed. The GMQE is given by the number between 0 and 1 and reflects the expected accuracy of the model built. Higher numbers reflect greater reliability. The QMEAN Z-score around zero indicates good agreement between the model structure and the experimental structure of similar size. Source: Moreira et al. (2022)

Conclusion
The work allowed the partial characterization of Lipoxygenase gene in the Black pepper (P. nigrum). The gene isolated has approximately 770 base pairs and identity with different plant species such as: Persea americana, Parasponia andersonii and Vitis vinifera. The gene encodes 258 amino acids and has the Plat-LH2 and Lipoxygenase domains, reported in the literature as important for plant defense against pathogens. The protein isolated is of the 9-Lipoxygenase type, important for activation of local defense, systemic resistance and activation of other plant defense responses. The results achieved represent a contribution regarding molecular data for P. nigrum, which can be considered important, as molecular information for this species is still scarce and these can help in the development of genetic improvement programs.