Comparative modeling, structure and active site prediction of Sclerotinia disease-resistant gene BnaMPK3 in Oilseed rape (Brassica napus)

Oilseed rape (Brassica napus L.) is an agronomically signifi cant crop that is developed around the world; including; Europe; North America; and South Asia. The noteworthy requirement for its effi ciency is sclerotinia disease brought about by fungal pathogen Sclerotinia sclerotiorum [1]. deBary; bringing about a huge misfortune in yield of seed: For instance; ten to twenty percent of yield misfortunes in a normal year; and round about eighty percent in seriously contaminated in fields of [2]. Surely, S. sclerotiorum is a massively damaging fungal plant pathogen which has host range of approximately four hundred eight plant species along with numerous economically signifi cant crops. More than sixty names have been utilized to indicating the symptoms by this pathogen of fungal in horticulture. As of now; cross-breeding of S. sclerotiorum resistance cultivars Abstract

of oilseed rape by utilizing conventional strategies is hard to manage; since no insusceptible or exceptionally resistance germplasm in Brassica napus is accessible [3]. The research activities are focused on activation of host defense system against S. sclerotiorum pathogen. Gene expression profi le of host, in response to pathogen, demonstrated that genes related with jasmonic acid (JA) and ethylene(ET) signals are prompted.
A few last investigations demonstrated that signaling of JA, together with SA and ET, adds to basal resistance against S. sclerotiorum [5]. These investigations summerise about the role of these defense effectors in protecting host from S. sclerotiorum, but not consistently. This information suggests that the activity of defense to the pathogen include various diffi cult organize of different signaling pathways.
It is reported that Mitogen-activated protein kinases (MAPK or MPK) assume signifi cant function in the transduction of signals because of ecological stresses and hormones [6]. An action of MAPK constrained through consecutive actuation of two different kinases protein by which a MAPK kinase activates another MAPK kinase [7]. The proof established that some MAPK relatives have been involved as a part of protection signaling pathways in plant defense. For instance; in Arabidopsis thaliana; microbe-associated molecular patterns (e.g. MAMP, the bacterial flagellum-inferred flg22 peptide; stimulates the initiation of AtMAPK involving AtMPK1; AtMPK3; AtMPK4; AtMPK6; AtMPK11; and AtMPK13 [8]. AtMPK4 was first designated as a negative regulator of plant resistance [9]. Though some others, e.g. AtMPK3 was acknowledged to accept a positive role in plant defenseless dependent on their fast initiation through flg22 and pathogen immunization [10].
Furthermore, MPK3 mutant from the Arabidopsis thaliana work loss presented importantly less bacterial titters of Pseudomonas syringe; and the genes expression that controlled by SA synthesis and signaling; either handle or flg22-treated AtMPK3 loss of function mutants have no dierent from AtMPK4 mutants that appear upgraded protection from biotrophic fungal pathogens Hyaloperonospora arabidopsis' and P. syringe [11]. In any case; another preliminary uncovered no reasonable vulnerable phenotype in the AtMPKs3 mutants when showering or infiltration-vaccinated with P. syringae [12].  [14]. In this investigation, we used the bioinformatics approach which is the best way to understand the function and characterize the BnaMPK3 protein. This article intended to 3D-model of the BnaMPK3 protein and anticipate its active binding site in-silico. The data accomplished will be utilized to distinguish the suitable effectors for BnaMPK3 protein fi rst in silico and afterward in vitro.

Amino acid sequence retrieval of BnaMPKs3
The sequence of the BnaMPK3 protein was obtained from the NCBI (National Center for Biotechnology Information); a large online (https://www.ncbi.nlm.nih.gov/) nucleotide sequence database (Accession # JQ708040.1).

Computational analysis of S-Nitrosylation active sites in BnaMPK3 Protein
For the computational prediction of different S-Nitrosylation active sites in BnaMPK3 protein, the GPS-SNO ( http://sno. biocuckoo.org/.) bioinformatics tool was used. The prediction analysis of S-Nitrosylation active site through using GPS-SNO was well with good accuracy, specifi city, and sensitivity, respectively. GPS-SNO predicted the reputed S-Nitrosylation sites of the different possibly S-Nitrosylated substrates for which the S-Nitrosylation sites were not predicted [15].

Comparative based modeling of BnaMPK3
Comparative modeling of BnaMPK3 proteins was done by utilizing the SWISS-MODEL web Bioinformatics tool (https:// swissmodel.expasy.org/). It is an online web computational tool used for the homology modeling of protein three-dimensional structure [16].

Evaluation of the three-dimensional structure of BnaMPK3
The 3D computational structure of the BnaMPK3 protein was

Amino acid sequence retrieval of BnaMPK3
An amino acid sequence of BnaMPK3 protein (Accession # JQ708040.1) retrieved from NCBI was consisted of 370 amino acids.

Analysis of the primary structure and Factional domain of BnaMPK3
Analysis of the primary structure of BnaMPK3 was

S-Nitrosylation plays an important character in biological
functions. Therefore, the prediction of S-Nitrosylation active sites through using bioinformatics tools is effi cient and quick.
The estimated analysis showed six different S-Nitrosylation active site through using GPS-SNO was well with good accuracy, specifi city and sensitivity, 75.80%, 53.57%, and 80.14%, respectively (Table 1).

Comparative based modeling of BnaMPK3 protein
The comparative modeling of the BnaMPK3 protein of oilseed rape (Brassica napus); which is encoded by Sclerotinia disease-resistant gene BnaMPK3 was done by using a bioinformatics tool "SWISS-MODEL". The 3D model was constructed using the amino acid sequence as a template for the BnaMPK3 protein. One best model was generated with maximum GMQE (Global Model Quality Estimation) from the Swiss model ( Figure 2, Table 2).

Evaluation of the three-dimensional structure of BnaM-PK3
The obtained best 3D-model was evaluated by ERRAT.
The best 3D-model of BnaMPK3 protein with a highly refi ned overall quality score 93.023%; which indicated the stability of the 3D-structure of BnMPK3 protein (Figure 3).

Active site prediction of BnaMPK3
Active sites of oilseed rape (Brassica napus) BnaMPK3 protein was attained using ADDS (Automated active sites detection; docking; and scoring). A total of 20 active sites were predicted fi rstly (Figure 4). The only best pocket with the highest score was selected for further analysis ( Figure   5). There are fi ve functionals domains are identifi ed in    Figure 2: The best-corrected 3D structure of BnaMPK3 protein predicted through using the SWISS-MODEL. of these fl agging are as yet not comprehended [21]. MPK6 was used as a template (76.88% identity) protein for prediction of the three-dimensional structure of BnaMPK3 Protein.
The BnaMPK3 protein has fi ve functional domains with the Leucine-Rich Region (LRR) and six S-Nitrosylation active sites make it effective against Sclerotinia sclerotiorum, LRR activated the innate immunity against pathogen so that known as fungus protein [22,23].

Conclusion
The main objective of this study is that to understand the