Bioinformatics analysis and pharmacological effect of Stevia rebaudiana in the prevention of type-2 diabetes

preference trend of these proteins was investigated using the sequence manipulation suite database. This information can be used for other research, including the transfer of these proteins.


Introduction
Sweeteners are compounds that have sweetening properties and are divided into two categories of natural and artificial sweeteners [1].In recent years, the development of natural sweeteners has greatly increased, especially natural sweeteners containing sucrose that is not absorbed in the digestive system and is not caloric, and are suitable for diabetes and obese people [2][3][4].One of these promising natural sweeteners is steviol glycosides (SGs).SGs are extracted from stevia leaves.Steviosides and ribodioside A are most abundant in leaf extract [5].
SGs are glycoside diterpene and have been identified in the extract of various components of the stevia plant body and contain various compounds [6].Among these compounds, ribodiozide A is more commercially important and the UGT family (UDP-glycosyltransferase) and especially the genes UGT85C2, UGT74G1, and UGT76G1 play an effective role in the conversion of SGs to ribodioside A [7]. Figure 1 shows the common skeletal structure of stevia glycosides with the structure of ribodioside A [8,9].In Stevia, in addition to Rebaudioside A, there are other types of Rebaudioside, such as Rebaudioside E, Rebaudioside D and Rebaudioside M [10,11].UGT76G1 is a key enzyme in the production of Rebaudioside.Research has shown that a single-point saturation mutation in the UGT76G1 gene converts Rebaudioside M to Rebaudioside E, which improves its performance and sweetening properties by 2 times [12].So far, more than 60 different steviolglycosyl and 68 different putative UGTs have been identified in stevia rebaudiana, which helps us better understand this key family in improving the sweetening properties of this plant [13].The latest medical research has shown that blood sugar can be treated with the use of stevia.It has also been found that the therapeutic effects of this plant are due to the presence of phenolic compounds in different parts of this plant, especially in the leaves and calluses, and have considered anticancer effects and treatment of diseases related to hypertension and hypoglycemia due to their free radical scavenging and antioxidant properties [14,15].Studies have shown that stevia has an antiviral effect, prevents high blood pressure and blood sugar, and is beneficial for patients with diabetes.Clinical trials have shown that the use of this plant and its extract as a real medicine is effective for patients with hypoglycemia.[16][17][18].This study aimed to compare the encoded regions, study the similarities and differences, and bioinformatics of UGT85C2, UGT74G1, and UGT76G1 genes in stevia to identify possible differences related to different reactions.

Materials and methods
First, the protein sequences of UGT85C2 (AY345978.1),UGT76G1 (KC631816.1),and UGT74G1 (AY345982.1)genes in stevia were obtained from NCBI (http://www.ncbi.nlm.nih.gov/).The lengths of these proteins were 528, 538, and 518 amino acids, respectively.Then these sequences were aligned using Vector-NTI software and protein domains in amino acid sequences of genes were identified using the Pfam database.The secondary protein structure of each sequence was determined using the SOPMA database and the three-dimensional structure of the proteins was determined using the MBC database.Hydropathicity analysis and determination of molecular weight and isoelectric point of proteins were performed using the ProtScale database.The codon preference trend of these proteins was investigated using the sequence manipulation suite database.Then the promoter region of the genes was analyzed using the PlantCARE database to identify putative CREs.

Results
The sequential results of protein sequences of UGT85C2 (AY345978.1),UGT76G1 (AY345974.1),and UGT74G1 (AY345982.1)genes in stevia showed that these sequences are very similar in primary structures Table 1.The percentage similarity of amino acid sequences is shown in Table 2

Secondary structure
Search results on the Pfam database indicate the presence of a common UDPGT protein domain in the UGT85C2, UGT76G1, and UGT74G1 sequences, indicating that the UDPGT protein domain is highly conserved in the UGT family.Also, the secondary structure of these sequences was very close to each other.Studies from the SOPMA database have shown that UGT74G1, UGT76G1, and UGT85C2 proteins are generally alpha-helix and random screws or loops associated with linear strands.Also, these proteins contain betahelix, which effectively stabilizes the structure of these proteins.

Three-dimensional structure
Molecular homologation modeling using SWISS-MODEL server in Expasy resulted in a three-dimensional structure of UGT85C2, UGT76G1, and UGT74G1 proteins based on sample 1a02 with the highest similarity, which these results it was consistent with previous findings of the high genomic similarity of these proteins (Figure 2).

Molecular weight, Isoelectric point and Hydropathicity
Also, determine the molecular weight (kDa), isoelectric point and hydropathicity analysis of these amino acid sequences were performed using the ProtScale program (Table 3).It was also found that due to the abundance of amino acids with negative hydropathicity in the sequences of UGT85C2, UGT76G1, and UGT74G1, these proteins are hydrophilic and are effective in creating resistance to drought stress.

Phylogenetic tree
Analysis of the phylogenetic tree of the UGTs family showed that genes UGT74G1 and UGT85C2 are most similar.Also, the similarity of UGTs genes in stevia is much greater than the genes of this family in other plants such as Triticum aestivum (Figure 3).

Codon usage
The codon preference trend of these proteins was investigated using the sequence manipulation suite database, the results for UGT85C2, UGT76G1, and UGT74G1 genes are shown in the Table 7 to 9.This information can be used for other research, including transferring these proteins.Also, we can use codon usage to assess whether a sequence shows a preference for particular synonymous codons (Table 7-9).

Discussion
UGTs genes constitute a large and important gene family in Stevia [19].Careful study of the structure and function of these genes is an essential step for a further indepth understanding of plant regulatory pathways.In this study, secondary structures, three-dimensional structures, and codon analyzes were performed to investigate the similarities and differences between these genes.Understanding these helps us better understand the transfer of these genes and other genetic analyzes.Examination of the three-dimensional structure of the UGTs genes showed that these structures contained a set of proteins bound to the active site with UDP and rebaudioside A, which also confirmed Lee et al evidence for the UGT76G1 gene [20].On the other hand, UGTs genes are very effective due to their significant effect on the synthesis of glycosides in plant sweetness, and understanding their structure is very important.Also, the close phylogenetic relationship between UGT74G1 and UGT85C2 genes showed that the structural similarities between these genes are very high.The promoter analysis suggested that UGTs gene expression is regulated by modulators, such as phytohormones, light, meristem, endosperm, and drought.In addition, the UGTs gene with diversified CREs may play a mediator to link different signaling pathways.Finally, we can say the careful study of these structures provides insight into glycosylation and a suitable model for steviol biosynthesis engineering.

Conclusion
In general, clinical results show that stevia consumption reduces blood sugar by increasing insulin sensitivity.A natural substance with antioxidant properties is effective in reducing and preventing kidney and liver damage in diabetic patients.Therefore, the consumption of stevia as a blood sugar controller and antioxidant is recommended for diabetic patients.

Fig. 1 .
Fig. 1.The image on the right shows the common skeleton structure of stevia glycosides and the image on the left shows the chemical structure of ribodioside A.

Fig. 2 .
Fig. 2. The image on the right shows the threedimensional structure of the UGT74G1 protein, the middle image shows the three-dimensional structure of the UGT76G1 protein, and the image on the left shows the three-dimensional structure of the UGT85C2 protein.

Table 3 .
Determination of molecular weight (kDa), isoelectric point, and hydropathicity evaluation of UGT85C2, UGT76G1, and UGT74G1 proteins using the ProtScale program.

Table 4 -
6).UGTs promoter contained several hormone-responsive CREs associated with activity and regulation of abscisic acid and auxin implying that UGTs may be involved in hormone crosstalk.

Table 4 .
The most important cis-elements in the promoter region of the UGT74G1 gene in Stevia rebaudiana

Table 5 .
The most important cis elements in the promoter region of the UGT76G1 gene in Stevia rebaudiana

Table 6 .
The most important cis-elements in the promoter region of the UGT85C2 gene in Stevia rebaudiana

Table 9 .
Results of codon usage for gene UGT85C2 (Results for 1586 residue sequence " UGT85C2"