شناسایی، جداسازی و تعیین خصوصیات برخی از اعضای خانواده ژنی snakin/gasa در سرخارگل (echinacea purpureae)

خانواده ژنی پیئیدهای ضد میکروبی snakin/gasa به دلیل نقش های کلیدی خود در فرآیندهای فیزیولوژیکی گیاهان از جمله تنظیم رشد پاسخ به تنش ها و دفاع ضد میکروبی به عنوان یکی از مهم ترین گروه از خانواده پیتیدهای ضد میکروبی شناخته شده اند. این پیتیدها در مسیرهای پیام رسانی متنوعی مشارکت دارند و در تنظیم بیان ژنها و القای مقاومت به تنش های زیستی نقش مهمی ایفا میکنند. در این مطالعه شش عضو از خانواده snakin gasa برای نخستین بار در گیاه دارویی echinacea purpurea شناسایی و مورد بررسی مولکولی قرار گرفتند. سرهم بندی نوپدید ترنسکریپتوم بر پایه داده های rna-seq ازبافت های مختلف سرخار گل منجر به شناسایی 27679 رونوشت شد. در میان آنها شش ژن با نامهای epu- epu-snakintsnakin6 از طریق تجزیه و تحلیل های بیوانفورماتیکی و تایید por شناسایی شدند. پروتئین های حاصل دارای وزن مولکولی کمتر از 12 کیلو دالتون و نقاط ایزوالکتریک بین 8/61 تا 9/28 بودند. در تمامی توالی ها پیتید سیگنال در ناحیه آمینی مشاهده شد و در پنج مورد نیز وجود حداقل یک مارپیچ گذرنده از غشا پیش بینی شد. پیش بینی های عملکردی بر پایه الگوریتم های یادگیری ماشین خاصیت ضد میکروبی قوی را برای تمامی پیتیدهای شناسایی شده تایید کرد تحلیل موتیفهای حفاظت شده و پیوندهای دی سولفیدی درون مولکولی ثبات ساختاری این پروتئین ها را نشان داد. علاوه بر این تجزیه و تحلیل فیلوژنتیکی این ژنها را در سه گروه اصلی طبقه بندی کرد که قرابت نزدیکی با ژنهای همولوگ در آفتابگردان داشتند. الگوی بیانی این سیلیکو ژنهای شناسایی شده نیز نشان داد که برخی به صورت گسترده در بافت های مختلف بیان میشوند در حالی که برخی دیگر دارای الگوی بیانی اختصاصی در بافت های گل یا رویشی هستند. نتایج این پژوهش دیدگاه تازه ای از ساختار و عملکرد ژنهای snakin/gasa در سرخارگل ارائه می دهد و کاربردهای بالقوه آنها را در توسعه داروهای گیاهی و زیست فناوری کشاورزی پیشنهاد می کند.

identification, isolation and characterization of some members of the snakin/gasa genes family in echinacea purpureae

introduction the snakin/gasa protein family in plants plays a crucial role in regulating plant growth, defense, and bacteriostasis. these proteins are involved in various signaling pathways that help plants respond to pathogens. additionally, they have been found to interact with other proteins to modulate gene expression and promote plant development. these peptides play a crucial role in protecting plants from pathogens by disrupting their cell membranes or interfering with their cellular processes. the snakin/gasa proteins also have the ability to induce systemic resistance in plants, enhancing their overall defense mechanisms against various threats. furthermore, it has been established that these peptides play a role in the development of plant adaptations to a wide range of abiotic stresses. overall, the multifunctional nature of snakin/gasa proteins highlights their importance in plant defense and adaptation mechanisms. their diverse roles make them valuable targets for further research in improving crop resilience and productivity in challenging environments. therefore, in this study snakin/gasa family members were identified and analyzed in echinacea purpurea. materials and methods to identify the members of the snakin/gasa gene family in the transcriptome of e. purpurea, rna sequencing data from different tissues and growth periods of e. purpurea were obtained from the ncbi database and after processing the data, transcriptome assembly was performed. the obtained transcripts were documented using the blastx tool against non-repetitive protein (nr) data. after mapping the clean reads with the reference assembly, the expression level of the transcripts was calculated in terms of fpkm (fragments per kilobase of transcript per million mapped reads: fpkm). in the next step, protein sequences related to the snakin/gasa antimicrobial peptide family of different plants were obtained from the ncbi database, and a consensus sequence was created using multiple alignments. then, the consensus protein sequence obtained from the multiple alignment, which was conserved in all snakin, was compared against the echinacea transcriptome assembly using the tblastn tool with an e-value threshold of less than 0.001. after bioinformatic confirmation of the identified sequences, rna extraction, cdna library preparation, amplification, and sequencing of target genes were performed to confirm the sequences of the identified genes and downstream analysis. also, gene and protein structure analysis, physicochemical properties, and evolutionary and phylogenetic relationships were performed for the target sequences. finally, the in silico relative expression of the identified snakin/gasa genes in different echinacea tissues was evaluated. results and discussion in the current study, a comprehensive de novo transcriptome assembly of e. purpurea was achieved using rna-seq data, yielding over 698 million raw reads from different tissues. after quality control and trimming, approximately 533 million clean reads remained, indicating that about 24% of the raw reads were discarded, highlighting the importance of data preprocessing in transcriptomic studies. the assembly generated 27,679 unique transcripts, with an n50 value of 1373 and the longest transcript being 8823 nucleotides. these metrics indicate high assembly quality. blastx annotation against the non-redundant protein database revealed that 81.16% of the unigenes (22,466) had significant similarity to known sequences, and 7,740 were functionally annotated with gene ontology (go) terms. the study identified six members of the snakin/gasa gene family in e. purpurea through tblastn and domain confirmation using interproscan, pfam, and cdd databases. these genes, designated as epu-snakin1 to epu-snakin6, ranged in sequence length from 530 to 700 nucleotides and showed gc content between 36% and 41%. amplification using specific primers confirmed the presence of full open reading frames (orfs), further supported by sequencing analyses. the physicochemical profiling revealed molecular weights between 9.59 and 11.86 kda, mostly under 12 kda, consistent with findings in other species such as olive and lettuce. the isoelectric points ranged from 8.61 to 9.28, and instability indices suggested that most proteins are potentially unstable in vitro except epu-snakin6. these proteins exhibited diverse aliphatic indices and gravy scores, indicating a range of solubility and structural properties. signal peptide analysis identified n-terminal signal sequences of 20–36 amino acids in all six proteins, suggesting their potential for extracellular localization or secretion. transmembrane helix predictions indicated that all except epu-snakin5 contained at least one transmembrane domain, implicating roles in signal transduction or membrane-associated processes. functional prediction using the camp database indicated strong antimicrobial properties for all six proteins, with high confidence from svm, rf, and ann algorithms. these results support the potential utility of e. purpurea snakin/gasa peptides in developing antimicrobial agents. conserved motif analysis using meme revealed three motifs across the family members, with only epu-snakin2 and epu-snakin6 containing all three. disulfide bond analysis revealed 5–6 intra-molecular disulfide bridges, consistent with the structural stability and biological activity roles attributed to snakin/gasa proteins. phylogenetic reconstruction using arabidopsis, sunflower, and e. purpurea snakin/gasa proteins placed the six genes into three main clades, reflecting both evolutionary and likely functional relationships. notably, epu-snakin2 and epu-snakin6 clustered with sunflower homologs, reinforcing the phylogenetic proximity observed in transcriptome-wide analysis. expression profiling revealed tissue-specific expression patterns. genes in group iii (epu-snakin1, 3, 4) showed broad expression across ten tissue types, suggesting fundamental roles in growth and development. in contrast, group i (epu-snakin2, 6) was flower-specific, and epu-snakin5 expressed solely in vegetative tissues.conclusion this study presents the first in-depth identification and characterization of six snakin/gasa genes e. purpurea. the structure and physicochemical properties of these genes, as well as how they are expressed in different tissues, show that they have a wide range of possible biological functions. the presence of key features such as gasa domains, signal peptides, transmembrane helices, and multiple disulfide bonds, along with strong predictions of antimicrobial activity, shows that these proteins could have many different functions. expression profiling analysis suggested both general and tissue-specific roles, particularly with group iii genes being expressed across most tissues, indicating involvement in core physiological processes, while other genes showed restricted expression patterns, hinting at specialized functions. phylogenetic analysis also placed these genes into distinct evolutionary clades, highlighting their divergence and potential for functional novelty. overall, the findings suggest that snakin/gasa proteins in e. purpurea may play important roles in plant development, defense mechanisms, and stress response. given their predicted antimicrobial activity, these proteins are promising candidates for pharmaceutical exploration and bioengineering applications in medicinal plants. more studies are suggested to test how well these proteins fight germs in lab tests, check how they work in different systems, and see how stable and effective they are in making plant-based medicines.