Transgenic Technology Research Articles

Application of an Efficient Enhancer in Gene Function Research

Although great progress has been made in transgenic technology, increasing the expression level and thus promising the expected phenotypes of exogenous genes in transgenic plants is still a crucial task for genetic transformation and crop engineering. Here, we conducted a comparative study of the enhancing efficiency of three putative translational enhancers, including Ω (natural leader from a plant virus), OsADH 5′ (natural leader from a plant gene), and ARC (active ribosomal RNA complementary), using the transient gene expression systems of Nicotiana benthamiana and Chirita pumila. We demonstrate that three tandem repeats of ARC (3 × ARC) are more efficient than other enhancers in expression. The enhancing efficiency of 6 × ARC is further increased, up to 130 times the expression level without the insertion of enhancers. We further evaluated the enhancing efficiency of 6 × ARC under agrobacterium-mediated transformation systems. In C. pumila, 6 × ARC significantly amplifies the phenotypic effect of CpCYC1 and CpCYC2 in repressing stamen development and yellow pigmentation. In Arabidopsis thaliana, 6 × ARC and the AtAP1 promoter work together to promote the accumulation of anthocyanin pigments in vegetative and reproductive organs. Most significantly, the fusion of 6 × ARC in a CpCYC1/2 transgenic system in C. pumila fully reveals that these genes have the complete function of repressing the yellow spots, displaying an advantage in manifesting the function of exogenous genes. This study highlights the application potential of the enhancer 6 × ARC in gene function research in plants.

Begomovirus disease of pumpkin crop in India and its management strategies possibility: a review

Pumpkin is an important commercial crop grown worldwide in tropical and subtropical regions. The whitefly-transmitted Pumpkin yellow vein mosaic disease seriously threatens pumpkin cultivation worldwide. The advent of transgenic technology in the 1980s revolutionized the possibilities for introducing virus resistance into agriculturally important plant species. It offered a powerful tool to enhance crop protection and provided a pathway to potentially unlimited sources of resistance against viral diseases. The ongoing research and development in this field continue to explore and refine conventional and non-conventional approaches for effective virus management in agriculture crops. This review focuses on developing transgenic resistance against begomoviruses and discusses possible management strategies to address these challenges.

CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects.

Abiotic stresses associated with climate change, such as heat, cold, salinity, and drought, represent a serious threat to crop health. To mitigate the risks posed by these environmental challenges, both transgenic technology and conventional breeding methods have been extensively utilized. However, these methods have faced numerous limitations. The development of synthetic nucleases as precise genetic tools allows for the targeted alteration of stress-responsive genes in crop improvement. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome-editing technique has transformed gene editing with its broad applicability, accessibility, adaptability, flexibility, and simplicity. Its application shows promise for the development of crop types that are more able to survive abiotic stress conditions. The present study presents recent scenario and application of CRISPR/Cas genome-editing technology in enhancing crop tolerance to a variety of abiotic stresses.

Revolutionizing livestock sustainability: Pioneering breeding strategies for superior forage biomass and quality

Livestock primarily rely on forage crops as a source of feed and nutrition. The milk productivity of a cow or meat production in goat/sheep could directly be associated with the availability of a sufficient quantity of quality green fodders with essential nutrients in a balanced ratio. Feeding the cereal/grass: legume fodders in the required proportion will not only improve productivity but also the reproductive capacity of animals. However, many countries of the world experience a huge gap between demand and availability of green fodder. In this context, emphasis should be placed on developing efficient forage genotypes with increased biomass and quality as per the requirements of animals, duly considering their digestibility. Breeding approaches encompassing required classical approaches, including wide hybridization to exploit natural genetic variability, biotechnological tools such as transgenic technology, marker-assisted selection, genomic selection, and various omics techniques alongside high-throughput phenotyping using multispectral cameras, would help to sustain livestock productivity by meeting out the present and future fodder requirements coupled with enhanced nutrients

Advances in bacterial artificial chromosome (BAC) transgenic mice for gene analysis and disease research.

Transgenic mice, including those created using Bacterial Artificial Chromosomes (BACs), are artificial manipulations that have become critical tools for studying gene function. While conventional transgenic techniques face challenges in achieving precise expression of foreign genes in specific cells and tissues, BAC transgenic mice offer a solution by incorporating large DNA segments that can include entire expression units with tissue-specific enhancers. This review provides a thorough examination of BAC transgenic mouse technology, encompassing both traditional and humanized models. We explore the benefits and drawbacks of BAC transgenesis compared to other techniques such as knock-in and CRISPR/Cas9 technologies. The review emphasizes the applications of BAC transgenic mice in various disciplines, including neuroscience, immunology, drug metabolism, and disease modeling. Additionally, we address crucial aspects of generating and analyzing BAC transgenic mice, such as position effects, copy number variations, and strategies to mitigate these challenges. Despite certain limitations, humanized BAC transgenic mice have proven to be invaluable tools for studying the pathogenesis of human diseases, drug development, and understanding intricate gene regulatory mechanisms. This review discusses current topics on BAC transgenic mice and their evolving significance in biomedical research.

AtZAT10/STZ1 improves drought tolerance and increases fiber yield in cotton.

Drought poses a significant challenge to global crop productivity, necessitating innovative approaches to bolster plant resilience. Leveraging transgenic technology to bolster drought tolerance in crops emerges as a promising strategy for addressing the demands of a rapidly growing global populace. AtZAT10/STZ1, a C2H2-type zinc finger protein transcription factor has shown to significantly improve Arabidopsis' tolerance to various abiotic stresses. In this study, we reports that AtSTZ1 confers notable drought resistance in upland cotton (Gossypium hirsutum), amplifying cotton fiber yield under varying conditions, including irrigated and water-limited environments, in field trials. Notably, AtSTZ1-overexpressing transgenic cotton showcases enhanced drought resilience across critical growth stages, including seed germination, seedling establishment, and reproductive phases. Morphological analysis reveals an expanded root system characterized by an elongated taproot system, increased lateral roots, augmented root biomass, and enlarged cell dimensions from transgenic cotton plants. Additionally, higher contents of proline, chlorophyll, soluble sugars, and enhanced ROS-scavenging enzyme activities are observed in leaves of transgenic plants subjected to drought, underscoring improved physiological adaptations. Furthermore, transgenic lines exhibit heightened photosynthetic rate, increased water use efficiency, and larger stomatal and epidermal cell sizes, coupled with a decline in leaf stomatal conductance and density, as well as diminished transpiration rates compared to the wild type counterparts. Transcriptome profiling unveils 106 differentially expressed genes in transgenic cotton leaves post-drought treatment, including protein kinases, transcription factors, aquaporins, and heat shock proteins, indicative of an orchestrated stress response. Collectively, these findings underscore the capacity of AtSTZ1 to augment the expression of abiotic stress-related genes in cotton following drought conditions, thus presenting a compelling candidate for genetic manipulation aimed at enhancing crop resilience.

Development of genetically modified rust resistant wheat: A breakthrough by dinted introgression of novel DREB2C and HSFA2 genes under stress induced expression

Wheat is a major staple food worldwide yet numerous yield limiting agents affect its productivity. Stripe rust is a major culprit in this context and efforts have been made to culminate this pathogen using conventional as well as advanced innovative techniques. Transgenic technology is of significant importance in this context and numerous success stories are evident to prove its worth. In the current study, two novel genes HSFA2 and DREB2C were expressed in an elite wheat genotype Akbar, Fakhre-e-Bhakhar under constitutive CAMV35S promoter and stress inducible rd29 Promoters. The shoot cut method was used for the Agrobacterium-mediated transformation and putative transformants were selected on kanamycin 50 mg/L. The resultant transformants were tested through PCR for transgene integration whereas expression analysis was carried out through realtime qPCR. Expression of both of the aforementioned genes was found to be higher under rd29 promoter as compared with transgene(s) expression under CAMV35S promoter. In the bioassay, transgenic wheat plants demonstrated significant tolerance to stress, exhibiting only minor spotting under constitutive expression conditions. Upon exposure to stress, these plants showed exceptional resistance to stripe rust, producing large, bold grains compared to individual trait expressions and negative controls. Subsequently, the DREB2C gene was knocked out to determine if stripe rust control was specifically attributed to this gene. Following the knockout, the onset of stripe rust was comparable to that of the negative control. This led to the conclusion that pyramiding the DREB2C gene with HSFA2 through dual expression represents a novel and highly effective strategy for controlling the widespread stripe rust in wheat. This approach also offers resistance to high temperatures (above 32 °C) from the pollination stage through to maturity.

Increased artemisinin production in Artemisia annua L. by co-overexpression of six key biosynthetic enzymes

Malaria remains a global health issue, especially in resource-limited regions. Artemisinin, a key antimalarial compound from Artemisia annua, is crucial for treatment, but low natural yields hinder large-scale production. In this study, we employed advanced transgenic technology to co-overexpress six key biosynthetic enzymes—Isopentenyl Diphosphate Isomerase (IDI), Farnesyl Pyrophosphate Synthase (FPS), Amorpha 4,11-diene Synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 oxidoreductase (AACPR) and artemisinic aldehyde D11 reductase (DBR2)—in A. annua to significantly enhance artemisinin production. Our innovative approach utilized a co-expression strategy to optimize the artemisinin biosynthetic pathway, leading to a remarkable up to 200 % increase in artemisinin content in T1 transgenic plants compared to non-transgenic controls. The stability and efficacy of this transformation were confirmed in subsequent generations (T2), achieving a potential 232 % increase in artemisinin levels. Additionally, we optimized transgene expression to maintain plant growth and development, and performed untargeted metabolite analysis using GC–MS, which revealed significant changes in metabolite composition among T2 lines, indicating effective diversion of farnesyl diphosphate into the artemisinin pathway. This metabolic engineering breakthrough offers a promising and scalable solution for enhancing artemisinin production, representing a major advancement in the field of plant biotechnology and a potential strategy for more cost-effective malaria treatment.

Screening and Functional Evaluation of Four Larix kaempferi Promoters.

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in Larix kaempferi (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed L. kaempferi transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the β-glucuronidase (GUS) reporter gene driven by these promoters. Recombinant vectors were introduced into the L. kaempferi embryogenic callus by means of the Agrobacterium-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring GUS expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: L. kaempferi Zhang Chen Yi-1 (LaZCY-1), Zhang Chen Yi-2 (LaZCY-2), Translationally Controlled Tumor Protein (LaTCTP), and ubiquitin (LaUBQ). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named pLaZCY-1, pLaZCY-2, pLaTCTP, and pLaUBQ. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive GUS expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the pLaUBQ promoter had higher activity than the other three L. kaempferi promoters and the CaMV35S promoter. Thus, the pLaUBQ promoter was suggested to be used in larch genetic transformation.

Plate centrifugation enhances the efficiency of polyethylenimine-based transfection and lentiviral infection

PurposeTo propose an efficient, reproducible, and consistent transgenic technology based on plate centrifugation, which is particularly useful for polyethylenimine (PEI) transfection and lentiviral infection. MethodsWe optimized multiple factors that could contribute to transfection efficiency, such as the dosage of the PEI or DNA, the working solution buffer used for diluting the PEI or DNA, the incubation time for the PEI/DNA complexes, and the transfection time. ResultsPlate centrifugation led to a 5.46-fold increase in the transfection efficiency of PEI-based transfection while maintaining the cell survival rate. Moreover, the average copy number of viral genes in each genome increased 4.96-fold with plate centrifugation. Plate centrifugation alters the spatial arrangement of the PEI/DNA complexes or lentiviruses, increasing the chances of these complexes or viruses coming into contact with target cells, ultimately resulting in improved transfection or infection efficiency. ConclusionsWe present a protocol based on plate centrifugation for transfection or lentiviral infection that is suitable for genetic modification of primary cells or stem cells.

Novel mechanism of MicroRNA408 in callus formation from rice mature embryo.

Mature embryos are the main explants of tissue culture used in rice transgenic technology. However, the mechanism of mature embryo callus formation remains unclear. In this study, a microRNA-mediated gene regulatory network of rice calli was established using degradome sequencing. We identified a microRNA, OsmiR408, that regulates the formation of the callus derived from the mature rice embryo. OsUCLACYANIN 30 (OsUCL 30), a target gene of OsmiR408, was the most abundant cleavage mRNA in rice callus. OsUCL17 was verified as a target gene of OsmiR408 using RNA ligase-mediated 5'-RACE. In analysis of the OsmiR408 promoter reporter line and pri-miR408 transcript level, the promoter activity and transcript level of MIR408 were increased dramatically during callus formation. In phenotypic observations, OsmiR408 knockout caused severe defects in mature embryo callus formation, whereas OsmiR408 overexpression promoted callus formation. Transcriptome analysis demonstrated that OsUCLs and certain genes related to the plant hormone signal transduction and phenylpropanoid-flavonoid biosynthesis pathway had different differential expression patterns between OsmiR408 knockout and overexpression calli. Thus, OsmiR408 may regulate callus formation mainly by affecting plant hormone signal transduction and phenylpropanoid-flavonoid biosynthesis pathway. Our findings provide insight into OsmiR408/UCLs module function in callus formation.

To what extent is transgenic technology beneficial to human beings?

Genetic engineering is a field which explores the possibility of modification or manipulation of genetic materials using various techniques and technologies. Broadly speaking, hybridisation, gene isolation and recombination, fall in this field. The most recent milestone development in this area would be CRISPR technology, which harnesses a component of the bacterial immune system. To elaborate, CRISPR acts as a precise molecular scissors to cut target sequences, thus allowing gene modification to take place. In the progression and advancement of this field, society has been carefully observing its consequences and impact. The utilisation of this technology has raised concerns such as the unknown detrimental impact of genetically modified food sources. On the other hand, the benefit of genetic engineering is self-evident. This article explores the various impacts of genetic engineering and concludes that the advantages of genetic engineering are overshadowed by its many benefits.

Biotechnological frontiers in harnessing allelopathy for sustainable crop production.

Allelopathy, the phenomenon in which plants release biochemical compounds that influence the growth and development of neighbouring plants, presents promising opportunities for revolutionizing agriculture towards sustainability. This abstract explores the role of biotechnological advancements in unlocking the potential of allelopathy for sustainable crop production and its applications in agriculture, ecology, and natural resource management. By combining molecular, genetic, biochemical, and bioinformatic tools, researchers can unravel the complexities of allelopathic interactions and their potential for sustainable crop production and environmental stewardship. The development of novel management methods for weed control is getting a lot of attention with the introduction of new genetic technologies such as Gene drive, Transgene technologies, Gene silencing, Marker-assisted selection (MAS), and Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). By strengthening competitive characteristics these tools hold great promise for boosting crops' ability to compete with weeds. Considering recent literature, this review highlights the genetic, transcriptomics, and metabolomics approaches to allelopathy. Employing allelopathic properties in agriculture offer sustainable benefits like natural weed management, pest management, and reduced chemical pollution, but challenges include environmental factors, toxicity, regulatory hurdles, and limited resources. Effective integration requires continued research, regulatory support, and farmer education​. Also, we aimed to identify the biotechnological domains requiring more investigation and to provide the basis for future advances through this assessment.

In-depth analysis of Bt cotton adoption: farmers' opinions, genetic landscape, and varied perspectives—a case study from Pakistan

BackgroundBt technology has played significant role in controlling bollworms and increasing cotton yield in earlier days of its introduction, a subsequent decline in yield became apparent over time. This decline may be attributed to various environmental factors, pest dynamics, or combination of both. Therefore, the present biophysical survey and questionnaire were designed to evaluate the impact of Bt cotton on bollworms management and its effect on reducing spray costs, targeting farmers with varied landholdings and educational backgrounds. Additionally, data on farmers' cultivated varieties and the prevalence of bollworms and sucking insects in their fields were recorded. Subsequently, about eleven thousand cotton samples from farmer fields were tested for Cry1Ac, Cry2Ab and Vip3A genes by strip test.ResultsIn this analysis, 83% of the farmers planting approved varieties believe that Bt technology control bollworms, while 17% hold contradictory views. Similarly, among farmers cultivating unapproved varieties, 77% agree on effectiveness of Bt technology against bollworms, while 23% disagree. On the other hand, 67% of farmers planting approved varieties believe that Bt technology does not reduce spray costs, while 33% agree with the effectiveness. Similarly, 78% of farmers cultivating unapproved varieties express doubt regarding its role to reduce spray costs, while 22% are in favour of this notion. Differences in opinions on the effectiveness of Bt cotton in controlling bollworms and reducing spray cost between farmers planting unapproved and approved varieties may stem from several factors. One major cause is the heavy infestation of sucking insects, which is probably due to the narrow genetic variation of the cultivated varieties. Additionally, the widespread cultivation of unapproved varieties (21.67%) is also an important factor to cause different opinions on the effectiveness of Bt cotton.ConclusionBased on our findings, we propose that the ineffective control of pests on cotton crop may be attributed to large scale cultivation of unapproved varieties and non-inclusion of double and triple transgene technologies in country’s sowing plan. On the basis of our findings, we suggest cotton breeders, regulatory bodies and legislative bodies to discourage the cultivation of unapproved varieties and impure seed. Moreover, the adoption of double and triple Bt genes in cottons with a broad genetic variation could facilitate the revival of the cotton industry, and presenting a promising way forward.

Integration of induction, system optimization and genetic transformation in Veratrum californicum var. vitro cultures to enhance the production of cyclopamine and veratramine

Cyclopamine, a compound found in wild Veratrum has shown promising potential as a lead anti-cancer drug by effectively blocking cancer signaling pathways. However, its complex chemical structure poses challenges for artificial synthesis, thus limiting its supply and downstream drug production. This study comprehensively utilizes induction, system optimization, and transgenic technologies to establish an efficient suspension culture system for the high-yield production of cyclopamine and its precursor, veratramine. Experimental results demonstrate that methyl jasmonate (MeJA) effectively promotes the content of veratramine and cyclopamine in Veratrum californicum var. callus tissue, while yeast extract (YE) addition significantly increases cell biomass. The total content of veratramine and cyclopamine reached 0.0638 mg after synergistic treatment of suspension system with these two elicitors. And the content of the two substances was further increased to 0.0827 mg after the optimization by response surface methodology. Subsequently, a genetic transformation system for V. californicum callus was established and a crucial enzyme gene VnOSC1, involved in the steroidal alkaloid biosynthesis pathway, was screened and identified for genetic transformation. Combined suspension culture and synergistic induction system, the total content of the two substances in transgenic suspension system was further increased to 0.1228 mg, representing a 276.69% improvement compared to the initial culture system. This study proposes a complete and effective genetic transformation and cultivation scheme for V. californicum tissue cells, achieving milligram-level production of the anticancer agent cyclopamine and its direct precursor veratramine for the first time. It provides a theoretical basis for the industrial-scale production of these substances.

In vivo spectrally unmixed multi-photon imaging of longitudinal axon-glia changes in injured spinal white matter

Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell–cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo.

Mechanism of emergency phytoremediation technology based on a 3D-QSAR pharmacological model.

The ability of transgenic plants to respond to sudden environmental pollution accidents has become viable. Nonetheless, there is a dearth of research regarding the mechanism by which transgenic plants degrade organic pollutants. Hence, this study aimed to elucidate the process of organic pollutant degradation by plants, offering theoretical support for the application of transgenic plant emergency phytoremediation technology. In this investigation, we developed a 3D-QSAR pharmacophore model to represent the collective impact of plant resistance and phytodegradation. This was achieved by employing integrated effect values following treatment with a sine function approach. Moreover, we have undertaken an inaugural exploration of the coregulatory mechanism involved in plant resistance and pollutant degradation within plants. Additionally, we applied virtual molecular modification techniques for analysis and validation, striving for a more indepth understanding of the molecular-level enhancement mechanism related to the degradation of pollutants within plant organisms. The mechanism analysis results of the Hypo 1 pharmacophore model were verified, indicating that hydrophobic characteristics affect the resistance and degradation of PCBs in plants, significantly affecting the degradation effect of pollutants in plants.

TT2023 meeting report on the 18th Transgenic Technology meeting in Houston, United States.

The 18th Transgenic Technology Meeting, held in Houston, Texas from November 12-15, 2023, was a vibrant international forum. It brought together nearly 400 delegates to discuss advances in transgenic technologies and the science these technologies support. Among them were 329 in-person and 70 remote delegates, representing 26 countries from 5 continents. The event, hosted by the International Society for Transgenic Technologies (ISTT), was set against the backdrop of the Hyatt Regency's panoramic views, reflecting the innovative spirit of the conference. A notable precursor to the main conference was the Allele Design Pre-conference Workshop, which fostered in-depth discussions on state-of-the-art methodologies. The main conference encompassed ten sessions, delving into diverse topics from Precision Animal Models of Human Disease to the use of transgenic animals in Space Biology. Eighty posters provided for a lively exchange of ideas, while the ISTT Prize and other awards highlighted the event's commitment to excellence. Beyond the conference halls, attendees had the opportunity to venture into Houston's Museum District, home to 19 museums in the downtown area, or indulge in unique dining experiences.

Gene therapy for retinal diseases: From genetics to treatment.

The gene therapy approach for retinal disorders has been considered largely over the last decade owing to the favorable outcomes of the US Food and Drug Administration-approved commercial gene therapy, Luxturna. Technological advances in recent years, such as next-generation sequencing, research in molecular pathogenesis of retinal disorders, and precise correlations with their clinical phenotypes, have contributed to the progress of gene therapies for various diseases worldwide, and more recently in India as well. Thus, considerable research is being conducted for the right choice of vectors, transgene engineering, and accessible and cost-effective large-scale vector production. Many retinal disease-specific clinical trials are presently being conducted, thereby necessitating the collation of such information as a ready reference for the scientific and clinical community. In this article, we present an overview of existing gene therapy research, which is derived from an extensive search across PubMed, Google Scholar, and clinicaltrials.gov sources. This contributes to prime the understanding of basic aspects of this cutting-edge technology and information regarding current clinical trials across many different conditions. This information will provide a comprehensive evaluation of therapies in existing use/research for personalized treatment approaches in retinal disorders.

Fibroinase plays a vital role in silk gland degeneration by regulating autophagy and apoptosis in the silkworm, Bombyx mori

The silkworm is an incredibly valuable insect that produces silk through its silk gland. Within this organ, Fibroinase has been identified and named due to its ability to fibroin degradation. The expression of Fibroinase in the silk gland significantly increases during the larval-pupal stage, which might be associated with the degeneration of the silk gland. In this study, Fibroinase was overexpressed and knocked down specifically both in the middle and posterior silk glands, respectively, using transgenic technology. The investigation of silk gland development in these transgenic silkworms showed that Fibroinase plays a direct role in accelerating silk gland degeneration. The staining analyses performed in the silk glands of transgenic silkworms suggest that Fibroinase is involved in the processes of autophagy and apoptosis during silk gland degeneration. Further experiments demonstrated that Fibroinase, acting as a lysosomal regulator, negatively regulates autophagy via the mTOR (mechanistic target of rapamycin) pathway. Moreover, during apoptosis, Fibroinase could activate Caspase3 by increasing the activity of BmCaspase1, ultimately accelerating the apoptosis process. These findings enhance our understanding of the physiological role of Fibroinase in promoting silk gland degeneration, which plays a role in breaking down proteins in the silk gland and coordinating the regulation of autophagy and apoptosis.