Measurement of SQSTM1 by flow cytometry.

Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type.

Autophagy-associated immune dysregulation and hyperplasia in a patient with compound heterozygous mutations in ATG9A.

ABBREVIATIONS: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

Protocols for the Analysis of microRNA Expression, Biogenesis, and Function in Immune Cells.

MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both in vitro and in vivo. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley & Sons, Inc.

The Pat1-Lsm complex prevents 3' to 5' degradation of a specific subset of ATG mRNAs during nitrogen starvation-induced autophagy.

Deregulation of macroautophagy/autophagy, a conserved catabolic recycling pathway, has been implicated in the onset and development of several diseases. While post-translational regulation of auto-phagy-related (Atg) proteins has been an important research focus leading to significant breakthroughs in understanding autophagy regulation, less is known about the post-transcriptional regulation of ATG transcripts. In a recent study we showed that, during nitrogen starvation, the RNA-binding complex Pat1-Lsm is involved in binding and preventing the 3' to 5' exosome-mediated degradation of a specific subset of ATG mRNAs. Dephosphorylation of Pat1 at residues S456 and S457 facilitates ATG mRNA binding, resulting in ATG mRNA accumulation, Atg protein synthesis and robust autophagy induction. In addition, we present evidence that these processes are conserved in human cells. These results further elucidate our understanding of the post-transcriptional mechanism necessary for efficient induction of autophagy during stress conditions.

The Pat1-Lsm Complex Stabilizes ATG mRNA during Nitrogen Starvation-Induced Autophagy.

Macroautophagy/autophagy is a key catabolic recycling pathway that requires fine-tuned regulation to prevent pathologies and preserve homeostasis. Here, we report a new post-transcriptional pathway regulating autophagy involving the Pat1-Lsm (Lsm1 to Lsm7) mRNA-binding complex. Under nitrogen-starvation conditions, Pat1-Lsm binds a specific subset of autophagy-related (ATG) transcripts and prevents their 3' to 5' degradation by the exosome complex, leading to ATG mRNA stabilization and accumulation. This process is regulated through Pat1 dephosphorylation, is necessary for the efficient expression of specific Atg proteins, and is required for robust autophagy induction during nitrogen starvation. To the best of our knowledge, this work presents the first example of ATG transcript regulation via 3' binding factors and exosomal degradation.

Transcriptional Profiling of Patient Isolates Identifies a Novel TOR/Starvation Regulatory Pathway in Cryptococcal Virulence.

Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.IMPORTANCECryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus.

Susceptibility to Cryptococcal Meningoencephalitis Associated With Idiopathic CD4+ Lymphopenia and Secondary Germline or Acquired Defects.

Idiopathic CD4+ lymphopenia (ICL) predisposes to opportunistic infections (OIs) but can often remain asymptomatic and does not have a strong association with monogenic mutations. Likewise, cryptococcal meningoencephalitis, the most common OI in ICL, is not strongly associated with monogenic mutations. In this study, we describe 2 patients with ICL plus an additional immune defect: one from an E57K genetic mutation in the nuclear factor-κß essential modulator, and the other with acquired autoantibodies to granulocyte-macrophage colony-stimulating factor. Thus, these cases may exemplify a "multi-hit model" in patients with ICL who acquire OIs.

Tor-dependent post-transcriptional regulation of autophagy: Implications for cancer therapeutics.

Paradoxically, both anticancer immunosurveillance and tumor progression have been associated with intact autophagy, which is regulated by the target of rapamycin (Tor1). Here, we describe the potential impact on the design of cancer therapeutics of a newly described highly conserved post-transcriptional mechanism whereby Tor regulates autophagy.

TOR-dependent post-transcriptional regulation of autophagy.

Regulation of autophagy is required to maintain cellular equilibrium and prevent disease. While extensive study of post-translational mechanisms has yielded important insights into autophagy induction, less is known about post-transcriptional mechanisms that could potentiate homeostatic control. In our study, we showed that the RNA-binding protein, Dhh1 in Saccharomyces cerevisiae and Vad1 in the pathogenic yeast Cryptococcus neoformans is involved in recruitment and degradation of key autophagy mRNAs. In addition, phosphorylation of the decapping protein Dcp2 by the target of rapamycin (TOR), facilitates decapping and degradation of autophagy-related mRNAs, resulting in repression of autophagy under nutrient-replete conditions. The post-transcriptional regulatory process is conserved in both mouse and human cells and plays a role in autophagy-related modulation of the inflammasome product IL1B. These results were then applied to provide mechanistic insight into autoimmunity of a patient with a PIK3CD/p110δ gain-of-function mutation. These results thus identify an important new post-transcriptional mechanism of autophagy regulation that is highly conserved between yeast and mammals.

A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy.

Autophagy is an essential eukaryotic pathway requiring tight regulation to maintain homeostasis and preclude disease. Using yeast and mammalian cells, we report a conserved mechanism of autophagy regulation by RNA helicase RCK family members in association with the decapping enzyme Dcp2. Under nutrient-replete conditions, Dcp2 undergoes TOR-dependent phosphorylation and associates with RCK members to form a complex with autophagy-related (ATG) mRNA transcripts, leading to decapping, degradation and autophagy suppression. Simultaneous with the induction of ATG mRNA synthesis, starvation reverses the process, facilitating ATG mRNA accumulation and autophagy induction. This conserved post-transcriptional mechanism modulates fungal virulence and the mammalian inflammasome, the latter providing mechanistic insight into autoimmunity reported in a patient with a PIK3CD/p110δ gain-of-function mutation. We propose a dynamic model wherein RCK family members, in conjunction with Dcp2, function in controlling ATG mRNA stability to govern autophagy, which in turn modulates vital cellular processes affecting inflammation and microbial pathogenesis.

The role of transcriptional 'futile cycles' in autophagy and microbial pathogenesis.

Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, whereby the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional 'futile cycle' where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novo synthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformans results in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a key role in the facilitation of microbial pathogenesis.

Microevolution during serial mouse passage demonstrates FRE3 as a virulence adaptation gene in Cryptococcus neoformans.

Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a "virulence acquisition gene" during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts. IMPORTANCE Cryptococcus neoformans is a significant global fungal pathogen that also resides in the environment. Recent studies have suggested that the organism may undergo microevolution in the host. However, little is known about the permitted genetic changes facilitating the adaptation of environmental strains to mammalian hosts. The present studies subjected environmental strains isolated from several metropolitan areas of the United States to serial passages in mice. Transcriptome sequencing expression studies identified the increased expression of an iron reductase gene, FRE3, in two strains that adapted in mice to become highly virulent, and overexpression of FRE3 recapitulated the increased virulence after mouse passage. Iron reductase in yeast is important to iron uptake in a large number of microbial pathogens. These studies demonstrate the capacity of C. neoformans to show reproducible changes in the expression levels of small numbers of genes termed "virulence adaptation genes" to effectively increase pathogenicity during the environment-to-mammal transition.

Overexpression of TUF1 restores respiratory growth and fluconazole sensitivity to a Cryptococcus neoformans vad1Delta mutant.

The yeast-like fungus Cryptococcus neoformans favours respiration as a mechanism of energy production, and thus depends heavily on mitochondrial function. Previous studies of a C. neoformans vad1Delta mutant revealed reduced expression of the mitochondrial elongation factor TUF1 and defects in glycerol utilization, consistent with mitochondrial dysfunction. In this study, we found that in trans expression of TUF1 in the vad1Delta mutant suppressed the mitochondrial defects, including growth on respiration-dependent carbon sources and fluconazole resistance, associated with VAD1 deletion. Tetracycline, an inhibitor of mitochondrial translation, was found to confer resistance to fluconazole in the wild-type and vad1Delta mutant, whereas the fluconazole susceptibility of the TUF1-overexpressing strain was unaffected by tetracycline treatment. In the presence of fluconazole, the vad1Delta mutant exhibited increased activation of the global transcriptional regulator Sre1. TUF1 overexpression failed to alter cleavage of Sre1 in response to fluconazole in the vad1Delta mutant, suggesting that TUF1 repression in the vad1Delta mutant is distal to Sre1, or that it occurs through an independent pathway.

PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans.

Autophagy is a process by which cells recycle cytoplasm and defective organelles during stress situations such as nutrient starvation. It can also be used by host cells as an immune defense mechanism to eliminate infectious pathogens. Here we describe the use of autophagy as a survival mechanism and virulence-associated trait by the human fungal pathogen Cryptococcus neoformans. We report that a mutant form of C. neoformans lacking the Vps34 PI3K (vps34Delta), which is known to be involved in autophagy in ascomycete yeast, was defective in the formation of autophagy-related 8-labeled (Atg8-labeled) vesicles and showed a dramatic attenuation in virulence in mouse models of infection. In addition, autophagic vesicles were observed in WT but not vps34Delta cells after phagocytosis by a murine macrophage cell line, and Atg8 expression was exhibited in WT C. neoformans during human infection of brain. To dissect the contribution of defective autophagy in vps34Delta C. neoformans during pathogenesis, a strain of C. neoformans in which Atg8 expression was knocked down by RNA interference was constructed and these fungi also demonstrated markedly attenuated virulence in a mouse model of infection. These results demonstrated PI3K signaling and autophagy as a virulence-associated trait and survival mechanism during infection with a fungal pathogen. Moreover, the data show that molecular dissection of such pathogen stress-response pathways may identify new approaches for chemotherapeutic interventions.

Analysis of autophagy during infections of Cryptococcus neoformans.

Cryptococcus neoformans is a yeastlike fungus that causes a lethal meningoencephalitis in a broad spectrum of immunocompromised patients and has become the most common cause of meningitis due to AIDS-related infections in Africa. Key to the development of new agents to control and prevent this infection is the identification of cellular mechanisms required for pathogenesis. Survival of the fungus within the hostile and nutrient-deprived environments of the host has recently been shown to depend on the induction of autophagy, whereby the cell recycles nutrients by slowly digesting itself in a regulated fashion. Further study of the role of autophagy during infection by C. neoformans requires the use of markers of autophagy that are specially adapted to the fungus within the mammalian host.

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans.

The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Delta strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice.

Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1Delta cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.

The Hsp70 member, Ssa1, acts as a DNA-binding transcriptional co-activator of laccase in Cryptococcus neoformans.

Hsp70 proteins are a well-known class of chaperones that have also been described to have roles in cellular regulation. Here, we show that a Cryptococcus neoformans Hsp70 homologue Ssa1 acts as a DNA-binding transcriptional co-activator of the fungal virulence factor, laccase, via binding to a GC-rich element within the 5'-UAS in response to glucose starvation, iron, copper, calcium and temperature. In addition, Ssa1 forms a regulatory complex with heat shock transcription factor and TATA-binding protein during laccase induction. Furthermore, deletion of Ssa1 results in reduced laccase and attenuated virulence using a mouse model. These results indicate that Hsp70 functions as a stress-related transcriptional co-activator required for fungal virulence.

Role of a VPS41 homologue in starvation response, intracellular survival and virulence of Cryptococcus neoformans.

Previous studies have demonstrated an important role for the vacuole in the virulence of the fungus Cryptococcus and studies in yeast have implicated the vacuolar protein Vps41 in copper loading of proteins such as iron transporters. However, our studies found that a cryptococcal vps41Delta strain displayed wild-type growth on media containing iron and copper chelators and normal activity of the copper-containing virulence factor laccase as well as almost normal growth at 37 degrees C and wild-type production of the virulence factor capsule. Despite these attributes, the vps41Delta mutant strain showed a dramatic attenuation of virulence in mice and co-incubation of mutant cells with the macrophage cell line, J774.16, resulted in a dramatic loss in viability of the vps41Delta mutant strain at 10 h compared with wild-type and complemented strains. Closer examination revealed that the vps41Delta mutant displayed a dramatic loss in viability after nutrient starvation which was traced to a failure to undergo G2 arrest, but there was no defect in the formation of autophagic or proteolytic vesicles. Our results indicate that VPS41 plays a key role in regulating starvation response in this pathogenic organism and that defects in cell cycle arrest are associated with attenuated pathogenic fitness in mammalian hosts.

[Heteologous expression of Mortierella isabellina delta6 -fatty acid desaturase gene in soybean].

Gamma-linolenic acid (GLA, C18:3delta(6, 9, 12) is one of nutritionally important polyunsaturated fatty acids in human and animal diets. Vast majority oilseeds crops do not produce GLA. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme, which catalyzes the conversion of linoleic acid (LA) and alpha-linolenic acid (ALA) to GLA and octadecatetraenoic acid (OTA). In this study, a delta6-fatty acid desaturase gene isolated from Mortierella isabellina was transformed into some soybean cultivars Jilin 35, Jilin 47, Suinong 10, Suinong 14 and Heinong 37 successfully by agrobacterium-mediated cotylendon node transformation system. The results of PCR analysis, Southern blotting and Northern blotting with transgenic plants indicated that target gene was integrated into transgenic soybean genome and expressed at the level of RNA. Meanwhile, total fatty acids were extracted from transgenic seeds and methyl-esterified. Analysis of gas chromatograph (GC) showed that a novel peak corresponding to the standard of GLA methyl ester was detected, the highest percentage of gamma-lenolenic acid to total fatty acids in transgenic seeds was 27. 067%, which indicated that Mortierella isabellina delta6-fatty acid desaturase gene was expressed in transgenic soybean. It is so far the first report on the expression of delta6-fatty acid desaturase gene in transgenic soybean in the world.