Convergent Regulation of Candida albicans Aft2 and Czf1 in Invasive and Opaque Filamentation.

Candida albicans is the most common fungal pathogen of mucosal infections and invasive diseases in immuno-compromised humans. The abilities of yeast-hyphal growth and white-opaque switching affect C. albicans physiology and virulence. Here, we showed that C. albicans Aft2 regulator was required for embedded filamentous growth and opaque cell-type formation. Under low-temperature matrix embedded conditions, Aft2 functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Moreover, deletion of AFT2 significantly reduced opaque cell-type formation under N-acetylglucosamine (GlcNAc) inducing conditions. Ectopic expression of CZF1 slightly increased the white-opaque switching frequency in the aft2Δ/Δ mutant, but did not completely restore to wild-type levels, suggesting that Czf1 at least partially bypassed the essential requirement for Aft2 in response to opaque-inducing cues. In addition, multiple environmental cues altered AFT2 mRNA and protein levels, such as low temperature, physical environment and GlcNAc. Although the absence of Czf1 or Efg1 also increased the expression level of AFT2 gene, deletion of CZF1 remarkably reduced the stability of Aft2 protein. Furthermore, C. albicans Aft2 physically interacted with Czf1 under all tested conditions, whereas the interaction between Aft2 and Efg1 was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate filamentous growth by antagonizing Efg1-mediated repression under matrix-embedded conditions.

Transcriptional regulation of desaturase genes in Pichia pastoris GS115.

Here we investigated the regulation of Pichia pastoris desaturase genes by low temperature and exogenous fatty acids in the late-exponential phase at the transcriptional level. Time-course studies of gene expression showed that mRNA levels of four desaturase genes were rapidly and transiently enhanced by low temperature and suppressed by exogenous oleic acid. Stearic acid showed no obvious repression of mRNA levels of Fad12 and Fad15 and a slight increase in Fad9A and Fad9B mRNA levels. Using a promoter-reporter gene construct, we demonstrated that the pFAD15 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by unsaturated fatty acids. Also, there was no absolute correlation between mRNA abundance and production of corresponding fatty acids. Disruption of Spt23 resulted in a decrease in transcript levels of Fad9A and Fad9B, but had little effect on the other desaturase genes. Consistent with these observations, a decrease in the relative amount of oleic acid (OLA) and an increase in the relative content of linoleic acid and ALA with different degrees were clearly observed in the stationary phase cells of ΔSpt23 mutant. Further analysis showed that the effect of low-temperature activation and OLA inhibition on expression of Fad9A and Fad9B seemed to disappear after disruption of the Spt23 gene, which indicated that Spt23p is essential for the expression of two Δ9-desaturase genes internally and probably involved in the regulation of Δ9-desaturase genes transcription in response to external stimuli, and thereby plays a role in the synthesis of OLA.

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences.

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ∆(12)-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ∆(15)-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ∆(9)-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.

Effects of different carbon sources on the growth, fatty acids production, and expression of three desaturase genes of Mortierella alpina ATCC 16266.

On the molecular and biochemical levels, the effects of different carbon sources on biomass production, fatty acid biosynthesis, and gene expression of three desaturases were investigated in Mortierella alpina ATCC 16266, at a stationary phase, which is an important filamentous fungus capable of producing various polyunsaturated fatty acids (PUFAs). The maximum mycelial biomass was achieved using sucrose as carbon source. However, the highest productivity of total lipids was shown to be no biomass associated. In addition, glucose was the preferred carbon source for the expression of three desaturase genes compared to others, but the change at the corresponding fatty acid product's level of these desaturase genes was not in accordance with the change measured at the mRNA level among those carbon sources that we utilized. Significant discrepancies between the mRNA expression and the product abundance may indicate post-transcriptional regulatory mechanisms of these desaturases.

Candida albicans ferric reductase FRP1 is regulated by direct interaction with Rim101p transcription factor.

The opportunistic pathogen Candida albicans develops different systems to acquire essential nutrients such as iron. The Rim101 pathway controls gene expression to adapt to different environments through transcription factor Rim101p. Ferric reductase genes are important for iron acquisition, but further work should be carried out to show about how ferric reductase genes are regulated. In this study we demonstrate that ferric reductase FRP1 expression is upregulated by alkaline pH and iron-limited conditions. Moreover, promoter deletion identified that the upregulated elements lie in -822 to 437 and 263 to -124 and the downregulated elements in -437 to -263 using a beta-galactosidase reporter system. Using electrophoretic mobility shift assay we also found that Rim101p can bind directly with two putative 5'-CCAAGAA-3' sites at the -157 and -629 sites. Interestingly, site-specific mutations show that only the -629 site, not the -157 site, plays an important role in FRP1 expression under iron-limited conditions.

[Characterization of pcr2 gene of Pseudomonas aeruginosa].

Type III secretion system (TTSS) is an important virulence factor encoding by pseudomonas aeruginosa. About 40 genes are involved and they function as structure proteins, chaperons, regulators, and effectors proteins, respectively. Although some genes have been studied previously, functions of many genes remained unknown. Pcr2 gene is the third gene of popN operon that is one of the five operons of the TTSS gene clusters. Its functions were investigated in this study. First, by characterization of the phenotypes of pcr(2-) mutant, we found that the abilities of secreting or translocating effectors proteins were significantly damaged in the absence of Pcr2 protein, suggesting that Pcr2 protein involved in both the secretion and translocation processes of TSS. Second, evidences were provided that no PopN protein was detectable in supernatant of pcr(2-) mutant culture. Combined with the data from the bacterial two-hybrid system, we can conclude that Pcr2 protein might function as part of a chaperone complex for the PopN protein. Third, Pcr2 protein was found secreted in a TTSS-dependent manner, suggesting that secreted Pcr2 may play a role in the TTSS needle biogenesis.

[Type III secretion study of popN- mutant of Pseudomonas aeruginosa and proteases degradation].

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.

[Protoplast transformation of Mortierella isabellina with hygromycin B resistance plasmid PD4].

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.

[The response to environmental pH of RIM101 pathway in Candida albicans].

Abstract: Candida albicans is the most common kind of human opportunistic pathogen and gets many attentions in recent years. A lot of research has been done. The sites this organism colonizes differ in pH. C. albicans must be able to response to the environmental changes for its survival and pathogenicity. Extracellular environment, especially pH changes,is critical for C. albicans morphological differentiation and virulence. RIM101 pathway is a conserved fungal signal transduction pathway and at least partly controls the cellular pH response through the activity of the zinc finger transcription factor Rim101p. This review concisely summarized recent research on RIM101 pathway, pH response and their relationship in C. albicans.

[A method using long primers for cloning the upstream sequence of delta-6 fatty acid desaturases gene of Thamnidium elegans by nested inverse PCR].

Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-fatty acid desaturase gene containing several putative regulatory elements including TATA. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.

Cloning and molecular characterization of delta12-fatty acid desaturase gene from Mortierella isabellina.

AIM: To clone delta(12)-fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo. METHODS: Reverse transcriptional polymerase chain reaction (RT-PCR) was used to clone the open reading frame of delta(12)-fatty acid desaturase gene (D12D) of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli (E.coli) strain BL21 using CaCl(2) method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain INVSc1. RESULTS: Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E.coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of delta(12)-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active delta(12)-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo. CONCLUSION: Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.

[Cloning and expression of delta6-desaturase gene from Thamnidium elegans in Saccharomyces cerevisiae].

A 459 bp DNA fragment was amplified from Thamnidium elegans As3.2806 with degenerate oligonucleotides primers designed based on the sequences information from the conserved histidine-rich motifs II and near III of fungal delta6-fatty acid desaturases genes by RT-PCR and sequenced. Gene specific primers designed according to this partial sequence were used for the amplification of the 3'- and 5'- end of this cDNA by RACE method, and sequence information coming from these two ends were used to design two GSPs to clone the 1504bp full-length cDNA. Sequence analysis showed this cDNA sequence had an open reading frame (ORF) of 1377bp encoding 458 amino acids of 52.4kD. The deduced amino acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprise the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA TED6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYTED6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees C. Total fatty acids were extracted from the induced cell and subjected to methyl-esterification. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 7.5%. Gas chromatography-mass spectrometry (GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the coding product of this sequence exhibited the activity of converting linoleic acid (LA) to gamma-linolenic (GLA), and indicated that amino-acid sequence exhibited delta6-fatty acid desaturase activity.

[Heteroexpression of Rhizopus arrhizus delta6-fatty acid desaturase gene in Pichia pastoris].

Delta6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a delta6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-fatty acid desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate fatty acid substrates including C16:1, C17:1, C18:1, linoleic acid and alpha-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this delta6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-fatty acid desaturase gene.

[Cloning and heterologous expression of a novel delta6 -desaturase gene from Rhizopus arrhizus NK030037].

A 593 bp DNA fragment was amplified from Rhizopus arrhizus NK030037 with degenerate oligonucleotide primers designed based on the sequences information for fungi delta6-fatty acid desaturase genes by RT-PCR and sequenced. Gene specific primers derived from this partial sequence were used for the amplification of the 3'- and 5'-ends of this cDNA by RACE method, and this lead to a full-length cDNA sequence of 1 482 bp was amplified. Sequence analysis showed this cDNA sequence had an open reading frame(ORF) of 1 377 bp coding 458 amino acids of 52 kD. The deduced amino-acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. The full-length cDNA sequence is a putative novel delta6-fatty acid desaturase gene. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA RAD6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L. Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters(FAME) were analyzed by gas chromatography(GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 3.85%. Gas chromatography-mass spectrometry( GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the transgenic product exhibited delta6-fatty acid desaurase activity, converting LA to GLA specifically.

[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.

[Progress on molecular biology of delta6-fatty acid desaturases].

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.

[Expression of delta6-fatty acid desaturase gene from Mortierella alpina in Pichia pastoris].

Gamma-linolenic acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.

[Expression of Mortierella isabellina delta6-fatty acid desaturase gene in gamma-linolenic acid production in transgenic tobacco].

Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta6.9.12.15) respectively have 19.7% and 3.5% of the total fatty acids in the transgenic plant. The presence of both GLA and OTA indicates that the delta6-fatty acid desaturase used both linoleic acid and a-linolenic acid (ALA, C18:3delta6.9.12.15) as substrates, and this may be responsible for the decrease in ALA observed in the transgenic line. That was the first report about the expression of M. isabellina delta6-fatty acid desaturase gene in tobacco. All results mentioned above have laid the foundation of the thorough studying on an breeding transgenic oilseeds containing GLA to change the fatty acid composition of conventional oilseeds, it is significant to study on regulation mechanism of fatty acid desaturase.