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The increased incidence of diabetes, coupled with the introduction of alternative insulin delivery methods that rely on higher doses, is expected to result in a substantial escalation in the future demand for affordable insulin. Plant-based systems offer a safe and economical method for producing pharmaceutical proteins. We used peanut (Arachis hypogaea L.) as bio-reactors to express biosafe, stable proinsulin. We designed two proinsulin analogues (FAIA and LAIA) with substitutions in their amino acid sequences. The fast-acting insulin analogue (FAIA) contains a Gly inserted between Cys19 and Gly20, as well as a Pro28Asp substitution, in the B chain. The long-acting insulin analogue (LAIA) contains a Gly inserted between Cys19 and Gly20 and two Arg residues inserted into the terminus of the B chain, as well as an Asn21Gly substitution in the A chain. Four plasmids were constructed: pROKII-Flag-FAIA, pROKII-Flag-LAIA, pCAMBIA2301-Oleosin-FAIA and pCAMBIA2301-Oleosin-LAIA. These plasmids were transferred into peanut to produce recombinant proinsulin. Western blot and GUS staining analysis indicated that some transgenic peanut successfully expressed exogenous proinsulin. Peanut seeds can act as insulin storage sites, which is the foundation for further production of recombinant proinsulin from peanut seeds.

Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world. However, biotechnological based improvement of peanut is far behind many other crops. It is critical and urgent to establish the biotechnological platform for peanut germplasm innovation. In this study, a peanut seed cDNA library was constructed to platform for peanut germplasm innovation. About 17 000 expressed sequence tags (ESTs) were sequenced and used for further investigation. Among which, 12.5% were annotated as metabolic related and 4.6% encoded transcription or post-transcription factors. ESTs encoding storage protein and enzymes related to protein degradation accounted for 28.8% and formed the largest group of the annotated ESTs. ESTs that encoded stress responsive proteins and pathogen-related proteins accounted for 5.6%. ESTs that encoded unknown proteins or showed no hit in the GenBank nr database accounted for 20.1 % and 13.9%, respectively. A total number of 5066 EST sequences were selected to make a cDNA microarray. Expression analysis revealed that these sequences showed diverse expression patterns in peanut seeds, leaves, stems, roots, flowers, and gynophores. We also analyzed the gene expression pattern during seed development. Genes that were upregulated ([gt]twofold) at 15, 25, 35, and 45 days after pegging (DAP) were found and compared with 70 DAP. The potential value of these genes and their promoters in the peanut gene engineering study is discussed. Key words: peanut (Arachis hypogaea L.), EST sequences. cDNA microarray. expression profiling. L'arachide (Arachis hypogaea L.) est l'une des oleagineuses les plus importantes dans le monde. Malgre cela, la contribution des biotechnologies a Amelioration genetique chez cette espece a ete beaueoup moindre que chez d'autres especes. Il est ainsi critique et urgent d'etablir une plateforme bioiechnologique permettant l'innovation au sein des ressources genetiques de l'arachide. Dans ce travail, une banque d'ADNc de graines de l'arachide a ete produite pour fonder l'assise d'une plateforme bioiechnologique d'innovation chez l'arachide. Environ 17 000 EST ont ete sequences et employes lors d'analyses subsequences. Parmi ceux-ci, 12,5% ont ete annotes comme etant lies au metabolisme et 4,6% des EST codaient pour des facteurs transcriptionals ou post-transcriptionnels. Les EST codant pour des proteines de reserve ou pour des enzymes impliquees dans la degradation des proteines formaient le plus grand groupe et representaient 28.8% des EST annotes. Les EST codant pour des proteines de reponse aux stress ou aux pathogenes constituaient 5,6% du total. Les EST codant pour des proteines inconnues et ceux codant pour des proteines sans homologues au sein de GenBank constituaient respective men t 20,1% et 13,9% du total. Une collection de 5066 sequences d'EST a ete retenue pour fabriquer une puce d'ADNc. Une analyse transcriptomique a revele que ces sequences montraient divers motifs d'expression chez les graines, les feuilles, les liges, les racines, les fleurs et les gynophores de l'arachide. Les auteurs ont egalement analyse l'expression genique au cours du developpement des graines. Des genes qui etaient sur-exprimes (>2 fois) a 15, 25, 35 et 45 jours apres fecondation par rapport au niveau enregistre a 70 jours ont ete identifies. Les auteurs discutent de l'utilite potentielle de ces genes et de leurs promoteurs pour des fins d'ingenierie genetique chez l'arachide. Mots-cles : arachide (Arachis hypogaea L.), sequences d'EST, puce d'ADNc, analyse transcriptomique., Introduction Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world. The cultivated peanut is an allotetraploid species (AABB, 2n = 4x = 40), which [...]

Abstract Background MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in the cell by negatively affecting gene expression at the post-transcriptional level. miRNAs have been shown to control numerous genes involved in various biological and metabolic processes. There have been extensive studies on discovering miRNAs and analyzing their functions in model species, such as Arabidopsis and rice. Increasing investigations have been performed on important agricultural crops including soybean, conifers, and Phaselous vulgaris but no studies have been reported on discovering peanut miRNAs using a cloning strategy. Results In this study, we employed the next generation high through-put Solexa sequencing technology to clone and identify both conserved and species-specific miRNAs in peanuts. Next generation high through-put Solexa sequencing showed that peanuts have a complex small RNA population and the length of small RNAs varied, 24-nt being the predominant length for a majority of the small RNAs. Combining the deep sequencing and bioinformatics, we discovered 14 novel miRNA families as well as 75 conserved miRNAs in peanuts. All 14 novel peanut miRNAs are considered to be species-specific because no homologs have been found in other plant species except ahy-miRn1, which has a homolog in soybean. qRT-PCR analysis demonstrated that both conserved and peanut-specific miRNAs are expressed in peanuts. Conclusions This study led to the discovery of 14 novel and 22 conserved miRNA families from peanut. These results show that regulatory miRNAs exist in agronomically important peanuts and may play an important role in peanut growth, development, and response to environmental stress.

In this study, a full-length cDNA of the acyl-ACP thioesterase, Ah FatA, was cloned from developing seeds of Arachis hypogaea L. by 3'-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50-70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that Ah FatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of Ah FatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli., 1. Introduction In higher plants, fatty acid biosynthesis is catalyzed by the action of a type II fatty acid synthase, located in plastids [1-4]. The reaction includes the condensation of [...]

Chemosensory proteins (CSPs) are small scavenger proteins that are mainly known as transporters of pheromone/odor molecules at the periphery of sensory neurons in the insect antennae and in the producing cells from the moth female pheromone gland. Sequencing cDNAs of RNA encoding CSPs in the antennae, legs, head, pheromone gland and wings from five single individual adult females of the silkworm moth Bombyx mori showed that they differed from genomic sequences by subtle nucleotide replacement (RDD). Both intronless and intronic CSP genes expressed RDDs, although in different rates. Most interestingly, in our study the degree of RDDs in CSP genes were found to be tissue-specific. The proportion of CSP-RDDs was found to be significantly much higher in the pheromone gland. In addition, Western blot analysis of proteins in different tissues showed existence of multiple CSP protein variant chains particularly found in the pheromone gland. Peptide sequencing demonstrated the occurrence of a pleiad of protein variants for most of all BmorCSPs from the pheromone gland. Our findings show that RNA editing is an important feature in the expression of CSPs and that a high variety of RDDs is found to expand drastically thus altering the repertoire of CSP proteins in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Summary: A known arabidopsis cDNA clone, the CRT/DRE binding factor 1 (CBF1), was isolated and introduced into tobacco plants. It has been reported that CBF1 is one member of CBF gene family related to low temperature and enhancing low temperature tolerance of plants. In the present work, the transcripts could be detected in the transgenic lines. The photochemical efficiency of PSII (F v/F m) and the photo-oxidizable P700 in the transgenic lines overexpressing CBF1 were higher than that in the wild type plants during the chilling stress under low irradiance. Similarly, the higher NPQ, higher qf, lower Φ NF, higher activity of SOD, and lower content of MDA were also detected in the transgenic tobacco lines. Additionally, higher expression levels of Nicotiana tabacum copper/zinc superoxide dismutase (Cu/Zn SOD) were also detected in the transgenic lines. These results suggest that CBF1 protein plays an important role in protection of PSII and PSI during the chilling stress under low irradiance. [Copyright &y& Elsevier]

Chemosensory proteins (CSPs) are small scavenger proteins that are mainly known as transporters of pheromone/odor molecules at the periphery of sensory neurons in the insect antennae and in the producing cells from the moth female pheromone gland. Sequencing cDNAs of RNA encoding CSPs in the antennae, legs, head, pheromone gland and wings from five single individual adult females of the silkworm moth Bombyx mori showed that they differed from genomic sequences by subtle nucleotide replacement (RDD). Both intronless and intronic CSP genes expressed RDDs, although in different rates. Most interestingly, in our study the degree of RDDs in CSP genes were found to be tissue-specific. The proportion of CSP-RDDs was found to be significantly much higher in the pheromone gland. In addition, Western blot analysis of proteins in different tissues showed existence of multiple CSP protein variant chains particularly found in the pheromone gland. Peptide sequencing demonstrated the occurrence of a pleiad of protein variants for most of all BmorCSPs from the pheromone gland. Our findings show that RNA editing is an important feature in the expression of CSPs and that a high variety of RDDs is found to expand drastically thus altering the repertoire of CSP proteins in a tissue-specific manner.

Background: MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in the cell by negatively affecting gene expression at the post-transcriptional level. miRNAs have been shown to control numerous genes involved in various biological and metabolic processes. There have been extensive studies on discovering miRNAs and analyzing their functions in model species, such as Arabidopsis and rice. Increasing investigations have been performed on important agricultural crops including soybean, conifers, and Phaselous vulgaris but no studies have been reported on discovering peanut miRNAs using a cloning strategy., Results: In this study, we employed the next generation high through-put Solexa sequencing technology to clone and identify both conserved and species-specific miRNAs in peanuts. Next generation high through-put Solexa sequencing showed that peanuts have a complex small RNA population and the length of small RNAs varied, 24-nt being the predominant length for a majority of the small RNAs. Combining the deep sequencing and bioinformatics, we discovered 14 novel miRNA families as well as 75 conserved miRNAs in peanuts. All 14 novel peanut miRNAs are considered to be species-specific because no homologs have been found in other plant species except ahy-miRn1, which has a homolog in soybean. qRT-PCR analysis demonstrated that both conserved and peanut-specific miRNAs are expressed in peanuts., Conclusions: This study led to the discovery of 14 novel and 22 conserved miRNA families from peanut. These results show that regulatory miRNAs exist in agronomically important peanuts and may play an important role in peanut growth, development, and response to environmental stress.

Abiotic stresses such as cold, drought and high salinity are common adverse environmental conditions that seriously influence plant growth and crop productivity worldwide. Some transcription factors (TFs) have been isolated and verified recently to play roles under abiotic stresses. Among them, the TF of DREB (Dehydration responsive element binding) can therefore regulate the expression of many stress-inducible genes in plants and play a critical role in improving abiotic stress tolerance of plants by interacting with specific cis-acting element named DRE/CRT, which is present in the promoter region of various abiotic stress-related genes. In this review, we summarized the current knowledge of DREBs in the structural and functional characters with emphasis on the regulation and mechanisms of DREBs on plant development, as well as new research approaches and complexity of the signal transduction pathway of DREBs. The practical and application value of DREBs in crop improvement engineering was also discussed.

NAC transcription factors are a family of functionally diverse proteins. They are unique to plants and play an important role in regulation of plant growth and development, hormone regulation and responses to various stresses. A cDNA encoding the NAC-like gene homologue was isolated from maize (Zea mays L.) by RT-PCR and designated ZmNAC1 (GenBank Accession No. EU224278). Sequence analysis showed that cDNA of ZmNAC1 was 1,029 bp long and contained a single open reading frame (ORF, 26 to approximately 907 bp). The predicted ZmNAC1 protein has 293 amino acids with an estimated molecular mass of 32.3 kDa and an isoelectric point of 8.65. RT-PCR analysis showed that the expression of ZmNAC1 was induced by low temperature, PEG, salt, and ABA, respectively. These results suggest that ZmNAC1 may play important roles in biotic and abiotic resistance pathways. This is the first NAC-like gene reported in maize.

Metallothioneins, which widely distribute in eukaryotic and prokaryotic organisms, are a class of cystein-rich and heavy metal-binding proteins with low molecular weights. Many genes encoding plant metallothioneins have been cloned in the past years. There is certain progress on the expression patterns, tissue specificities and structures of these genes such as the loci of promoters and introns in chromosomes, however, their exact functions remain unknown. The expressions of many plant metallothionein genes under environmental stresses suggested that they may function in both metal chaperoning and scavenging of reactive oxygen species with their large number of cysteine residues to protect plants from oxidative damage. The classification, characteristics, gene structure of plant metallothioneins, and recent advances in heavy metal detoxification are summarized in detail.

Alpha-linolenic acid(ALA, C18:3delta9,12,15 ) is an essential fatty acid which has many sanitary functions to human. However, its contents in diets are often not enough. In plants, omega-3 fatty acid desaturases(FAD) catalyze linoleic acid(LA, C18:2delta9,12) into ALA. The seed oil of Glycine max contains high level of ALA. To investigate the functions of Glycine max omega-3FAD, the cDNA of GmFAD3 C was amplified by RT-PCR from immature seeds, then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 throuth LiAc method. The positive clones were screened on the CM(Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 degrees C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in strain Kp4GFAD3C was detected,which was not detectable in control, Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid is ALA. The content of ALA reached to 3.1% of the total fatty acid in recombinant strain, the content of LA correspondingly decreased from 22% to 16.2% by contrast. It was suggested that the protein encoded by GmFAD3 C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at delta15 location. In this study, we expressed a Glycine max omega-3 fatty acid desaturase gene in S. cerevisiae; An efficient and economical yeast expressing system(K601-p416 system) which is suitable for the expression of FAD was built.

Wolbachia are common and maternally inherited bacteria found in reproductive tissue of a wide range of arthropod species. A tremendous amount of progress on their manipulating reproduction of their host has been made over the past 30 years. Recent surveys have found that they could effect the fitness of their hosts. The recent advances on Wolbachia distribution, locality and their effects on the fitness of hosts are reviewed, and the significance and potential implications of the fields are discussed.