مجله بیوتکنولوژی کشاورزی
سال چهارم شماره 1 (پیاپی 7، بهار و تابستان 1391)

Periwinkle (Catharanthus roseus) has been considered as an important medicinal plant because it contains many alkaloids such as vincristine and vinblastine. In vitro culture of periwinkle provides new tissue sources such as callus, cell suspension and seedlings to produce secondary metabolites. As for the mass production of callus and cell suspension in order to optimize the extraction of secondary metabolites have been few studies, the present study describes two callus production optimization procedures and one cell suspension experiment. The first trail was a factorial experiment with three explants (roots, shoots and leaves) on Murashige and Skoog (MS) medium supplemented with different concentrations of BAP and 2,4-D. The second trial was a factorial experiment with six explants (roots, shoots, in-vitro grown leaves, ex-vitro grown leaves, seeds and nodes) and ten different hormonal combinations. The cell suspension experiment was conducted with six hormonal and one control (without hormone) treatments based on completely randomized design. Comparison of means showed that the maximum callus production was obtained from leaf explants, the root and shoot explants were in the second orders. In overall, 0.5 mg /l BAP + 1 mg/l 2,4-D and 0.5 mg /l KN + 1 mg/l 2,4-D concentrations proved to be optimal for the production of maximum callus. The best combination for suspension culture was 1 mg/l 2,4-D and/or 2 mg/l 2,4-D+1 mg /l BAP according to the dried cell weight.

Phytase has a hydrolysis function on phytic acid which yields inorganic phosphate. Using phytase enzymes in domestic animals have a positive effect on optimal diet intake. Bacillus species can only produce thermostable alkaline phytase. Accordingly extracellular phytase gene was isolated from Bacillus subtilis ATCC 12711 (phyc) using linker primers containing restriction sites of BamHI and HindIII. The isolated fragment was then inserted into pTZ57R/T vector for cloning and nucleotide characterization. Molecular weight of phytase protein was estimated about 42 kDa. On the other hand the model of phytase secondary structure showed the potential of thermostable characterization of enzyme.

Thioredoxins (Trxs) are ubiquitous, low-molecular-mass proteins with two cysteins in their active site WC(G/P)PC which are involved in reversible reduction of disulfide bonds. Plants contain several forms of Trxs. Thioredoxin f, m, x and y are found in the chloroplast, Trxo is localized in mitochondria and Trxh is typically cytosolic. Trxh isoforms play important roles in plants development. In this study, we describe isolation and cloning of three cDNAs encoding different Trx h isoforms, stated OsTrx1, OsTrx20 and OsTrx23. Three Trx h were heterologously expressed in Rosetta (DE3) - a strain of Escherichia coli. After inducing T7 promoter with IPTG, proper amount of recombinant proteins were produced. The recombinant proteins were purified by affinity chromatography. The recombinant Trxh isoforms were analyzed for their ability to reduce Insulin – an electron receptor substrate for Trxs- and DTT, as a primary reducing agent. All three isoforms were active, but the rate of reduction varied among different reactions. The existence of observed variations can reveal different functions for thioredoxins in plant system.

Silymarin is a flavonoid compound derived from milk thistle plant (Silybum marianum) seeds comprising several pharmacological applications. chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids, thereby identification of CHS gene in milk thistle plant can be of great importance. Seeking that, the available sequences of CHS genes from different plants collected, aligned and the conserved regions detected. Then, 4 degenerate primers designed based on the CHS consensus sequences. The fragments of CHS genes from Borazjan's native genotype and Majar's modified genotype were amplified by polymerase chain reaction and then, cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences lead to identification of two different members of CHS gene family from Silybum marianum. 6 specific primers were designed based on the diverged regions of each member for amplification of related cDNA from majar's total RNA in the RACE system. Amplified fragment of cDNA in 3'RACE and 5'RACE were cloned and sequenced. Full length cDNA was identified by overlapping the 3'RACE and 5'RACE sequences of member 1 whose open reading frame contains 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp) encoding 63 and 349 amino acid residues, respectively. Altogether, analysis of the resulting nucleotide and deduced amino acid sequences lead to identification of three different members of chalcone synthase from Silybum marianum containing the conserved chalcone synthase C-terminal and N-terminal domains.

Tomato aspermy virus (TAV) has a wide host range and usually infects ornamental plants. To identify and characterize molecular and biological properties of the virus, 436 samples were collected from various provinces of Iran. TAV was detected in three samples of ornamental plants through double antibody sandwich ELISA test using specific polyclonal antibody. The 687 bp segment including the coat protein (CP) gene was amplified by RTPCR, cloned and sequenced. Nucleotide sequences of the CP gene of Iranian TAV isolates were compared with available GenBank isolates. Phylogenetic analysis showed that TAV isolates were classified into two groups I and II, and the group II was divided in two subgroups IIA and IIB. All the Iranian TAV isolates were classified in subgroup IIA and shared the high nucleotide and amino acid sequence identities (more than 99%). The results of this study also showed a wide host range of the Iranian TAV isolates among test plants. This study is the first report of TAV occurrence in Iran and the first natural occurrence of TAV on Petunia hybrida in the world.

The purpose of this study was to identifying the M. avium paratuberculosis (MAP), investigating its outbreaks and optimizing a fast, accurate and simple method based on PCR and RFLP for detection of this subspecies and their strains. This pathogen cause a chronic infection called Johne's disease. Johne's disease a ruminant chronic infection and economically, is one of the most important disease in livestock industry. 243 fecal samples and 56 raw milk samples of suspected cattle, from some farms of Mashhad were gathered. After DNA extraction, in order to identifying contaminated samples, a specific MAP insertion segment called IS900, with length of 413bp was amplified with PCR using P90 and P91 primers. Then, in order to determining the cow and sheep strains of MAP, 268bp fragment of insertion segment IS1311 was amplified with PCR using M56 and M94 primers. The resulting fragments were digested with HinfI restriction enzyme. 107 of 243 stool samples (44%) and 10 of 56 raw milk samples (18%) were identified as infected with MAP. Among 56 Milk and feces samples taken from the same animal, 19 stools and 10 milk samples were identified as infected (18% vs. 34%). Results of enzymatic digestion also showed that all detected MAP were from bovine isolates.

Inbreeding and Heat stress have negative effects on reproductive performance of dairy cattle. Two suggested strategies to overcome to these factors are to use crossbreeding and crossbreeding with thermo tolerant genotype, respectively. The aims of present study were to determine the effects of inbreeding and crossbreeding on cleavage and blastocyst rates at early stages of in vitro development in inbreed (Holstein) and crossbreds (Sistani-Holstein) embryos during normal and heat shock stress. In vitro-matured (IVM) Holstein oocytes (n=623) were inseminated with either Holstein or Sistani spermatozoa, and after incubation period, presumptive embryos were collected and assigned to control (38.5°C) or heat shock at 96 h post insemination (hpi; 41°C for 12 h) treatments. The presumptive embryos were cultured in vitro for 8 days and evaluated for cleavage and blastocyst formation rates per cleaved embryo. Data were analyzed using logistic regression. The results indicated a cleavage at 48 hpi was influenced by the species of the sperm (embryo) genotypes, where percentage of Holstein oocytes cleaving after insemination with Holstein spermatozoa were significantly higher than Holstein oocytes inseminated with Sistani spermatozoa (207/314; 66% vs. 130/309; 42%), (P≤0.05); respectively. moreover, the overall blastocyst production rate during heat shock stress was clearly affected by the crossbreeding, with Holstein oocytes were inseminated with Sistani spermatozoa having a greater blastocyst yield in comparison with Holstein inbreed embryos during heat shock stress (4/49; 8.1% vs. 1/84; 1.1%), (P≤0.05) respectively. In conclusion, the present results indicate that Sistani-Holstein crossbreed embryos are more resistant to heat shock than purebred Holstein at early stages of in vitro development. Moreover, according to this criterion, crossbreeding with Sistani spermatozoa may result in higher pregnancy rates in Holstein cows during heat stress condition.