JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2018 | Volume:10 | Issue:1 |Papers Count:9

Human basic Fibroblastic Growth Factor (hbFGF) is a valuable protein with a wide variety of clinical and research applications. Currently, recombinant production of this growth factor in conventional platforms of microbial and insect cells is accompanied by several problems causing high cost production that limited its applications. Rice cell suspension culture is an efficient, safe and low cost platform for recombinant protein production. In this study we prepared a suitable construct for high level expression of an optimized version of bFGF coding sequence under control of ramy3D gene expression sequences in rice cell suspension culture. The codon usage and GC content of bFGF coding sequence was optimized for expression in rice and then synthesized. Following optimization, the Codon Adaptation Index and GC content were upgraded from 0. 76 to 0. 82 and from 52. 65% to 55. 45%, respectively. The ramy3D regulating sequences were amplified from genomic DNA in two separate fragments and then cloned. The ramy3D regulating fragments were assembled in the binary vector pCAMBIA1304. Then the bFGF optimized CDS was inserted in the construct between signal peptide CDS and 3′ UTR. Ramy3D expression system has several advantages including high level expression of transgene, inducibility and simple protein purification through protein secretion into the medium.

Today, demand for production of recombinant proteins in different tissues of transgenic plants is increasing day by day. Meanwhile, hairy roots are under consideration due to their high levels of biomass and high genetic stability as appropriate target tissue. Among pharmaceutical proteins, regulatory glycoproteins, are proposed as one of the most important growth factors with therapeutic and research applications in tissue engineering. Selection of proper host species, plant tissue and desirable gene, are of the most important factors to elevate the levels of recombinant protein production. In order to achieve this goal, in the present study, production of recombinant protein BMP2 in transgenic hairy roots of tobacco and canola were investigated. For optimal transgene expression, TMV (5’-UTR) and KOZAK sequences were inserted upstream the BMP2 gene to increase expression level, furthermore, six-histidine sequence for protein purification and KDEL sequence as ER retention signal protein were inserted downstream of the target gene. The transgene was designed to be under the control of CaMV 35S promoter. One-month old tobacco leaf explants and six-day-old cotyledons of canola were transformed with Agrobacterium rhizogenes carrying the construct. Transformed hairy roots that grew well in the medium were confirmed by gPCR analysis for transgene integration. Moreover, to verify mRNA transcription as well as protein production, RT-PCR, ELISA and western blot analyses were performed. The results of this study demonstrated the efficiency of the current systems for the production of rhBMP2 is tobacco hairy roots suggesting them as an efficient bioreactor.

Citrus bacterial canker is a disease caused by Xanthomonas citri subsp. citri (Xcc). The bacterial type III secretion system (TTSS) consist of an extracellular filamentous structure mediating transfer of bacterial proteins into the plant cytosols. The main part of the pilus is pilin protein (HrpE) that is encoded by the HrpE gene. Production of specific antibodies against the pilin protein can be implemented for detection of infected plants as well as to develop a source of genetic resistance against disease. Inhibition of HrpE protein through antibody therapy leads to suppression of disease in the plant. The objective of the present study was to isolate, clone and express the HrpE gene. To do this, the HrpE gene was amplified by PCR using gene-specific primers and cloned to the pTZ57R/T vector. Then, it was cloned to the pET28a(+) bacterial expression vector and expressed in the E. coli strain Rosetta as host. The protein production procedure was optimized for incubation time and temperature as well as the concentration of inducer. The greatest amount of the recombinant protein was yielded at 1 mM IPTG at 30° C for 16 h. Purification of HrpE recombinant protein was done via metal affinity chromatography. The results of both SDS-PAGE and Western blotting assays confirmed the expression accuracy and purity of recombinant pilin protein. The recombinant protein can be used as an antigen to develop monoclonal and polyclonal antibodies.

The Septoria tritici blotch disease (STB) caused by Zymoseptoria tritici is one of the most important wheat diseases in the world as well as Iran. Genetic resistance is one of the most efficient and economical strategies to control this disease. Identification of resistance sources in wheat genotypes is necessary to control STB. In this study, 33 spring bread wheat genotypes were evaluated against five Z. tritici isolates at seedling stage. Of 33 genotypes, 10 genotypes showed resistance to one or more isolates., Nogal, Arta and N-92-9 genotypes were highly resistant to all isolates tested that show these genotypes possess known or novel effective resistance genes that can be used as resistance sources to the STB in wheat breeding programs. Seven genotypes were moderately resistant against one or more of isolates. The other wheat genotypes were susceptible to isolates tested. The five isolates studied in this study had varying degrees of virulence and invasion on wheat genotypes, which indicates difference in avirulence genes of this pathogen. The isolate BK94 was the most virulent and invasive isolate on wheat genotypes, and the isolate BK56 had the least virulence and invasion.

In order to mapping main and epistatic qtl and their interaction with environment for biological yield using a RILs population of wheat, comprising 148 recombinant inbred lines derived from a cross between two winter wheat cultivars, ‘ YecoraRojo’ and ‘ No. 49’ , was evaluated in two locations in Iran (Miandoab and Mahabad) during 2014-2016. A linkage map including 177 microsatellite and 51 retrotransposon markers was used in this study. Quantitative trait loci (QTL) were determined using QTL Cartographer 2. 5 and QTL Network 2. 0 software based on the CIM and mixed-linear method. In the present study, the estimated heritability for biological yield in normal, water deficit and average of two conditions were 26. 52, 26. 91 and 16. 09%. Also, the highest genetic gain for biological yield was observed in normal conditions. Results of QTL analysis showed. In normal condition, one QTL (R2 A= 2. 46), one QTL×E (R2 AE= 5. 46), 2 additive × additive epistatic effects (R2 AA= 3. 06) and 7 QTL × QTL×E interactions (R2 AAE= 14. 06) were significant. In water deficit condition, one QTL (R2 A= 8), 3 additive × additive interactions (R2 AA= 2. 04) and 3 QTL × QTL × E interactions (R2 AAE= 24. 74) were identified. In average of two conditions, two QTL (R2 A= 7), 3 QTL×E (R2 AE= 4. 66), 5 additive × additive epistatic effects (R2 AA= 4. 67) and 8 QTL × QTL × E interactions (R2 AAE= 24. 20), were significant. However, a little QTL was observed for biological yield, but in all three conditions, the role of the 7B chromosome in control of biological was significant and a stable QTL was located adjacent to the 'Cfa2174. 1-' Wms573 markers, which can be used in marker assisted selection for biologically selective.

The induction of polyploidy is a useful tool in plant breeding, as important characteristics such as new forms and colors, resistance to both drought and low temperatures can be achieved through chromosome doubling. Several methods and materials were used to obtain polyploid varieties but in some cases these substances have unusual effects. The main purpose of the present study was to regenerate polyploidy plants. In this research, Fritillaria raddeana calluses were treated by colchicine at four different concentrations 0. 005, 0. 001, 0. 05 and 0. 01% for 24, 48 and 72 h to induce polyploidy. The experiment was laid out with three replications in completely randomized design. Due to its slow growth rate, survival percent were identified after 3 months. Root tip chromosome counting, stomata and flow cytometry analysis shown that dosages higher than 0. 01% and exposure time of colchicine treatment higher than 48 h cause more explants lethality. Although the growth rates have been increased and stomata density decreased, neither chromosome counting nor flow cytometry analysis indicated any polyploid plantlets in colchicine treated explants.

To identify single nucleotide polymorphisms (SNPs) in eugenol O-methyl transferase (EOMT) and Chavicol O-methyl transferase (CVOMT) genes, cleaved amplified polymorphic sequence (CAPS) markers and direct sequencing were used in different basil populations. Fragments with sizes of 571 and 908 bp of coding regions of both genes were amplified and digested with restriction enzymes Pst1 and MseI. In silico digestion of the coding region of the genes by Pst1 produced fragments of 480 and 91 bp in EOMT and 621 and 287 bp in CVOMT. However, no polymorphic restricted patterns were produced in 80 basil individuals using Pst1 digestion. In silico MseI digestion of EOMT and CVOMT gene sequences produces fragments of 59, 135 and 377 bp, and 275, 302 and 331 bp, respectively. Digestion of the amplified fragments of both genes generated polymorphic banding patterns in studied basil genotypes. One out of each different restriction patters which is produced for both genes in basil genotypes, was selected for sequencing. Sequences obtained, were aligned for both genes using Clustal Omega and SNPs were identified. The results of EOMT alignment revealed transition mutations T<->C and A<->G, but no transversion was observed in this gene. Mutations A<->G, T<->C, A<->C and A<->T were found in CVOMT gene with the highest frequency of A<->G. In conclusion, the results of the current investigation revealed low polymorphism in coding regions of the studied genes and demonstrated the conservity of the coding regions of both genes during basil evolution.

Red Seedless grape is one of the most important and qualitative Iranian grape varieties which has been welcomed by farmers recently. Given the importance of the mass propagation of this plant, producing healthy seedlings, and particularly free of grapevine crown gall disease or cancer, in the present study the effects of various hormonal concentrations treatments were studied on proliferation and rooting of red seedless grape, with the aim of giving a suitable instruction for in vitro culture of the plant. For this purpose, the MS medium containing various concentrations of benzyl amino purine (BAP) (0. 25, 0. 5, 1, and 2 mg L-1) with 0. 2 mg L-1 indole acetic acid (IAA) were used for proliferation and various concentrations of indole butyric acid (IBA) (0, 0. 8, and 1. 6 mg L-1) were used for rooting of the samples in a completely randomized design. The results showed that the studied hormones had the significant effects on the characteristics such as node number, nodal length, shoots number, length, diameter, fresh and dry weight, leaf number, leaf area, chlorophyll index, root number, length, fresh and dry weight. There was also different between concentrations of the used hormones. According to the results the highest proliferation and rooting was achieved at 0. 25 mg L-1 of BAP and 0. 8 mg L-1 of IBA, respectively. According to the results, these hormonal treatments can be useful for proliferation and rooting of red seedless grape.

One of the most important approaches in animal breeding is identification of structural variations responsible for economical important traits. Next generation sequencing technologies are provided accurate tools for this purpose. In the present study, Fars province native poultry genome was analyzed using genome sequencing approach. Paired-end sequencing of the whole genome was conducted by Illumina Company in China. Data quality was determined by FastQC software. Short reads of sequences were mapped with the reference genome with the BWA program. Single nucleotide polymorphisms and small deletions and insertions were identified with the GATK program. The short sequences were compared with the reference genome of over 99% and with the mean depth of the X8 coverage. In this study, 8858153 single nucleotide polymorphisms and 895378 small deletions and insertions were identified with the lowest counts in the exon region. Since the results of identifying the variants showed that the most frequent variant is related to single nucleotide polynomials، in this study, heterozygous loci were higher than homozygous loci indicating the high diversity of the population under study. Given the history of several thousand years of chicken domestication in Iran, one can expect that adaptation to the environment has occurred in the population studied. As a result of genomic changes in order to adapt to the environment, it has created a diversity in the population.