مجله زیست فناوری گیاهان زراعی
پیاپی 12 (زمستان 1394)

As a rule the reference genes used in gene expression analysis have been selected for their housekeeping roles, but the variation observed in most of them is a major obstacle to their effective use. It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. In this study, suitability of seven wheat housekeeping genes for normalization of mRNA expression in wheat leaves infected by Mycosphaerella graminicola was investigated. Expression level of Actin, Rubisco, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Translation Elongation Factor 1α (TEF-1α), α-Tubollin, eukaryotic release factors 1 and 3 (ERF1 and ERF3) genes were examined by reverse northern dot blot method. Expression stabilities of the reference genes were statistically analyzed by Ecxel and SAS softwares. α-Tubolin, TEF-1α and Actin were the three most stable genes whereas the expression of Rubisco and GAPDH had the least stability. The presented comprehensive data on changes in expression of various wheat housekeeping genes in wheat- M. graminicola phatosystem facilitate selection of reference genes for Reverse northern dot blot method.

Sesame (Sesamum indicum L.) is one of the oldest oilseed crops that has been a main food source providing beneficial nutritional outcomes to human health due to its high contents of antioxidants and secondary metabolites. The importance of sesame arises from presence of antioxidant sesamin. Over recent decades, due to being used in cancer cases, there have been numerous efforts aimed at better understanding of the genes underlying sesamin biosynthesis and increasing their expression. In this research, expression levels of the key genes in sesamin biosynthesis pathway including CYP81Q1, CYP81Q2, CYP81Q3 and C3H genes were measured in 10 sesame cultivars seeds (Yekta, early Palestinian, Naz tak shakheh, Naz chand shakheh, Oltan, Jiroft, Dashtestan 5, Dashtestan 2, Varamin and Karaj 1) by QRT-PCR technique and 18S rRNA gene as reference gene. Our findings showed that the level of CYP81Q1 gene expression was low in Early Palestinian that has low sesamin content. Therefore, this might not be considered as a qualitatively desired oil cultivar compared to others, whereas Yekta had the highest level of CYP81Q1 expression the best varieties for oil production both qualitatively and quantitatively. The highest expression level of CYP81Q2 was observed in Naz chand shakheh, while the lowest CYP81Q2 expression was found in Early Palestinian. Karaj1, Yekta and Dashtestan 5 showed the highest whereas Oltan had the lowest expression level of C3H. Karaj 1, Yekta and Dashtestan 5 were relatively from the late mature group that was reported to have higher contents of oil and sesamin than early ones.

Abiotic stresses are among major factors limiting crop yields, and SnRK2 protein kinases are one of the key regulators of plant response to abiotic stresses. Due to the economic importance, cultivation area, and tolerance of barley to the abiotic stresses, identification and characterization of SnRK2 family members in barley is performed in present research. SnRK2 conserved sequences were used as a query for tBLASTn analysis in different databases such as NCBI and international barley sequencing consortium against all of the reported barley sequences. As a result, 10 members were identified (HvSnRK2.1 to HvSnRK2.10) which 8 of them were not yet reported. These HvSnRK2 members were aligned with AtSnRK2s and OsSnRK2s and a phylogenetic tree was constructed. Detection of chromosomal localization, promoter analysis and gene structure determination was also performed. Half of the family members were located on chromosome 2 and the rest on chromosomes 1, 4, 5 and 6. Number of introns in the gene family members varied from 0 to 8. Totally, 19 sorts of cis elements including abiotic stress responsive elements were found in HvSnRK2s promoter sequences. Expression pattern of the family members were evaluated in different tissues, treatments and genotypes, based on the microarray data. Expression of HvSnRK2.6 was up-regulated by drought, salt and cold stresses implementing its important role in signal transduction of these stresses and tolerance induction to them. It is expected that this gene could be used in plant manipulation and breeding programs aimed for tolerance enhancement to abiotic stresses especially drought.

In order to investigate genetic diversity of sunflower genotypes, TRAP markers were used with six fixed and arbitrary primers. Nineteen primer combinations generated a total of 116 bands in which 109 of them were polymorphic. Restorer inbred lines with a mean of 22.76 polymorphic bands had the highest polymorphic loci (84.48 %), and Iranian hybrids with a mean of 2.97 polymorphic bands had the lowest polymorphic loci (40.52 %). Maximum and minimum genetic distances were between restorer lines and Iranian hybrids (0.151) and foreign hybrids and open pollinated cultivars (0.064), respectively. Maximum genetic similarity was between restorer and CMS lines (0.066). AMOVA analysis revealed that 87 % of total variance was within groups, and 13 % was between groups. Using UPGMA method of clustering and principal coordinate analysis, three distinctive groups were identified. Minimum similarity coefficient (0.472) was observed between R42 and CMS328 inbred lines. Results showed that TRAP marker was useful in genetic diversity estimation of sunflower genotypes. Higher similarity coefficient (0.755) for the studied genotypes indicated a narrow genetic base suggesting increasing genetic diversity of sunflower germplasm and selection of high diversity of new inbred lines in the future sunflower breeding programs.

Molecular analysis is often required in many genome extracted from various tissues. In much molecular analysis of genetic studies of microbial communities, often require direct extraction of DNA from soil. In this study, after careful study of the various methods proposed three methods (chemical enzyme), (chemi-mechanical) and (chemical mechanical enzymatic), in order to extract DNA from soil, a separate project was studied to most identify different methods of extraction. The comparison between the same conditions, a significant difference in the DNA product of the average concentration of 2/32 to 6/1 ng per µl DNA was diluted and also on the basis of information obtained from Nanodrop and electrophoresis device and review of the DNA extracted protein product DNA, methods (chemical, mechanical, enzymatic) better performance at higher concentrations, remove humic acid proteins and other contaminants was superior to other methods. The molecular detection of pathogens that extraction is important applications; the aim of this study is molecular detection of pathogen Fusarium oxysporum f. sp lycopersici and Fusarium oxysporum f. sp. radicis lycopersici of soil microbial community by molecular techniques to identify the special primer evaluated. Molecular detection results showed that there is tomato Fusarium infection in soil samples S1.

The monoterpenoids comprise a family of structurally and pharmaceutically diverse alkaloids. Strictosidine synthase is a key enzyme in monoterpenoid indole alkaloid biosynthesis pathway. In spite of the apparent lack of complex alkaloids in Arabidopsis, a gene family called Strictosidine synthase like (SSL) has been found in the genome. SSL6, a member of SSL family, has been induced significantly by various stresses and signaling molecules. An overexpressed mutant is a powerful tool for functional characterization of an unknown gene. In view of that, SSL6 overexpressed mutant have been generated in order to study the possible role of the gene in Arabidopsis defense against biotic and abiotic stresses. The open reading frame from SSL6 was amplified and cloned into intermediate pJET vector before subcloning into pPZPY122 plant vector. Plant transformation was made by floral dip method using Agrobacterium tumefaciens strain GV3101 (PMP90). The putative transgenic plants were isolated on selective MS medium containing gentamycin. Transgene integration was further analyzed by PCR using SSL6 and gentamycin resistance gene specific primers. The transcription level of SSL6 in the T2 plants was measured using q-PCR and indicated an overexpression in transgenic compared to wild type Col-0 plants. Segregation ratio of plants in T2 and T3 generation on selection medium proved that some of the genotypes contain single T-DNA insert. The SSL6 Expression level in response to salt stress was measured in Col-0 and indicated an up-regulation of the gene 3, 6 and 12 hrs after treatment.