مجله فنآوری زیستی در کشاورزی
سال هجدهم شماره 2 (پاییز و زمستان 1398)

In this research, the effect of chitin binding domain was investigated on the enhancement of chitinase activity Chit42 enzyme tested on the last instar larvae of Ephestia kuehniella Zell. Chimeric chitinase containing chitin binding domain from Trichoderma atroviride 18-10 chitinase and Chit42 were cloned in pET 26b(+) and over expressed in Escherichia coli. The expressed protein was observed and verified by SDS-PAGE method. Hydrolytic activity of the chimeric and native enzymes was measured on the dry powder of 10 last instar larvae of E. kuehniella. The effect of 3 different concentrations of each enzyme including 30, 45 and 60μg/ml was evaluated on the mortality of 30 E. kuehneilla last instar larvae (each 10 larvae as 1 replication) at 28ºC, 50% RH and 16:8h L:D. Larvae were treated by protein solution lacking enzymes in control. After 24h, the number of dead larvae was counted in treatments and control. Mortality was calculated by Abbott’s formula. The chimeric chitinase and native enzymes activity on the larval chitin (substrate) was calculated to be 15.47 and 9.16U/ml, respectively. Bioassay tests demonstrated that the chimeric chitinase and Chit42 enzymes imposed 65 and 47% mortality to E. kuehniella larvae, respectively. Finally, microscopic observation demonstrated higher amount of chitin degradation in treated larvae with chimeric chitinase compared with those treated by native Chit42.

Bacteria are an important compatible group of microorganisms which live within insect gut. Most of these microorganisms are useful to help the digestive system to work better. The aim of this study was to identify the symbionts bacteria associated with Plodia interpunctella and Ephestia kuehniella guts to use in future biotechnology studies. The larva from the two above-mentioned insects wereprepared and their guts organ were used to isolate symbiotic bacteria. The bacteria were isolated and purified on the enriched nutrient agar medium and their frequency was recorded. Based on the phenotypic characteristics the isolated bacteria were preliminary characterized. Then after comparing of their total soluble proteins by electrophoresis, they were kept for further studies. Identification of bacteria were done based on phenotypic characteristics with standard bacteriological methods as well as use of 16SrRNA encoding gene sequence and its comparison with NCBI data bank. Results indicated that the dominant symbiont bacteria in the Ephestia kuehniella gut were Bacillus toyonensis, Serratia marcescence and in Plodia interpunctella gut were Bacillus safensis, Bacillus zhangzhouensis and Bacillus wiedmannii. The accuracy of the results for phenotypic determination was verified by molecular analyzes using DNA sequences of the 16SrRNA gene comparission via NCBI sequences database. This is the first study on gut symbionts of Ephestia kuehniella and Plodia interpunctella in Iran.

Because of to the importance of wheat, it seems necessary to identify the proteins involved in different stages of its growth. This is for breeding more capable plants, which have more production and are resistant to adverse environmental conditions. In this study, proteomics was used to identify and compare the protein pattern of bread wheat in developmental stages of leaves and tillering stages. For this purpose, Bread wheat cultivar Azar2 was cultivated in greenhouse conditions and samples were taken at different stages of leaf development (single-leaf, two-leaf and three-leaf) and three stages during the tillering period. Protein extraction was performed by TCA-acetone method and proteins were separated by 2D electrophoresis. By analyzing the resulting gels by Image master 6.0 software, 360 reproducible protein spots were identified in the leaf development stages and 400 reproducible protein spots in the tillering stages. Of these, 31 protein spots in different stages of leaf development and 31 protein spots in different stages of tillering showed significant changes. Finally, protein spots were identified based on their molecular weight, isoelectric point, spot shape, and by searching related articles and the Uniprot site. The results showed that in these stages, a wide range of metabolic activities including photosynthesis, respiration, protein biosynthesis, protein transport, protein assembly, and packaging, carbohydrate biosynthesis, message transmission system, toxin degeneration and basic genetic processes (replication, transcription and translation) were changed during different stages of wheat plant growth.

In this study, fourteen tomato landraces and cultivars were evaluated using ten RAPD primers and ten ISSR primers. In RAPD marker, the most polymorphism index (0.41) has belonged to TIBMBA02 and TIBMBB17 primers and the lowest polymorphism index (0.19) has belonged to TIBMBC12 primer, and in ISSR marker, the most polymorphism index (0.42) has belonged to UBC808 primer and the lowest polymorphism index (0.23) has belonged to UBC864 primer. In the total merged data of the two markers, the most percent of polymorphism (80.72), Nei diversity index (0.44), Shannon diversity index (0.48) and effective allele number (1.6) belonged to Iranian Anbar cultivar. The lowest similarity index (0.368) was belonged to Iranian Anbar cultivar and also Y Keshideh. The diversity of inter and intra landraces and cultivars was shown 79 percent of all the diversity in total variances based on analysis of molecular variance. Cluster analysis, based on the total merged data of the two markers, was grouped all landraces and cultivars into 4 groups but they were not grouped according to the geographical zone. To obtain a superior cultivar, it is recommended that the Iranian Anbar cultivar can be cross-fertilized with either Y or US107 cultivars from Italy and the United States, which are higher in production than Iranian cultivars.

The expression of nitrogen and ammonium transfer genes in wheat roots in response to drought stress is mainly unknown. A greenhouse experiment was conducted to investigate the expression of nitrogen transfer genes (TaNRT1.1) and ammonium transfer genes (TaAMT2.1) in wheat roots in response to drought stress under limited nitrogen conditions (no nitrogen added) and sufficient nitrogen (0/3 g nitrogen per kg soil). Three genotypes (Gonbad, Morvarid and Falat) were used and water stress was applied in the vegetative stage of wheat growth. The expression of genes was determined using real-time PCR technique. The results showed that with application of N fertilizer, shoot dry weight, root and nitrogen content increased significantly under N application conditions and similar conditions were observed for drought treatments under complete irrigation conditions. Due to the different response of cultivars to the expression of the studied genes, cultivars had different responses in terms of gene expression. Generally, expression of both genes under nitrogen application was increased in line with nitrogen supply and in Pearl and Plateau cultivars expression of these genes was increased under drought stress and N application was higher. In general, Pearl cultivar was better for all traits under drought and nitrogen deficiency treatments.

Abiotic stresses, such as drought, affect the growth and development of plants like common bean (Phaseolus vulgaris L.) and plant responses to them are associated with large variations in complex gene networks. In the present study, changes in gene expression pattern under drought and normal conditions of common bean were studied through bioinformatics analysis of ESTs (expressed sequence tags) data from the NCBI database. After initial splicing, EST sequences were clustering and assembling, resulting in 8983 unigene under normal and 6665 unigene of drought stress conditions. Grouping and genetic enrichment analysis the common bean library EST sequences under drought stress based on biological processes, molecular function and cellular component at 23, 16 and 20 different functional groups, respectively. Protein-protein interaction network showed that genes encoding antioxidant enzymes (CAT1, APX1, APX3, APX1B, GSTU25), Proline-rich proteins (P5CS1, P5CS2), heat shock proteins (ATHSP70, HSP70b, ATHSP90.1, ATHSP90.5) and ubiquitin family proteins (UBQ3, UBQ11) account for a large share of the regulatory network in starvation water. Additionally, we found that the expression of photosynthetic genes decreased. In this study, provides a list of transcription factors (including: RAP2.10, DEAR2, NF-YC9, WRKY40, ARF1, CDF2, ATCTH, ERF-1) as one of the excellent point that have not been reported with so many detail in the common bean so far and it can be very important and applicable. The genes identified in this study could be suitable candidates for drought resistance and marker-assisted selection in common bean.