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العنوان
Evaluating the expression of some insecticidal
protein coding genes during seeds storage /
المؤلف
Abdelgaber, Rasha Essameldin Saber.
هيئة الاعداد
باحث / رشـــا عصام الدين صابر عبد الجابر
مشرف / هدى محمد صبحي بركات
مشرف / مواهب محمود زيور
مشرف / محمد عبد الستار حسن علي
تاريخ النشر
2022.
عدد الصفحات
225 P. :
اللغة
الإنجليزية
الدرجة
الدكتوراه
التخصص
علوم النبات
تاريخ الإجازة
1/1/2022
مكان الإجازة
جامعة عين شمس - كلية العلوم - قسم النبات
الفهرس
Only 14 pages are availabe for public view

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from 225

Abstract

6. Summary
Seed considers the basic and crucial input for agricultural production. During storage the quality of seeds get deteriorated in several ways of which infestation by the storage pests contribute a bulk share. Maintenance of high seed or grain quality from harvest until planting is of almost importance in a seed production programs.
Vicia faba L. (Family Fabaceae) and Triticum durum L. (Family Poaceae) are from the most important crops in Egypt. They are subjected to both quantitative and qualitative losses due to infestation by several insect species. The most injurious stores insects are the bean beetle (Callosobuchus maculatus F.) and the lesser grain borer, (Rhyzopertha dominica).
Plants developed different defense mechanisms in order to reduce insect attack, such as the induction of defensive proteins. Several different classes of plant proteins have been shown to be insecticidal towards a range of economically important insect pests. Amongst the genes encoding insecticidal proteins are those that encode lectins protein, therefore studying the genes conferring partial resistance against insect pests such as lectin genes, could provide a more sustainable control strategy.
The present work was designed to evaluate the efficacy of three protectants; deltamethrin (delta-tox) as synthetic insecticide, Beauveria bassiana (biosect) as bioinsecticide and phosphate fertilizer on storage pests. Also, this study was designed to highlight the expression levels of some defense related genes as resistant factors during storage condition. Quantification the expression of lectin gene, that encoded lectin as insecticide protein and the genes encoding pectin, pectin-esterase and pectin methyl esterase inhibitor were evaluated at the transcriptional level in Vicia faba and wheat plants against insecticide stress.
According to the result obtained from the number of emerging insects (F1-progeny) for each cultivar, it can be concluded that the imported V. faba and the T. durum Beni-Suef5 cultivars were the most sensitive and will be more susceptible to infestation by C. maculatus and R. dominica respectively, because they have a greater number (mean of three replicates) of emerging insect (30.4 and 17.5 F1-progeny) as compared with Noubaria1 and Suhag5 cultivars which appeared to be less susceptible and more resistant to these insects since they recorded only 5 and 5.6 F1-progeny respectively.
The mortality percentage of C. maculatus and R. dominica was increased with increasing the concentrations of the tested materials; and there is a potential linear relationship between the rate of treatments and mortality percentages. The mortality percentage of C. maculatus showed similar value in both Vicia faba cultivars; the highest value recorded 85.33% after treatment seeds with 0.05 ml/Kg of deltamethrin; 100.00% after treatment with 9.6 x1010 cell/Kg of B. bassiana and 84.00 % with 1.8 gm/Kg of the phosphate fertilizer. For R. dominica insect, the mortality percentage showed similar value in both cultivars; recorded 81.33% when wheat grains treated with 5x10-4 ml/Kg of deltamethrin, 97.33% with 1.28 x1010 cell/Kg of B. bassiana and 84.00% with 1.20 gm/Kg of the fertilizer.
The residual activity of lethal concentrations (LC95) of the three tested materials was evaluated by calculating the mortality percentage of C. maculatus and R. dominica adults at initial treatment and every two weeks till the storage period (3 months) was ended. They showed 97.33% and 98.67% mortality at initial treatment, then 100% up to three months in both V. faba cultivars treated with LC95 deltamethrin or LC95 B. bassiana respectively, indicating the high residual effect of these insecticides, while a sharp decline in the efficiency of the fertilizer was recorded in which the mortality percentage of C. maculatus recorded 96% at initial treatment, then recorded 20% at the 4th week and no effect was recorded after 8th week.
On the other hand, 98.67% mortality to R. dominica adult were recorded at initial treatment, then 100% at the second week up to the end of the storage period (3 months) with T. durum (Suhag5 or Beni-Suef5) treated with LC95 of B. bassiana, indicating the high residual effect of this bio-insecticide. On the other hand, 97.30% mortality percentage was recorded at initial treatment and 90% or 89.33% by the end of the storage period when of R. dominica exposed to T. durum cultivars treated with LC95 deltamethrin. A sharp decline in the efficiency of the phosphate fertilizer occurred after the six weeks which recorded 48 and 46.67 % with the cultivars Suhag5 and Beni-Suef5, respectively. from the previous data it can be concluded that a sharp decline in the activity of the fertilizer on the insect infestation which had not any effect after 8th week and hence it is not included in the further analyses.
Considerable variations in protein banding patterns were observed as compared with control in which some bands were disappeared and new were appeared in both V. faba (Noubaria1 and imported cv) treated with LC95 deltamethrin or with B. bassiana. In Noubaria1 treated with deltamethrin, one protein band with molecular weight of 75 KD was disappeared and one new band 48 KD was appeared, while no change in band number were observed after treatment with B. bassiana. On the other hand, in the imported cultivar, one protein bands were disappeared with molecular weight of 92 KD after treatment with B. bassiana and band 75 KD after treatment with deltamethrin, while two new bands 110 and 48 KD were induced in samples treated with deltamethrin and band with 32 KD was induced in sample treated with B. bassiana. Band with molecular 48 KD was induced in both V. faba cultivars treated with deltamethrin.
The most visible changes in the protein band patterns of T. durum (Suhag5 and Beni-Suef5) treated previously with LC95 of deltamethrin and B. bassiana and stored for three months, were the disappearance of band with molecular weight of 48 KD in the cultivar Beni-Suef5 and the band 42 KD in the cultivar Suhag5. Four new bands were appeared, band with 60 KD in cultivar Suhag5 after treatments with deltamethrin or B. bassiana and bands with 60 and 42 KD appeared with cultivar Beni-Suef5 treated only with B. bassiana.
Eight primers were used for screening alteration in Vicia faba and T. durum genomes in response to treatments with LC95 of the synthetic insecticide deltamethrin and LC95 of the bio-insecticide Beauveria bassiana after the storage periods. The ratio of polymorphism and Genomic Template Stability (GTS) were calculated. A maximum number of loss and gain bands in both Vicia faba cultivars treated with LC95 deltamethrin were recorded 21 and 31 bands as compared with 13 and 26 bands which recorded with B. bassiana. In general, the polymorphism value (total value of disappeared and appeared bands) recorded in imported cultivar is larger (57 bands) than that recorded with Noubaria1 (34 bands) and in general, the polymorphism in response to deltamethrin (52) was greater than that recorded with the bio-insecticide (39).
On the other hand, decreasing trend of the GTS index in deltamethrin was much greater than the GTS recorded with B. bassiana. In Vicia faba cultivars (Noubaria1 and imported) treated with deltamethrin, the GTS value are recorded 72.4% and 64% respectively as compared with GTS values of these cultivars treated with B. bassiana which recorded 82.8% and 69.8%. from these data, it can be recorded that the highest GTS value was recorded with cultivar Noubaria1 treated with the bio-insecticide (82.8%).
For T. durum cultivars, the SCoT pattern revealed by eight primers showed different profiles that target various regions in the separated DNA. GTS values in T. durum cultivars Suhag5 and Beni-Suef5 treated with deltamethrin are recorded 48.21% and 55% respectively as compared with these cultivars treated with B. bassiana which recorded 40.07% and 63.34% respectively. from these data, it can be recorded that the highest GTS value was recorded with Beni-Suef5 cultivar treated with the bio-insecticide B. bassiana (63.34%) and GTS values of Suhag5 cultivar treated with the two test materials recorded the lowest values as compared with the cultivar Beni-Suef5.
The gene expression of three key defense related genes (Vf_Lectin, Vf_Pectin and Vf_ Pectinesterase) were investigated in Vicia faba and Td_Pectin-methyl-esterase inhibitor gene was investigated in both T. durum cultivars, after the storage period by quantitative real time PCR (qRT-PCR) using three specific primers for broad bean and one specific primers for wheat cultivars.
The expression level of Vf_Lectin gene in both Vicia faba cultivars treated with deltamethrin was increased as compared with the control. The upregulation of this gene in Noubaria1 and imported cultivars were 1.21 and 1.42 respectively compared with control (1.0). On the contrary, after treatment Noubaria1 and imported cultivars with the bio-insecticide, the expression of Vf_Lectin gene appeared to be significantly down-regulated in both cultivars (0.34 and 0.25) respectively, as compared with the control (1.0). The upregulation of this gene appeared to be contributing to deltamethrin stress. These data indicated that the change in the expression of this gene is not associated with the resistance or the susceptibility of these cultivars against C. maculates and may be related to insecticide stress.
The expression level of Vf_Pectin gene in response to deltamethrin was significantly upregulated in both Vicia faba cultivars in which the expression level was 4.6 and 2.4-fold for Noubaria1 and imported cultivar respectively, compared to non-treated seeds. Interestingly, the expression level in response to B. bassiana has exhibited the highest level by 44-fold for imported cultivar compared to Noubaria1 that exhibited slightly up-regulation by 1.8-fold. Application of B. bassiana enhanced the constitutive expression of Vf_Pectin gene in the imported cultivar, which previously showed in this study susceptibility to infestation by the C. maculatus, but 100% mortality was recorded after treatment this cultivar with the bioinsecticide till the end of storage period. The higher of expression of this gene in the imported cultivar appeared to be contributing to the bioinsecticide stress and to the character of this cultivar.
The expression levels of Vf_ Pectin-esterase gene in Vicia faba previously treated with LC95 deltamethrin or LC95 B. bassiana and stored for three months, were investigated and the result showed that the expression of this gene was downregulated (0.203) in cultivar Noubaria1 treated with deltamethrin as compared with the control. On contrary, the expression of this gene in the Vicia faba imported cultivar treated with deltamethrin was increased by 6.90-fold. On the other hand, up-regulation in this gene in both cultivars treated with B. bassiana was observed and recorded similar values by 1.40-fold.
To gain insights of the responses T. durum against the effect of the two insecticides, the change in the expression of Td_Pectin-methyl-esterase Inhibitor (PMEI) gene was studied. The expression level of this gene in both T. durum cultivars (Suhag5 and Beni-Suef5) in response to LC95 deltamethrin was significantly upregulated in which the expression level was 4.01 and 3.23-fold for Suhag5 and Beni-Suef5 cultivars, respectively compared to non-treated grains. Interestingly, the expression level in response to B. bassiana has exhibited highest level by 12-fold for Beni-Suef5 compared to Suhag5 that exhibited slightly up-regulation by 2.69-fold.