Republic of Iraq Ministry of Higher Education and Scientific Research University of KUFA College of Agriculture

ANALYSIS GENE EXPRESSION FOR FIVE GENES WHICH ARE MAINLY RESPONSIBLE FOR FATTY ACID DESATURATASES FOR TEN OLIVE VARIETIES (Olea europaea L.)
Doctoral Dissertation

By

Ali Saeed Atiyah AL-Janabi

Supervised by Assistant Prof. Dr. Muslem.A .A. Abdulhussein Assistant Prof. Dr. SALWA. J. ALawady 2013

Summary
In this study analyzed gene expression for five genes which are mainly responsible of fatty acid desaturases production FADs is (FAD2.1 , FAD2.2 , FAD3 , FAD6, FAD7) for 10 Olive varieties (Olea europaea L.) (Khaderi , Qaysi , Manzenillo , Baashiqi , Arabqween , Nabali , Labeeb , Dahkan , Shami and Sorani) . Different plant organis as plant materials (transplants leaves , leaves for fruitefull trees and fruits flesh)used for analysis. transplants leaves were collected from Al-Zafrania and Mosul Agriculture Stations which belongs to the Ministry of Iraqi Agriculture , While leaves for fruitful trees and fruit were collected from two different locations orchards , first location was Baashiqa / Mosul / Iraq where Iraqi varieties (Baashiqi and Dahkan) were collected, and others varieties were collected from second location from Al-Mshaqer station / Madaba / Jordan (about 52 km north west Aman) were collected 8 varieties except Iraqi varieties . Two methods for analysis gene expression were used, first method was called Semi quantitative (used only on mature leaves and fruit) and second method was called relative quantitative (used on seedling leaves , mature leaves and fruits) used in relative method (Real time PCR and Actine gene as Housekeeping gene). On the other hand chemical analysis was used on fruits like moisture % , oil D.W% , oil F.W and addition to saturated and unsaturated fatty acid (%) based on Gas chromatography. The results revealed the following :

1- Analysis of relative quantitative for gene expression fatty acid desaturases in the transplants leaves.

Shami variety showed high relative expression levels of the (FAD2.1 , FAD2.2 and FAD7)which were (1200 , 24 and 6.9 fold) respectively as compared with the other varieties , on the other hand the Khaderi variety showed high relative expression in FAD3 gene (about 100 fold) , while FAD6 was showed high level relative gene expression in Arabqween variety (about 8- fold) and shami variety (4.75 - fold) as compared with the other varieties . 2- Analysis of gene expression for fatty acid desaturases in mature leaves olive varieties Semi quantitative analysisfor FADs genes in mature leaves for olive varieties revealed that Iraqi varieties (Baashiqi , Dahkan) were the least varities expression for FADs genes , that was clear on bands for these varieties which were not found or weaked in most replication cDNA per programs with primers genes and special in 25 cycle PCR programs for semi quantitative analysis . Other verities were with high intensity or medium specially Shami , Manzenillo and Sorani varieties which had clear bands in most Semi quantitative PCR programs , and this refers to their high gene expression for FADs genes in mature leaves of olive varieties. Relative quantitative gene expression for FADs genes in mature leaves showed high levels of gene expression for (FAD2.1 , FAD6 and FAD7) in Dahkan variety which were (61.1 , 85.0 , 32.5 and 52.5 fold) respectively as compared with the other varieties . Arabqween variety had high gene expression level for FAD2.2 gene about (17.5 fold) , while Baashiqi variety was in the second gene expression level for most of studied genes and on the other hand that results showed decrease in

gene expression levels for the other varieties for genes studies because this varieties becams they are early matured varieties and ended the Biocollection for fatty acid unsaturated and oil. 3- Analysis of gene expression for fatty acid desaturases in fruits of olive varities. Semi quantitative analysis for FADs gene in olive varieties fruits showed differences between studied genes and semi quantitative PCR programs (25 , 35) . Shami variety had intensity and clear bands to most FAD3 genes and cycles except 25 cycle for (FAD2.2 , FAD7)which were genes bands were weak as compared with the others varieties . On top of Khaderi variety had intensity bands to most Semi quantitative cycles for most genes. While Semi quantitative gene expression for Iraqi variety (Baashiqi) was high intensity and clear bands to (FAD2.2 , FAD7) genes and to two cycles (25 , 35) , but had low intensity band for otheres genes . While Dahkan variety had was weaking not found in most cycles and Semi quantitative programs except 25 cycle for FAD7 gene which had clear band. Analysis of the relative quantitative gene expression of the FADs genes in olive fruit varieties revealed differences in gene expression levels between genes , which showed the highest relative quantitative gene expression for FAD2.1 gene in Khaderi variety which reached about (60- fold) , while Iraqi varieties (Baashiqi and Dahkan) had reached to (25 , 21 fold) respectively . While highest level of FAD3 gene expression was in Manzenello fruit variety which reached about (21.5 fold) . For FAD6 gene expression there were convergent levels in fruits of varieties except Sorani variety which are lowest level for FAD6 gene expression

reached about (1-fold) . The highest level of FAD7 gene expression in Dahkan fruit variety which was (36 fold). 4- Chemical analysis of the fruits of olive varieties. Dahkan variety showed highest moisture percentage in fruits with significant difference reached (71.3%) , while the lowest moisture percentage in Khaderi variety fruit. Arabqween variety fruit contained high percentage of oil in dry weight reached (57.5%) and Baashiqi fruit had low percentage of oil dry weight reached (15.5%). Percentage of oil in fresh weight showed which highest significant value in Khaderi variety which was (37.4%) , while the lowest percentage in Iraqi varieties (Baashiqi and Dahkan) reached (4.9 and 6.6%) respectively . The chemical analysis saturated fatty acids percentage (FAS) showed high percentage in the Iraqi varieties fruits Baashiqi and Dahkan reached (3.41 , 3.39) respectively , where the least percentage in fruits of varieties were in Khaderi and Shami varieties reached (2.31 , 23.4) respectively . Chemical analysis also showed highest significant difference in Shami , Khaderi in fruits varieties were reached (12.67 , 12.01) respectively , where as the lowest significant difference of FAD in Iraqi Dahkan variety which reached (8.66) For the ratio of percentage of unsaturated fatty acids to the percentage of saturated fatty acids was high in Shami and Khaderi varieties which were (5.42 , 5.19) respectively . Where was the lowest ratio was in Iraqi Baashiqi and Dahkan varieties which were (2.65 , 2.55) respectively.