المواقيت المكانية: دراسة تحليلية موضوعية

إعداد الباحث: بندر بن أحمد بن ثابت عاتي: قسم الكتاب والسنة، كلية الدعوة وأصول الدين، جامعة أم القرى، المملكة العربية السعودية الملخص: إن من جملة المناسك ما حدَّه النبي  من المواقيت المكانية التي لا تجوز مجاوزتها بغير إحرام منها لمريد الحج أو العمرة، وإن المواقيت المكانية ذات أهمية كبيرة؛ ويقع الكثير من المسلمين في حرج وإشكال؛ لعدم فقههم بمسائل الحج والعمرة، فربما لا يتم حجه، وربما تجب عليهم الفدية، وهذا تحديداً ما يحاول الباحث قراءته بشكل تحليلي، ولذلك فقد قام الباحث بتحليل الأحاديث الواردة في المواقيت المكانية ، مع بيان المسائل الفقهية ودراستها دراسةً موضوعيةً، وخرجت الدراسة بعدة نتائج منها: وجوب الإحرام من المواقيت، ومن تركه عليه دم. وأنَّ المواقيت توقيفية لا تجوز مجاوزتها لمريد النسك. ومشروعية المحاذاة وأنها معتبرة. وأنَّ مدار الخلاف في مسألة هل جدة ميقات، هو بيان المقصود بالمحاذاة. أن جدة ليست ميقاتًا إلا لأهلها ومن حاذاها من الغرب. وأن المواقيت لأهلها ولمن مرَّ عليها من غير أهلها. جواز الإحرام من الطائرات والسفن بالمحاذاة. الكلمات المفتاحية: أركان الإسلام، الحج، مناسك الحج، المواقيت المكانية

High Throughput Search of Drought Tolerant Genes in Aagave Sisalana L.

Agave sisalana, a hard succulent CAM (crassulacean acid metabolism) plant along with other land plants like cactus, pineapple and vanilla orchids, has evolved over millions of years to drive a different kind of photosynthesis that allows the plants to survive in semiarid environments where water isn‟t always readily available. This process is known as CAM and it is the main area of interest for the scientists all over the world to develop drought resistant plants. Modern genomic approaches like gene identification, their characterization and expression under various abiotic stresses lead to the crafting of genetically resistant crops. The genomes of a number of different CAM plants have been sequenced in the past two years but Agave sisalana L. has received limited attention by many researchers around the globe as far its ability to survive under drought conditions is concerned. Therefore present study was aimed to develop drought stressed cDNA library and to find out potentially up-regulated genes expressed under drought in leaf epidermal tissue of Agave sisalana. Agave sisalana plant selection under drought stressed conditions was done on the basis of their physio-biochemical and water related attributes. All the physiological parameters including photosynthetic and transpiration rate, stomatal conductance and water use efficiency showed decreasing trend with increasing drought stressed conditions. Biochemical analyses of control and drought stressed Agave sisalana plants also played an important role in the adaptation of plants to adverse environment conditions. Plant under drought stress produces certain osmolytes like proline and products of reactive oxygen species which triggers the regulatory signaling pathways involved in the plant defense mechanism. In the present study the correlation coefficients (r) among various physiological, biochemical and water related factors under drought stress conditions also indicated that decreasing photosynthesis, transpiration, total chlorophyll content and other parameters are well monitored by the accumulation of osmolytes produced in response to abiotic traumas. In case of water related measuring parameters, the values of relative water content decreases with the drought stress compared to control where it remain significantly high. Microscopic examination of epidermal tissues of leaves of Agave sisalana showed partially and fully closed stomata upon the induction of drought stress. Stomatal conductance also decreased and showed accordance with the closed stomata under drought stress. Instantaneous water use efficiency showed contradictory results of showing decreasing trend with abiotic stress. It has been reported in many cases that it varies with different plant species as has been explained in the discussion part of the dissertation. All the biochemical assays performed in Agave plants confirmed the accumulation of macro molecules that could lead to support the idea of searching drought tolerant functional EST in leaves of Agave sisalana L. A cDNA library from drought stressed epidermal tissue of leaves of Agave sisalana L. has been constructed. Ten thousands clones were randomly picked, replicated and PCR amplified. The inserts size was found in a range of 100-1000bp. One hundred and five (105) clones (submitted to NCBI GenBank (JZ892707 - JZ892811) were sequenced and annotated. As there were no reported sequences on Agave sisalana L. under drought stress so the homology search was done in comparison with already reported sequences in land plants. Blast ( EST, nucleotide and protein) database in NCBI GenBank was used for homology search. Approximately 4% clone sequences didn‟t show homology at selection criteria (e < 1.0) whereas maximum homology was found in Elaeis guineensis (African palm 16%) followed by Phoenix dactylifera (date palm 8%), Musa acuminate (Banana 5%), Solanum species (5%), Medicago tranculata (5%), Populus trichochorpa (desert poplar 4%), Vitis vinifera (Grapes 4%), Nicotiana species (3%) and Agave species (3%) respectively. The clones (amplified PCR products) were printed in duplicate at an expected ratio of 9,408 spots per microarray chip. The labeled cDNAs were prepared from total RNAs of control and drought stressed leaves of Agave sisalana L. These labeled cDNAs were hybridized to cDNA chips, scanned and data were analyzed. Ten (10) clones were found to be differentially expressed on cDNA microarray platform. The microarray results were validated by real time PCR. EST sequences of potential candidates for drought stressed genes were analyzed through BLAST2GO programe. Out of ten (10) ESTs, maximum homology was found in Phoenix dactylifera plant. Six EST‟s which gives the best homology with drought tolerant genes in other land plants on nucleotide BLAST (nr) and protein BLAST (p) were further evaluated with real time PCR along with bioinformatics studies. Real time data analysis showed highest expression in EST‟s (JZ892752 and JZ892726) whereas EST (JZ892743, JZ892761 and JZ892787) showed low expression of drought tolerance in Agave sisalana L. The differentially expressed EST JZ892778 showed no expression or upregulated activity in stressed leaves of Agave sisalana L. Similar expression of two up-regulated ESTs (JZ892726 and JZ892752) was observed in expression behavior and fold change when compared with K means in cluster analysis of microarray. This new EST collection for the first time in Agave sisalana L. indicated an important step towards the identification of many molecular markers that aided the selective hybridization and biotechnological approaches to further improve Agave sisalana L. as drought resistant plant. Furthermore, the public availability of the novel up regulated cDNA clone sequences generated in this study will enable testing of the biological function of the genes represented and the development of transgenic plants in future.