پديد آورندگان :
چوپاني، ادريس دانشگاه زنجان - دانشكده كشاورزي , باقري، خديجه دانشگاه زنجان - دانشكده كشاورزي - بخش زراعت و اصلاح نباتات , ملكي زنجاني، بهرام دانشگاه زنجان - دانشكده كشاورزي - بخش زراعت و اصلاح نباتات
چكيده فارسي :
جهت اجتناب از مضرات پروموترهاي عمومي، شناسايي و جداسازي پروموترهاي قوي و اختصاصي بافت امري بسيار مهم و ضروري در مهندسي ژنتيك است. يكي از پروموترهاي اختصاصي و قوي بذري، پروموتر بتافازئولين لوبيا مي‌باشد كه بيان حدود 50درصد پروتئين‌هاي بذر لوبيا را كنترل مي‌كند. استفاده از اين پروموتر براي بهينه‌كردن توليد پروتئين‌هاي بذري در لوبيا و گياهان ديگر و همچنين توليد پروتئين‌هاي نوتركيب مفيد خواهد بود. آناليز بيوانفورماتيكي پروموترها براي پيشگويي در مورد قدرت و ضعف آن‌ها، جداسازي صحيح و همچنين سنتز پروموترهاي مصنوعي كمك خواهد كرد. ابتدا به‌وسيله نرم‌افزارهاي آناليز پروموتر توالي‌هاي پروموتري بتافازئولين گياه لوبيا مورد تجزيه و تحليل قرار گرفت. نتايج حاصل نشان داد كه بيش از 20فاكتور سيس از جمله مهم‌ترين آن‌ها G-box، E-box، فاكتور RY،ACGTSEED2، جعبه لگومين، جعبه اندوسپرم و...در توالي پروموتر بتافازئولين وجود دارد كه در بيان بالا و اختصاصيت آن نقش دارند. با توجه به نتايج آناليز بيوانفورماتيكي پرايمرهاي اختصاصي اين پروموتر طراحي شد و با استفاده از آن‌ها توالي پروموتر موردنظر از DNA ژنومي لوبيا تكثير و با توجه به اندازه قطعه تكثيرشده، صحت آن مورد تأييد قرار گرفت. در مرحله بعدي توالي پروموتر موردنظر در ناقل pTZ57R/T كلون شد و با استفاده از واكنش‌هاي PCR و هضم آنزيمي مورد تأييد قرار گرفت. از آنجايي‌كه هدف از اين تحقيق جداسازي پروموتر اختصاصي بذر بتافازئولين از لوبيا و استفاده از آن در تهية سازه‌هاي ژني مي‌باشد، لذا قطعة موردنظر در ناقل بياني گياهي pBI121 ساب‌كلون شد و نتايج آن با هضم آنزيمي و PCR تأييد شد.
چكيده لاتين :
Introduction
Constitutive promoters such as CaMV 35S, which is usually widely used in the plant genetic engineering express downstream genes in the all stages of plant life and in all tissues and if a transgene expressed at incorrect tissue and time perhaps unexpected results would be seen in plant growth. To avoid disadvantages of constitutive promoter function identification and isolation of tissue-specific and strong promoters is very important in genetic engineering. Beta phaseolin is one of the strong seed-specific promoters that controls the expression of about 50% bean seed proteins. Extensive studies in bean (Phaseolus vulgaris) and transgenic tobacco have revealed that the promoter for the beta-phaseolin storage protein gene (phas) is stringently regulated. Expression is very high during embryogenesis and microsporogenesis but is absent in vegetative tissues. The utilization of this promoter to optimize the production of seed proteins in bean sand other recombinant proteins in other plants would be will be useful. According to the above-mentioned advantages about seed specific promoter, this study has been conducted to identify and isolate the beta phaseolin gene promoter from bean (phaseolus vulgaris) and clone it in pBI121 plants vector.
Materials & Methods
Bioinformatic analysis help the prediction of promoter intensity, proper separation and synthesis of artificial promoter. According to bioinformatic analysis, specific primers using gene runner, vector NT and primre 3 softwares were designed and by using these primers, promoter sequence was amplified from bean genomic DNA, Due to the size of amplified fragment, its authenticity was confirmed. In the next step, the desired sequence ligated into the cloning vector pTZ57R /T and by using PCR and digestion reactions was confirmed. The aim of this study was to isolate beta phaseolin of bean and its use in the preparation of gene constructs. For this reason, we subcloned fragment in plant expression vector (pBI121) and cloning was confirmed by colony PCR and digestion.
Results & Discussion
The results showed more than 20 factors cis such as ACGTSEED2, opaque-2,E-box, legumin box, endosperm box and etc are in beta phaseolin promoter that play a role in the high expression and specificity. ACGTSEED2 factor that is unique in the phaseolin promoter has major role in the expression of specific genes in the seeds of beans. G-box sequence is one of important cis factors. This factor is of responding to abscisic acid, promoter activity is reduced to 2.6% by removing the G-box. Functional E-site may be necessary to complement the G-box-mediated promoter activation, hence acting as a coupling element. The vicilin core sequence (GCCACCTCA) was initially described as a part of a large vicilin boxThe vicilin box (henceforth, this term refers only to the core sequence) is found in the promoters for many seed storage proteins. There is also three TATA box is activated as the promoter core and Plays an important role in being a strong promoter and high expression. Previous studies and portions of the current study confirm that 295 bp from beta phaseolin promoter is relevant seed-specific expression that consists of three parts; 1- Sequence 68 bp (227 to -295) that known as Seed Specific Enhancer (SSE) 2- The middle part (109 to -127) 3- base promoter (+20 to -109). SSE, base promoter and the middle part simultaneously causes increase in gene expression. OSE (Organ specific elements) factors are in the legumes promoters like beans, soybeans, alfalfa, etc, and related to the symbiosis of these plants with the Rhizobium bacteria. Therefore, in this study isolated completely sequence of the beta phaseolin promoter were shown. But it is advisable that if there are restrictions on the size of the promoter and the promoter artificial synthesis can be separated 295 bp of the beta phaseolin promoter sequence.
Conclusion
The promoters are important part of gene constructs and necessary for production of recombinant proteins in genetically modified plants beta phaseolin promoter is a strong and seed-specific promoter, so it has ability for the production of recombinant proteins and building gene constructs.
