بررسی بیوانفورماتیکی و جداسازی پروموتر بتافازئولین از لوبیا (Phaseolus vulgaris)

نوع مقاله : مقالات پژوهشی

نویسندگان

• ادریس چوپانی ¹
• خدیجه باقری ²
• بهرام ملکی زنجانی ³

¹ دانشگاه زنجان

² دانشگاه تربیت مدرس

³ انستیتو تحقیقات کشاورزی هند- دهلی نو

10.22067/ijpr.v8i2.50075

چکیده

جهت اجتناب از مضرات پروموترهای عمومی، شناسایی و جداسازی پروموترهای قوی و اختصاصی بافت امری بسیار مهم و ضروری در مهندسی ژنتیک است. یکی از پروموترهای اختصاصی و قوی بذری، پروموتر بتافازئولین لوبیا می‌باشد که بیان حدود ۵۰‌درصد پروتئین‌های بذر لوبیا را کنترل می‌کند. استفاده از این پروموتر برای بهینه‌کردن تولید پروتئین‌های بذری در لوبیا و گیاهان دیگر و همچنین تولید پروتئین‌های نوترکیب مفید خواهد بود. آنالیز بیوانفورماتیکی پروموترها برای پیشگویی در مورد قدرت و ضعف آن‌ها، جداسازی صحیح و همچنین سنتز پروموترهای مصنوعی کمک خواهد کرد. ابتدا به‌وسیله نرم‌افزارهای آنالیز پروموتر توالی‌های پروموتری بتافازئولین گیاه لوبیا مورد تجزیه و تحلیل قرار گرفت. نتایج حاصل نشان داد که بیش از ۲۰‌فاکتور سیس از جمله مهم‌ترین آن‌ها G-box، E-box، فاکتور RY،ACGTSEED2، جعبه لگومین، جعبه اندوسپرم و...در توالی پروموتر بتافازئولین وجود دارد که در بیان بالا و اختصاصیت آن نقش دارند. با توجه به نتایج آنالیز بیوانفورماتیکی پرایمرهای اختصاصی این پروموتر طراحی شد و با استفاده از آن‌ها توالی پروموتر موردنظر از DNA ژنومی لوبیا تکثیر و با توجه به اندازه قطعه تکثیرشده، صحت آن مورد تأیید قرار گرفت. در مرحله بعدی توالی پروموتر موردنظر در ناقل pTZ57R/T کلون شد و با استفاده از واکنش‌های PCR و هضم آنزیمی مورد تأیید قرار گرفت. از آنجایی‌که هدف از این تحقیق جداسازی پروموتر اختصاصی بذر بتافازئولین از لوبیا و استفاده از آن در تهیة سازه‌های ژنی می‌باشد، لذا قطعة موردنظر در ناقل بیانی گیاهی pBI121 ساب‌کلون شد و نتایج آن با هضم آنزیمی و PCR تأیید شد.

کلیدواژه‌ها

• پروموتر اختصاصی
• پروموتر بذری
• آنالیز بیوانفورماتیکی

---

عنوان مقاله [English]

Bioinformatics analysis and isolation of beta-phaseolin promoter from beans (Phaseolus vulgaris)

نویسندگان [English]

• edris choupani ¹
• khadijeh bagheri ²
• bahram maleki ³

¹ zanjan

² tarbiat modares

³ Indian Agriculture Research Institute - Delhi

چکیده [English]

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.

کلیدواژه‌ها [English]

• Bioinformatic analysis
• Seed promoter
• Specific promoter

مراجع

1. Baumlein, H., Nagy, I., Villarroel, R., Inz, D., and Wobus, U.1992. Cis- analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential fortissue-specific expression of a legumin gene. Plant Journal 2: 233-239.

2. Bustos, M.M., Begum, D., Kalkan, F.A., Battraw, M.J., and Hall, T.C. 1991. Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter. The EMBO Journal 10(6): 1469-1479.‏

3. Cahoon, E.B., and Shanklin, J. 2000. Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils. Proceedings of the National Academy of Sciences 97(22): 12350-12355.

4. Chandrasekharan, M.B., Bishop, K.J., and Hall, T.C. 2003. Module‐specific regulation of the β‐phaseolin promoter during embryogenesis. The Plant Journal 33(5): 853-866.‏

5. Chern, M.S., Eiben, H.G., and Bustos, M.M. 1996. The developmentally regulated bZIP factor ROM1 modulates transcription from lectin and storage protein genes in bean embryos. The Plant Journal 10(1): 135-148.

6. De Jaeger, G., Scheffer, S., Jacobs, A., Zambre, M., Zobell, O., Goossens, A., and Angenon, G. 2002. Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nature Biotechnology 20(12): 1265-1268.

7. Ezcurra, I., Ellerström, M., Wycliffe, P., Stålberg, K., and Rask, L. 1999. Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression. Plant Molecular Biology 40(4): 699-709.‏

8. Fehlberg, V., Vieweg, M.F., Dohmann, E.M., Hohnjec, N., Pühler, A., Perlick, A.M., and Küster, H. 2005. The promoter of the leghaemoglobin gene VfLb29: functional analysis and identification of modules necessary for its activation in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots. Journal of Experimental Botany 56(413): 799-806.

9. Goto, F., Yoshihara, T., Shigemoto, N., Toki, S., and Takaiwa, F. 1999. Iron fortification of rice seed by the soybean ferritin gene. Nature Biotechnology 17(3): 282-286.

10. Hall, T.C., Chandrasekharan, M.B., and Li, G. 1999. Phaseolin: its past, properties, regulation and future. In: Seed Proteins Springer Netherlands P. 209-240.

11. Kawagoe, Y., Campell, B.R., and Murai, N. 1994. Synergism between CACGTG (G-box) and CACCTG c/s-elements is required for activation of the bean seed storage protein ß-phaseolin gene. The Plant Journal 5(6): 885-890.‏

12. Keeler, S.J., Maloney, C.L., Webber, P.Y., Patterson, C., Hirata, L.T., Falco, S.C., and Rice, J.A. 1997. Expression of de novo high-lysine α-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants. Plant Molecular Biology 34(1): 15-29.

13. Li, G., Chandler, S.P., Wolffe, A.P., and Hall, T.C. 1998. Architectural specificity in chromatin structure at the TATA box in vivo: nucleosome displacement upon β-phaseolin gene activation. Proceedings of the National Academy of Sciences 95(8): 4772-4777.‏

14. Ma, Y., and Bliss, F.A. 1978. Seed proteins of common bean. Crop Science 18(3): 431-437.

15. Muren, E., and Rask, L. 1995. Processing in vitro of pronapin, the 2S storage-protein precursor of Brassica nupus produced in a baculovirus expression system. European Journal of Biochemistry 227: 316-321.

16. Shintani, D., and DellaPenna, D. 1998. Elevating the vitamin E content of plants through metabolic engineering. Science 282(5396): 2098-2100.

17. Thomas, T.L. 1993. Gene expression during plant embryogenesis and germination: an overview. The Plant Cell 5(10): 1401-1410.‏

18. Van der Geest, A.H., and Hall, T.C. 1997. A 68 bp element of the β-phaseolin promoter functions as a seed-specific enhancer. Plant Molecular Biology 32(4): 579-588.

19. van der Geest, A.H., and Hall, T.C. 1997. The β-phaseolin 5′ matrix attachment region acts as an enhancer facilitator. Plant Molecular Biology 33(3): 553-557.‏

20. Van Droogenbroeck, B., Cao, J., Stadlmann, J., Altmann, F., Colanesi, S., Hillmer, S., and De Jaeger, G. 2007. Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proceedings of the National Academy of Sciences 104(4): 1430-1435.

21. Van Ooyen, A.J.J., Rietveld, K., Quax, W.J., Pen, J., Hoekema, A., Sijmons, P.C., and Verwoerd, T.C. 2006. Production of Enzymes in Seeds and their use.U.S. Patent No. 7,033,627. Washington, DC: U.S. Patent and Trademark Office.

22. Ye, X., Al-Babili, S., Klöti, A., Zhang, J., Lucca, P., Beyer, P., and Potrykus, I. 2000. Engineering the provitamin a (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287(5451): 303-305