เอกสารการประชุมวิชาการและเสนอผลงานวิจัย มหาวิทยาลัยทักษิณ ครั้งที่ 19 2552 - page 313

„µ¦ž¦³»¤ª·µ„µ¦Â¨³Áœ°Ÿ¨Šµœª·‹´¥ ¤®µª·š¥µ¨´¥š´„¬·– ‡¦´ÊŠš¸É 19 ž¦³‹Îµže 2552
Abstract
Rubber elongation factor (REF) is a key gene in rubber biosynthesis in Hevea brasiliensis. The Hevea
REF (HbREF) had been shown higher expression in high latex yielding Hevea clone than the low one. The
objective of this research is to express the HbREF protein in E.coli. The HbREF gene was directionally cloned
into expression vector pSUMO and the resulting recombinant plansmid pSUMO–HbREF was transformed into
E. coli BL21 plus (DE3) RIL. The positive clone was selected and the open reading frame of HbREF was
determined by PCR and sequencing analysis. The factors, including IPTG concentration, incubation temperature
and induction time were optimized during the induction of target protein. The proteins were analyzed by SDS-
PAGE. The results showed that, selected positive clone was contained recombinant plansmid pSUMO–HbREF
and corrected open reading frame. The Hb REF protein was successfully expressed by E .coli BL21 plus (DE3)
RIL/pSUMO– HbREF. The optimal conditions for Hb REF expression were IPTG concentration of 0.1 mM,
incubation at 37 qC for 6 hours. However, recombinant protein was expressed as inclusion body by all induced
conditions.
Keywords: rubber elongation factor (REF); recombinant protein; Hevea brasiliensis
‡Î µœÎµ
‹µ„„µ¦ª· Á‡¦µ³®r®µ¥¸œš¸É¤¸„µ¦Â—Š°°„˜„˜nµŠ„´œ¦³®ªnµŠ„¨»n¤µ¥¡´œ›»r¥µŠÄ®oŸ¨Ÿ¨· ˜œÊε¥µŠ¼ŠÂ¨³˜Éε
—oª¥Áš‡œ·‡ cDNA-AFLP ‡´—Á¨º°„ transcription-derived fragments (TDFs) š¸É ¤¸‡ªµ¤Á o¤¤µ„Äœ„¨»n ¤¥µŠµ¥¡´œ›»rÄ®o
Ÿ¨Ÿ¨· ˜¼ ŠÅžª· Á‡¦µ³®r®µ¨Î µ—´ÁÂ¨³Áž¦¸ ¥Áš¸¥„´ ¨Î µ—´ Á °Š¥¸ œÄœ“µœ o °¤¼ ¨ (GenBank) ¡ªnµ
TDFLH363 ¤¸ ¨Îµ—´ÁÁ®¤º°œ„´¥¸œ REF (rubber elongation factor) š¸É ¦µ¥ŠµœÅªo×¥ Priya ¨³‡–³ (2007)
(GenBank accession no. AY120685) REF Áž}œÃž¦˜¸ œš¸É¤¸šµšÎµ‡´Äœ„¦³ªœ„µ¦´ŠÁ‡¦µ³®r cis -1,4-
polyisoprene ×¥šÎ µ®œoµš¸É¦nª¤„´ cis-prenyl transferase Ĝ„µ¦˜n°®œnª¥ isoprene Ä®o„´µ¥ cis-1,4 polyisoprene
(Dennis ¨³ Light , 1989; Dennis ¨³‡–³, 1989) ¥µŠ¡µ¦µš¸ÉÁžd—„¦¸—¨o ª°µ¥»ž¦³¤µ– 17 že ¤¸„µ¦Â—Š°°„ °Š
¥¸œ REF Ĝ„¨»n¤µ¥¡´œ›»r Ä®oŸ¨Ÿ¨·˜œÊÎ µ¥µŠ¼Š¤µ„„ªnµ„¨»n ¤µ¥¡´œ›»rÄ®oŸ¨Ÿ¨· ˜˜Éε°¥nµŠ´—Á‹œ (Priya ¨³‡–³,
2007) 宦´ Äœ„¨oµ¥µŠ°µ¥» 4 Á—º°œ„µ¦Â—Š°°„ °Š¥¸ œ REF Ĝ„¨»n¤µ¥¡´œ›»rÄ®oŸ¨Ÿ¨·˜œÊε¥µŠ¼Š (4 µ¥¡´œ›»r)
¨³„¨»n ¤µ¥¡´œ›»rÄ®o Ÿ¨Ÿ¨·˜œÊε¥µŠ˜Éε (4 µ¥¡´ œ›»r) Ťn˜„˜n µŠ„´œ Á¤ºÉ°„¨oµ¥µŠ°µ¥» 18 Á—º °œ „¨»n¤µ¥¡´œ›»rÄ®o
Ÿ¨Ÿ¨·˜œÊÎ µ¥µŠ¼Šš´ÊŠ®¤—¤¸„µ¦Â—Š°°„ °Š¥¸ œ REF ¤µ„„ªnµ„¨»n¤µ¥¡´œ›»rÄ®oŸ¨Ÿ¨· ˜œÊε¥µŠ˜Éε‹Îµœªœ 3 µ¥¡´œ›»r
(ª´œÁ¡È ¨³‡–³, 2552) ®µ„°µ¥»°Š¥µŠÁ¡·É¤ ¹Êœ„µ¦Â—Š°°„ °Š¥¸œ REF œnµ‹³µ¤µ¦™‹ÎµÂœ„¡´œ›»r¥µŠ„¨»n¤µ¥
¡´ œ›»rÄ®oŸ¨Ÿ¨·˜œÊε¥µŠ¼Š°°„‹µ„„¨»n¤Ä®oŸ¨Ÿ¨·˜œÊε¥µŠ˜Éεŗo °¥nµŠ´—Á‹œš»„µ¥¡´ œ›»r Ž¹É Š°µ‹‹³µ¤µ¦™Äo¥¸œ REF
Áž}œÁ‡¦ºÉ°Š®¤µ¥Äœ„µ¦‡´—Á¨º°„¡´ œ›»r¥µŠÄ®oŸ¨Ÿ¨·˜œÊÎ µ¥µŠ¼ŠÄœÃž¦Â„¦¤„µ¦‡´—Á¨º °„¡´œ›»r¥µŠÅ—o ˜nÁœºÉ°Š‹µ„ª· ›¸
˜¦ª‹ª´—„µ¦Â—Š°°„ °Š¥¸ œ—oª¥Áš‡œ·‡ RT-PCR ¤¸®¨µ¥ ´Êœ˜°œÂ¨³˜oœš»œ‡n°œ oµŠ¼Š ‹¹Šœnµ‹³¡´•œµÁž} œª·›¸ „µ¦
enzyme linked immunosorbent assay (ELISA) Ž¹É Š¤¸‡nµÄn‹nµ¥š¸É˜É優nµÂ˜n˜o°ŠÄo Þ¦˜¸œ °Š REF Áž}œÁ°œ˜· Á‹œÄœ
„µ¦¦oµŠÂ°œ˜· °—¸