【摘要】：為了揭示二酰甘油�；D(zhuǎn)移酶(DGAT)在小球藻油脂合成過程中的作用,對單細(xì)胞小球藻Chlorella variabilis NC64A的二酰甘油酰基轉(zhuǎn)移酶進(jìn)行原核克隆表達(dá)及功能初步研究。結(jié)果表明,其編碼序列為894bp,編碼297個(gè)氨基酸,表達(dá)蛋白的表觀分子量為33 k Da,p I 9.48。保守結(jié)構(gòu)域分析表明,該蛋白屬于Lysophospholipid acyltransferases(LPLATs)超家族,具有二酰甘油�；D(zhuǎn)移酶活性,序列位點(diǎn)H68,L71,F76,R94,I97和GAA(144?146)組成特定的�；荏w結(jié)合口袋,能夠結(jié)合�；�?酰基載體蛋白(ACP)或者�；o酶A上的�；�,催化三酰甘油合成的最后一步。表達(dá)蛋白與Ss PDAT和At PDAT的序列相似性分別為32%和24%,表明該蛋白可能具有磷脂酰甘油�；D(zhuǎn)移酶(PDAT)活性,能利用磷脂上的�；铣扇８视�。因此,采用薄層層析方法以L-α-磷脂酰膽堿和1,2-二油酰-sn-甘油為底物,檢測到其確實(shí)具有PDAT活性,表明限氮條件下NC64A中DGAT的PDAT活性可能促進(jìn)了膜脂降解耦合三酰甘油的合成。
[Abstract]:In order to elucidate the role of diacylglyceryl transferase (DGAT) in the synthesis of Chlorella oil, the prokaryotic expression and function of diacylglyceryl transferase (Chlorella variabilis NC64A) of Chlorella sp. Chlorella variabilis NC64A were studied. The results showed that the encoding sequence was 894 BP, encoding 297 amino acids, and the apparent molecular weight of the expressed protein was 33k Da,p I 9.48. The conserved domain analysis showed that the protein belongs to the Lysophospholipid acyltransferases (LPLATs) superfamily and has the activity of diacylglyceryl transferase. Can bind acyl groups? Acyl carrier protein (ACP) or acyl coenzyme A catalyzes the last step in the synthesis of triacylglycerol. The sequence similarity between the expressed protein and Ss PDAT and At PDAT were 32% and 24% respectively, which indicated that the protein might have the activity of phosphatidyl glyceryl transferase (PDAT) and could synthesize triacylglycerol from phospholipid acyl group. Therefore, using L- 偽 -phosphatidylcholine and 1- 2-dioleyl-sn- glycerol as substrates, it was found that L- 偽 -phosphatidylcholine and 1 ~ (2 +) -dioleyl-sn- had PDAT activity by thin-layer chromatography. The results showed that the PDAT activity of DGAT in NC64A could promote the synthesis of lipide-coupled triglycerides under nitrogen limit.
【作者單位】： 農(nóng)業(yè)生物技術(shù)國家重點(diǎn)實(shí)驗(yàn)室中國農(nóng)業(yè)大學(xué)生物學(xué)院;
【基金】：863計(jì)劃(2013AA065802) 農(nóng)業(yè)生物技術(shù)國家重點(diǎn)實(shí)驗(yàn)室2014年開放課題(2014SKLAB06)資助
【分類號】：Q943.2

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