CAJ | 학술논문

近年来,人们发现丝氨酸羧肽酶类蛋白(SCPL)参与植物次生代谢产物的酰基转移过程,即具有转酰基功能.本研究采用 RT-PCR 技术,获得了3个茶树丝氨酸羧肽酶基因的全长序列;生物信息学分析表明,3个 CsSCPL蛋白均包含了1个底物结合位点、3个催化作用保守区和多个 N-糖基化位点,及其 Ser-Asp-His 三联体催化中心等 SCPL 家族的典型特征;进化树分析表明,3个 CsSCPL 可能具有酰基转移酶的功能.实时荧光定量 PCR 结果表明3个基因在芽叶茎根中都有表达,其中,CsSCPL1和 CsSCPL3在叶中的相对表达量明显高于茎和根,而CsSCPL2则在根中高表达.本研究成功地将 CsSCPL 重组到表达载体 pET32a(+)上进行原核表达,并对诱导时间及诱导温度进行了优化;经 IPTG 诱导、SDS-PAGE 检测,目标蛋白条带分子量为70 kD,与预测大小相符.
근년래,인문발현사안산최태매류단백(SCPL)삼여식물차생대사산물적선기전이과정,즉구유전선기공능.본연구채용 RT-PCR 기술,획득료3개다수사안산최태매기인적전장서렬;생물신식학분석표명,3개 CsSCPL단백균포함료1개저물결합위점、3개최화작용보수구화다개 N-당기화위점,급기 Ser-Asp-His 삼련체최화중심등 SCPL 가족적전형특정;진화수분석표명,3개 CsSCPL 가능구유선기전이매적공능.실시형광정량 PCR 결과표명3개기인재아협경근중도유표체,기중,CsSCPL1화 CsSCPL3재협중적상대표체량명현고우경화근,이CsSCPL2칙재근중고표체.본연구성공지장 CsSCPL 중조도표체재체 pET32a(+)상진행원핵표체,병대유도시간급유도온도진행료우화;경 IPTG 유도、SDS-PAGE 검측,목표단백조대분자량위70 kD,여예측대소상부.
In recent years, serine carboxypeptidase-like proteins (SCPL) have been found that they are involved in plant secondary metabolites with the function of transferring acyl. The full-length cDNA of three CsSCPL were cloned from Camellia sinensis by RT-PCR technology. Bioinformatics analysis showed that the three CsSCPL proteins contained SCPL family's characteristic structures, such as one substrate binding and three catalysis conserved regions, a number of N-glycosylation sites and a conserved catalytic triad Ser-Asp-His amino acid active catalytic site and so on. The phylogeny analysis showed that CsSCPL probably possessed acyltransferase function. Quantitative RT-PCR analysis showed that the CsSCPL genes expressed in bud, leaf, stem and root. The relative expression of CsSCPL1 and CsSCPL3 in the leaves was significantly higher than that in stems and roots, while CsSCPL2 was highly expressed in the root. The CsSCPL genes were constructed into expression vector pET-32a(+) for over expression in prokaryotic cells and optimal inducing conditions including time, temperature were studied. The SDS-PAGE showed that recombinant proteins with formula weight 70 kD were induced successfully by IPTG, which coincided with the prediction.