Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(...Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism.展开更多
文摘Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism.
