泡桐丛枝植原体胸苷酸激酶的原核表达、纯化及酶活性测定  

Prokaryotic Expression,Purification and Enzyme Activity Assay of Thymidylate Kinase of the Paulownia Witches'-broom Phytoplasma

作　　者：宋传生[1] 胡佳续[2] 林彩丽[1] 任争光[1] 耿显胜[3] 田国忠[1] 

机构地区：[1]中国林业科学研究院森林生态环境与保护研究所,国家林业局森林保护学重点实验室,北京100091 [2]天津出入境检验检疫局,天津300457 [3]中国林业科学研究院亚热带林业研究所,浙江富阳311400

出　　处：《林业科学研究》2014年第6期786-793,共8页Forest Research

基　　金：国家自然科学基金(30872025和31170602)

摘　　要：胸苷酸激酶是d TTP从头合成和补救途径的关键酶,催化d TMP形成d TDP,在DNA复制和生物的生存中发挥着必不可少的作用。本文在前期研究的基础上,对从泡桐丛枝(Pa WB)植原体中获得的的3个同源蛋白TMK-a-1、TMK-a-2及TMK-b与已报道的小麦蓝矮(WBD)、洋葱黄化(OY-W)植原体的TMK-a、TMK-b的氨基酸序列进行了比对和相似性分析,结果显示Pa WBPS TMK-b与WBD TMK-2和OY-W TMK-b之间的相似性分别为95.65%和99.03%;Pa WBPS TMK-a-1与Pa WBPS TMK-a-2的相似性为90.57%,且二者与WBD TMK-1和OY-W TMK-a之间的相似性为87.32%-94.26%;而Pa WBPS、WBD、OY-W三种植原体的TMK-a与TMK-b之间的相似性仅为22.22%-25.95%。构建了Pa WBPS TMK-b、TMK-a-1、TMK-a-2的p ET28a原核表达载体,对Pa WBPS TMK-b、TMK-a-1、TMK-a-2 3种蛋白进行了原核表达,经Ni-NTA柱纯化后,利用双酶法进行了胸苷酸激酶催化活性测定,结果表明,Pa WBPS TMK-b具有较高的胸苷酸激酶活性,为85.96±0.74 U·mg-1,而Pa WBPS TMK-a-1和TMK-a-2几乎没有胸苷酸激酶活性。本文为进一步研究胸苷酸激酶在植原体繁殖过程中的作用机理奠定了基础。Thymidylate kinase catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis,and it is crucial for DNA replication and life survival.In previous study,the tmk-a-1,tmk-a-2 and tmk-b genes of Paulownia witches＇-broom Pingshan strain （PaWBPS） were obtained.On the basis of previous study,the amino acid sequence alignment and similarity analysis were conducted among PaWBPS TMK-a1,PaWBPS TMK-a-2,PaWBPS TMK-b,onion yellow wild-type line （OY-W） TMK-a,OY-W TMK-b,white blue dwarf （WBD） TMK-a and WBD TMK-b.The amino acid sequence similarity value was 96.65% between PaWBPS TMK-b and WBD TMK-2,99.03% between PaWBPS TMK-b and OY-W TMK-b,90.57% between PaWBPS TMK-a-1 and PaWBPS TMK-a-2,ranged from 87.32% to 99.26% among PaWBPS TMK-a-1,PaWBPS TMK-a2,WBD TMK-1 and OY-W TMK-a,and from 22.22% to 25.95% among TMK-a and TMK-b of PaWBPS,OY-W and WBD phytoplasma.The pET28a system was used to generate a polyhistidine （polyHis）-tagged TMK fusion protein.The polyHis-tagged TMKs were expressed in Escherichia coli BL21 （DE3） cell and purified under native conditions by Ni-NTA chelating column.Then,the TMK activity was measured using enzyme-coupled assay.Results suggested that the TMK-b had thymidylate kinase activity （85.96 ± 0.74 U · mg^-1） and the TMK-a-1 and TMK-a2 had hardly any activity.

关 键 词：泡桐丛枝植原体 胸苷酸激酶 原核表达 酶活性 

分 类 号：S792.43[农业科学—林木遗传育种]