烟草NtTkr尾部点突变对卷曲螺旋结构及与靶蛋白相互作用的影响

doi:10.13560/j.cnki.biotech.bull.1985.2018-0957

摘要/Abstract

摘要： NtTkr是个在烟草分裂旺盛组织中优势表达的驱动家族新成员,尾部有3个卷曲螺旋（Coiled-coil,CC1-3）,已知位于CC3 1084位的T缺失或点突变会影响其分布,推测该改变可能受与其结合的蛋白底物的影响。为确定T1084缺失或点突变对NtTkr与候选蛋白互作的影响,通过重叠延伸PCR在烟草NtTkr尾部引入T1084缺失（T1084d）和T1084A点突变,克隆入pGBKT7获诱饵表达载体,转入AH109并分别与携带pGADT7-H2A/Sulfer/NT3的Y187接合后进行双杂交检测。结果表明T1084A与T相近,但先于T显蓝色,且颜色较T深;T1084d明显不如T;一级结构和Coiled-coil软件分析表明T1084处于CC3的a位,属极性亲水氨基酸,将其替换为非极性疏水的丙氨酸可增强七肽重复序列结构域（HR结构域）的疏水性,并造成七肽重复序列增长,T1084缺失则造成其缩短。表明增加HR结构域的疏水性和七肽重复序列的长度可增强NtKrp与候选蛋白的互作,而缩短卷曲螺旋的长度则造成其相互作用的减弱。

关键词: NtTkr, 卷曲螺旋, 定点突变, 蛋白相互作用

Abstract: As a novel member of kinesin family,NtTkr is predominantly expressed in the vigorous division tissues of tobacco,and in it there are 3 coiled-coils（CC1,CC2 and CC3）. It is known that the point mutation and T deletion of T1084 in the conventional CC3 motif of the tail may affect the distribution of NtTkr,and which is inferred to be affected by the protein substrate binding with it. In order to determine the effects of deletion and point mutations of T1084 on the interaction with candidate proteins,T1084 deletion（T1084d）and T1084A point mutations were introduced into the tobacco NtTkr tail by overlap extension PCR,cloned into pGBKT7 to construct bait expression vector,and transferred to AH109 and mated to Y187 with pGADT7-H2A/Sulfer/NT3 for two-hybrid analysis. The results showed that T1084A was similar to T,but bluing earlier and the color was darker than T;while T1084d did not perform as T did. The primary structure and Coiled-coil software analysis showed that T1084 was located in the ‘a’ position of CC3 and was a polar hydrophilic amino acid. Substituting it with non-polar hydrophobic alanine enhanced the hydrophobicity of heptapeptide repeat domain（HR domain）,and resulting in the prolongation of repeated sequence of heptapeptide,while the deletion of T1084 resulted in its shortening. The results indicate that increasing the hydrophilicity of HR domain and the length of heptapeptide repeat sequence may enhance the interaction of NtKrp with the candidate protein,while shortening of coiled coil may result in a decrease of their interaction.

Key words: NtTkr, coiled-coil, site-directed mutagenesis, protein interaction

徐林娜, 胡孟可, 童文艳, 李芬. 烟草NtTkr尾部点突变对卷曲螺旋结构及与靶蛋白相互作用的影响[J]. 生物技术通报, 2019, 35(5): 64-69.

XU Lin-na, HU Meng-ke, TONG Wen-yan, LI Fen. Effects of T1084d and T1084A Point Mutations in the NtTkr Tail of Nicotiana tabacum on Coiled-helix Structure and Interaction with Target Proteins[J]. Biotechnology Bulletin, 2019, 35(5): 64-69.

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