白桦E-box元件的载体构建及与BplMYB46转录因子的互作分析

Construction of E-box Motifs and the Analysis of Its Interaction with BplMYB46 Transcription Factor of Betula platyphylla

MYB转录因子是植物中最大的转录因子家族成员之一,主要参与植物次生代谢调控、激素和环境因子的应答,在植物的生长发育中起着至关重要的作用。已有研究发现,E-box的核心序列为CANNTG(N:A/G/C/T),是一类与光响应和苯丙氨酸生物合成途径相关的元件。在前期研究中,先构建顺式作用元件库,然后以转录因子为中心的酵母单杂交技术筛选发现,白桦的BplMYB46转录因子能够与核心序列为CAAATG的E-box顺式作用元件结合。但是,是否能与E-box的其他核心序列结合还不清楚。本研究将每一种E-box顺式作用元件的核心序列分别进行双链DNA的复性并连接到pHIS2载体上,然后通过酵母单杂交技术筛选与BplMYB46转录因子能够特异结合的E-box顺式作用元件。结果显示,当E-box的核心序列第3个碱基为A/T/C、第四个碱基为A/G/C时,酵母单菌落能在三缺培养基TDO/3AT上生长,表明与BplMYB46转录因子结合的E-box顺式作用元件的特异性序列为CA(A/T/C)(A/G/C)TG。为后续通过BplMYB46转录因子与E-box顺式作用元件的结合来分析BplMYB46转录因子对下游基因的调控,以及为筛选优良下游基因改良白桦的遗传性状奠定数据基础。

MYB transcription factor is one of members in the largest family of transcription factors in plants;it is mainly involved in the regulation of plant secondary metabolism,the response of hormones and environmental factors,and thus plays a crucial role in plant growth and development.A study reveals that the core sequence of E-box is CANNTGN(N:A/G/C/T),which is a cis-element associated with the response to light and phenylpropanoid biosynthesis pathway.A library of cis-acting elements was constructed in our previous study,and results by a transcription factor-centered yeast one-hybrid technology showed that the BplMYB46transcription factor bound to the E-box cis-acting element with a core sequence of CAAATG in birch;however,it is unclear whether BplMYB46may bind to other core sequence of E-box.Therefore,in this study,the renaturation of the double-stranded DNA of every core sequence of E-box cis-acting element was performed and connected to the pHIS2vector.Further,the yeast one-hybrid technique was used to select E-box cis-acting elements that specifically bound to the BplMYB46transcription factor.The results showed that the yeast colony grew on TDO/3AT medium when the third nucleotide of the core sequence of E-box was A/T/C and the fourth was A/G/C,respectively,indicating that the specific sequence of E-box cis-acting element bound by BplMYB46transcription factor was CA(A/T/C)(A/G/C)TG.This study provides a data basis for improving the genetic traits of birch by analyzing the regulation of BplMYB46transcription factor to downstream genes and screening downstream genes via the binding of BplMYB46transcription factor with E-box cis-acting element.