目的 克隆地黄多药及有毒化合物外排(multidrug and toxic compound extrusion,MATE)转运蛋白基因RgMATE6,并进行亚细胞定位与时空表达分析,为深入研究其在地黄转运次生代谢产物中的分子功能奠定基础.方法 利用地黄转录组数据库候选基因序列,利用实时荧光PCR (RT-PCR)克隆RgMATE6基因;利用生物信息学软件对其编码蛋白的结构、理化性质、同源性和进化树进行分析;通过与绿色荧光蛋白(GFP)融合表达进行亚细胞定位;利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)的方法测定该基因的时空表达模式.结果 克隆获得1892bp的RgMATE6基因序列,RgMATE6基因编码524个氨基酸、具有2个典型的MatE结构域和12个跨膜结构;RgMATE6蛋白与越橘的VcMATE6蛋白亲缘关系最近;RgMATE6-GFP载体瞬时表达的结果发现RgMATE6蛋白定位在液泡膜上;qRT-PCR分析发现RgMATE6基因在地黄不同时期的根中表达最高,尤其是块根膨大前期.结论 RgMATE6蛋白定位在液泡膜上,在地黄块根膨大期的根中表达较高,初步表明RgMATE6可能参与地黄体内次生代谢产物向液泡转运的过程.

[目的]克隆TaARF20基因,并分析其表达特性,为解析小麦生长素响应因子(ARF)基因家族成员的调控机制提供理论依据.[方法]以普通小麦为材料,利用同源克隆技术获得TaARF20基因的cDNA全长序列,利用生物信息学软件分析其序列特征,并通过亚细胞定位试验明确TaARF20蛋白的作用部位.将TaARF20基因与表达载体Pcold-TF连接,转化大肠杆菌中进行原核表达.采用实时荧光定量PCR检测TaARF20基因在普通小麦不同组织及普通小麦和多子房小麦不同发育时期幼穗中的表达模式.[结果]TaARF20基因包含1个内含子和2个外显子,编码区(CDS)长度为1116 bp,编码371个氨基酸残基,蛋白分子量约40.38 kD,理论等电点(pI)为4.96,脂溶性系数为19.80,疏水性系数为0.985,不稳定系数为50.51,属于不稳定蛋白,定位在细胞核中,含有ARF家族蛋白的保守结构域和B3 DNA结合域,主要由α-螺旋(18.06％)、无规则卷曲(59.57％)和延伸连(22.10％)组成.启动子区含有激素响应、光响应和低温响应等多个顺式作用元件.TaARF20蛋白与二粒小麦ARF20的氨基酸序列相似性最高,亲缘关系也最近.TaARF20融合蛋白在原核系统中成功表达.TaARF20基因在小麦的根、叶、幼穗和籽粒中均有表达,但在幼穗中的相对表达量最高.对于长度为3和4 cm的幼穗来说,普通小麦TaARF20基因的相对表达量低于多子房小麦,但对于长度为5、6、8和9 cm的幼穗,普通小麦TaARF20基因的相对表达量高于多子房小麦.[结论]TaARF20基因在小麦穗生长发育中发挥重要调控作用,推测其是调控穗型、穗大小或穗粒数的关键基因.

The continuous cultivation of Rehmannia glutinosa is problematic due to its release of auto toxic allelochemicals into the rhizosphere. Phenolics have been implicated as potential agents of this autotoxicity in R. glutinosa. Although Multidrug and Toxic Compound Extrusion Transporters (MATEs) are known to mediate the release of certain toxins in some plants, the identification of MATEs, which contribute to the release of phenolics, which have not been explored in R. glutinosa. Here, we scanned R. glutinosa transcriptome sequences and identified 66 RgMATE transcripts. An in silico analysis of the RgMATEs identified 9-12 transmembrane domains which predicted that most of them were deposited on plasma membrane and function as transporters. The phylogeny and homology analysis implied that ten of the RgMATEs were potentially associated with the release of phenolics. The transient expression showed that RgMATE33 and RgMATE46 were both localized in plasma membrane. Positive correlations were established between the expression abundance of eight RgMATE candidate genes in hairy roots and total phenolics content in their exudation. The conclusion was that the eight RgMATE efflux transporters likely contributed to the release of phenolics into R. glutinosa rhizosphere. Our study will lay the foundation for revealing the molecular basis of autotoxicity formation. (C) 2020 Friends Science Publishers

Following publication of the original article [1], authors reported Pro. Gang Zhao has to be considered as another corresponding author, according to his important contribution.