大豆结荚习性控制基因GmTFL1b启动子解析

Soybean (Glycine max) possesses two types of stem growth habits, indeterminate and determinate. Stem growth habit is one of the most important factors that has direct impact on soybean yield. In our previous study, we isolated GmTFL1b, the responsible gene for Dt1. The GmTFL1b transcript accumulated in the SAMs (shoot apical meristems) during early vegetative growth in both the determinate and indeterminate lines but thereafter was abruptly lost in the determinate line, and this difference was closely associated with the formation of stem growth habits, the determinate lines may condition the determinate habit via the earlier loss in GmTFL1b expression concomitant with floral induction. Many far-red light-related cis-acting elements such as SORLIP1、G-box、I-box、the LAMP-element were identified in GmTFL1b promoter region; And the indeterminate lines possessed two SORLIP1 cis-elements: at 1,487 and 1,306 nucleotides upstream of the start ATG codon, respectively. The first SORLIP1 was absent in the three determinate lines due to the SNP in the motif. Light signals, the length and key area of the promoter, the cis-acting elements and coordinating role between the cis-acting elements, all may affect gene expression pattern. In this study, we will compare the expression of GmTFL1b by qRT-PCR after or without far-red light induction , so as to determine that whether far-red light has a regulation role in GmTFL1b expression; Previous results showed 2047bp upstream translational start site of the GmTFL1b can fully recapitulate the pattern of expression of the endogenous GmTFL1b transcript, we will further delimit the upstream regulatory region of GmTFL1b and generate the reporter constructs, according to the expression pattern of the reporter gene in SAMs, the key sections of the promoter can be determined. To identify putative cis-acting elements that might contribute to the expression pattern of GmTFL1b, electrophoretic mobility shift assays (EMSAs) in combination of competition assays will be performed using the SAMs for whole-cell plant extracting and radiolabeled fragments of the GmTFL1b promoter. To test the role of the putative cis-acting elements in regulation of GmTFL1b gene expression, point mutations that can disrupt protein binding in vitro will be identified, and mutations will be therefore tested in vivo for its effect on GmTFL1b expression. We will reveal the mechanisms of GmTFL1b expression pattern based on functional analysis of the cis-acting elements.

大豆结荚习性直接影响大豆单株产量。我们前期克隆了大豆结荚习性基因GmTFL1b，无限型中GmTFL1b在顶端分生组织中表达相对于有限型持续时间更长，这种差异与无限、有限结荚习性的形成密切相关；GmTFL1b启动子中有很多与远红光相关的顺式作用元件；并且无限型大豆GmTFL1b启动子区有两个完整SORLIP1元件，而有限型中只有一个。光信号、启动子长度及作用区域、顺式作用元件及元件之间的协调作用，对基因表达调控至关重要，本研究拟通过qRT-PCR的方法比较远红光诱导和非诱导下GmTFL1b的表达，分析远红光对基因表达的影响；将无限型中GmTFL1b启动子分区与报告基因连接，依据转基因拟南芥中报告基因是否在顶端分生组织中表达及表达的持续性，确定启动子关键区段；并通过竞争结合和点突变实验确定出关键顺式作用元件及对GmTFL1b表达的影响。最终从顺式作用元件角度揭示GmTFL1b被调节表达的机制。

大豆结荚习性直接影响大豆单株产量。我们前期克隆了大豆结荚习性基因GmTFL1b，无限型中GmTFL1b在顶端分生组织中表达相对于有限型持续时间更长，这种差异与无限、有限结荚习性的形成密切相关。我们获得、分析了包含GmTFL1b启动子有效范围的2388bp全长序列，精细的确定了GmTFL1b启动的有效区域，初步确定GmTFL1b启动子中起关键调控作用的元件可能在p582-p416之间，初步确定p582-p416之间“GATA”box和“SORLIP2AT”motif可能同时协同调控了该启动子的功能，而且“SORLIP2AT”motif在其中起的作用可能更大。在长日下，有限型大豆出苗后25d天时表达量已经减弱，而无限型大豆中出苗35d时依旧表达表达；在短日情况下，有限型大豆出苗后25d天时表达量已经几乎没有表达，而无限型大豆中出苗30d时依旧有微弱表达。而且在短日照情况下GmTFL1b的表达明显低于在长日照情况下的表达。大豆是短日照作物，长日条件会抑制大豆开花，因此有限结荚习性的形成可能就是通过GmTFL1b 在顶端分生组织中提前终止表达从而诱导开花。在添加远红光下GmTFL1b的表达在有限结荚型大豆与无限结荚型大豆中呈现截然相反的模式，且发现有限型大豆Misuzudaizu(MI)及无限型大豆Moshidou Gong 503(MO)中GmTFL1b基因在伤害及JA处理后表达量随时间显著上调，且MO中该基因响应JA处理的程度或响应的速度显著高于MI中。而MI中与MO中响应JA基因差异很可能与二者启动子中的差异密切相关。本研究将有助于从从顺式作用元件角度及激素调控角度揭示GmTFL1b被调节表达的机制，对基因资源、分子元件的发掘，及分子设计育种等领域的研究都具有重要的科学意义和应用价值。