摘要
脂肪族芥子油苷是一类含氮和硫的植物次生代谢产物,其侧链来源于甲硫氨酸。在拟南芥中存在三种正向调控脂肪族芥子油苷生物合成的R2R3-MYB家族转录因子,分别是MYB28、MYB29和MYB76、由于这三个转录因子之间存在很强的相互作用,因此其独立的功能尚未被完全解析。在MYB28和MYB29基因的双敲除突变体myb28myb29中,三个转录因子的基因表达水平均急剧下降,脂肪族芥子油苷的含量也接近于零,这为解析三个转录因子的独立功能提供了绝佳的突变背景。在以往芥子油苷基因功能解析的研究中,过表达和恢复表达的转基因工作多采用组成型的强启动子。然而基因自身的启动子是其转录调控的一部分,在转录因子的研究中采用自身的启动子来控制MYB的转基因表达更能反映真实的调控状态。
我们以野生型拟南芥DNA为模板,分别克隆了包含自身启动子的MYB28、MYB29和MYB76基因片段并将其转导至myb28myb29突变体中,建立了三个基因的自启动子转基因植株ProMYB28:MYB28、ProMYB29:MYB29和ProMYB76:MYB76。通过检测ProMYB:MYB转基因植株中脂肪族芥子油苷的含量及其合成基因的表达水平并与野生型和双突变体进行比较,判断脂肪族芥子油苷合成能力的恢复水平,从而解析独立的MYB转录因子的功能。
结果显示,在ProMYB2B:MYB28转基因植株中,MYB28能够激活脂肪族芥子油苷合成途径的大部分基因并恢复短链脂肪族芥子油苷的合成。MYB29出能够激活相同的途径,但是对位于上游的侧链延长循环反应相关基因的诱导能力弱于MYB28,因此ProMYB29:MYB29转基因植株中脂肪族芥子油苷含量的恢复水平较低。由于MYB28和MYB29都不能激活MAM3基因的表达,ProMYB28:MYB28和ProMYB29:MYB29植株均无法合成长链脂肪族芥子油苷,表明独立的MYB因子缺乏对长链脂肪族芥子油苷的诱导能力。MYB76不能单独激活任何脂肪族芥子油苷的合成,并且MYB76的基因表达不能被自身所激活,而需要由MYB28或MYB29来诱导其表达。缺硫胁迫实验表明,MYB28基因的表达受到硫缺乏的诱导,MYB29和MYB76比MYB28对环境变化更加敏感,MYB28的表达受到MYB29和/或MYB76的抑制。
综上,本实验解析了独立的MYB转录因子在自启动子的调控下如何调控脂肪族芥子油苷的生物合成。
Aliphatic glucosinolates (AGSLs) are nitrogen-and sulfur-containing plant secondary metabolites which are derived from methionine. Arabidopsis transcription factors, MYB28, MYB29and MYB76, positively regulate aliphatic glucosinolate biosynthesis. The interaction among these MYBs is complex, and their individual functions have not been fully elucidated. In a functional multiple-knockout by myb28myb29, the expression of all three MYBs was repressed and no AGSL was detected, providing a ideal transgenic background for elucidating individual MYB function. In previous studies, overexpression and complementary of MYBs was generally driven by constitutive strong promoters. However, own promoter of a gene consists an important part of transcriptional regulation. In study of transcription factors, control of MYBs driven by their own promoters can reflect more realities.
Using wild-type Arabidopsis DNA as template, three MYB genes containing their own promoters were cloned and transformed to myb28myb29, and individual transgenic lines of three MYB genes driven by their own promoters were. We analyzed the AGSL contents and expression levels of AGSL biosynthetic genes.
MYB28expression activated most of the biosynthetic genes and rescued accumulation of short-chain AGSLs. MYB29also activated the same pathway, but induction of genes involved in side-chain elongation was weaker than that by MYB28, resulting in a minor rescue of short-chain AGSLs. Neither MYB28nor MYB29recovered accumulation of long-chain AGSLs, probably due to the inability to induce MAM3expression. MYB76could not solely activate AGSL biosynthesis, and MYB76expression was induced by MYB28and MYB29. A sulfur deficiency experiment suggested that the expression of MYB28was induced by sulfur deficiency, and expression of MYB29and MYB76responds more sensitively to sulfur stress than MYB28. Furthermore, MYB29and/or MYB76inhibited the induction of MYB28expression.
We illustrate the unique functions of individual MYB transcription factors driven by their own promoters.
引文
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