夜间低温诱导ABA抑制设施番茄叶片气孔张开的miRNA作用机制研究

The protected vegetables in winter and spring seasons cultivation easily suffer from low night temperature(LNT),which inhibit the photosynthesis.Stomatal limitation closely related to ABA is the key factor of photosynthesis inhibition.Studies show that microRNA(miRNA) is the negative factor in the gene expression and plays an important role in regulating stomatal opening of plant leaf inhibited by ABA. Therefore, it is significance to parse the mechanism of the inhibition in photosynthesis under LNT by clarifying the mechanism of miRNA on stomatal limitation of tomato leaf induced by LNT.Wild type tomato (RR) and ABA-deficient mutant (sit) treated by LNT(6 ℃) will be selected as the research matierials in this study. Firstly, we will identify miRNAs ,which regulate stomatal opening of tomato leaf inhibited by ABA, by high-throughout sequencing technology and the bioinformatics methods.Then screen the target miRNAs by analysis of differential gene expression with qRT-PCR.Finally,we will over-express the target miRNAs and use artificial microRNA(amiRNA)technology,construction vector and get over-expressed and silencing transgenic tomato plants to verify the function of miRNAs.It is expected to clarify the mechanism of miRNA on stomatal opening inhibited by ABA under LNT, thereby to establish a scientific foundation for clarifying the mechanism of the inhibition of the photosynthesis of tomato under LNT,as well as,providing a new method for regulating photosynthesis.

设施冬春茬蔬菜极易遭受夜间低温而导致光合抑制，其抑制的关键因素是与ABA密切相关的气孔限制；有研究表明miRNA作为基因表达中的一类负调控因子，在调控ABA抑制气孔张开中起重要作用。因此，弄清miRNA在夜间低温导致番茄叶片气孔限制中的作用机制，对于解析番茄光合的低夜温抑制具有重要意义。本研究以低夜温处理的野生型番茄（RR）和ABA缺失突变体（sit）为试材，通过高通量测序技术及生物信息学方法，鉴定出调控ABA抑制番茄叶片气孔张开的miRNA，并结合qRT-PCR技术明确基因表达差异，筛选出目的miRNA；进一步通过目的miRNA超表达和人工miRNA技术，构建载体，获得过表达和沉默的转基因植株，进行目的miRNA基因功能验证,从而阐明夜间低温诱导ABA抑制番茄叶片气孔张开的miRNA作用机制，为探明夜间低温抑制番茄光合作用的机理及其调控新方法奠定基础。

采用solexa高通量测序4个小RNA库及生物信息学方法，将两两样品之间的表达量存在显著性差异的已知miRNA进行统计，初步筛选出在各两两样品均存在差异表达的miR156/160a/162/166b/1919/5300以及预测的novel-m0005/m0088-3p，并通过qRT-PCR进一步验证，其结果与高通量测序的结果基本保持一致；采用qRT-PCR技术检测叶片中6个miRNA（miR160、miR162、miR166、miR1919、miR397 和miR6026）及其预测的靶基因在夜间低温和喷施外源ABA逆境胁迫下的表达情况，结果表明，在15 ℃夜温（对照）下，sit 的ABA含量是60ng•g-1，RR是120ng•g-1，sit叶片的保卫细胞和气孔的开度都明显大于野生型RR，ABA 缺失突变体sit中miRNA 的表达量与野生型RR相比都显著下调，其靶基因除miR1919外，表达量都上调；夜间低温（6 ℃）处理12 h 后，所有 miRNA 在sit中比RR中的表达量都显著增加；喷施80mg•L-1的ABA后，sit 中miR162、miR166、miR1919及miR397比0mg•L-1ABA处理的表达量显著上调。推测miR160、miR162和miR6026 通过依赖ABA信号途径参与夜间低温胁迫；最后我们选择miR162进行载体构建和遗传转化，获得T0代，进行功能验证。