叶用莴苣高温抽薹相关蛋白的鉴定及其互作分析

The high temperature in summer usually caused earlier bloting,even markedly declined its quality and lossed production of lettuce belonged to the plants with high temperature vernalization, which was a serious problem in cultivation.However, its mechnism was unclear. Therefore, it is important to declear the mechanism of high temperature induced bloting for regulating plant bloting. In this study, it is designed high temperature treatment to induce bloting of two lettuce which was easy or hard to bloting in response to high temperature.Firstly,the differences of bloting characters are observed using microscope between two lettuce subjected to high temperature treatment in order to determine bloting period. Secondly,the differentially expressed proteins between two lettuce are analysed based on proteome using 2D DIGE and iTRAQ technology. Then,the interaction network of known differentially expressed proteins is analysed by bioinformatics in order to define some relative main proteins,and unknown proteins are sequencd.Then these proteins are tested by western blotting and their function are analysed by RNAi method. Integered with the results of western blotting and transgene, the key bloting-associated proteins are determined. Finally, the function pattern of key bloting-associated proteins, including action locus and amount, are disccused using ICP. According to the above analyses, it is expected to determine the key bloting-associated proteins and reveal the molecular mechanism of bloting induced by high temperature, thereby to provide theoretic basis for regulating bloting, as well as resolving the problem of earlier bloting in summer.

叶用莴苣（"高温春化"型）常因夏季高温造成"先期抽薹"而严重影响其品质甚至大面积绝收，是困扰生产的一大难题。但是造成此现象的机理尚不清楚，因此弄清高温诱导"高温春化"型植物抽薹的相关机制对于调控抽薹期具有重要意义。本项目拟选用耐抽薹和易抽薹叶用莴苣品种，通过显微观察比较高温处理后品种间抽薹特性差异，明确抽薹期；在此基础上采用2种蛋白质组学（2D DIGE差异荧光双向电泳和iTRAQ技术）研究手段，互补分析，比较抽薹前后品种间的蛋白质组差异，寻找与抽薹相关的差异表达蛋白；再借助生物信息学对已知蛋白进行功能网络分析来确定一些相对重要的蛋白质,并对未知蛋白从头测序；之后验证这些蛋白的表达，并采用转基因手段分析蛋白质功能来明确几个关键蛋白；最后探讨关键蛋白的互作模式。研究可望明确参与高温抽薹的蛋白质及其相互作用关系，进而揭示"高温春化"型植物高温抽薹的分子机制，为调控其抽薹期，避免"先期抽薹"现象

叶用莴苣常因夏季高温造成“先期抽薹”而严重影响其品质，致使周年生产困难，然而有关高温抽薹的分子机理尚不明确，因此明确“高温春化”型植株抽薹的机理为生产中避免“先期抽薹”提供理论依据。本试验选用2个易抽薹品种进行外观形态和细胞学观察，明确抽薹的具体时期，并对其进行糖组分和内源激素的分析，随后选择1个易抽薹品种利用iTRAQ技术对未抽薹和抽薹期的叶用莴苣进行差异蛋白分析，并对部分的差异蛋白进行荧光定量PCR分析，明确差异转录水平与翻译水平的相关性，通过以上分析，为进一步探讨“高温春化”型植物高温抽薹的内在机制奠定基础。本实验研究结果如下：.1.在高温处理第8 d开始进入抽薹始期，32 d 显著抽薹。.2.在叶片和茎中进入抽薹始期的含量低于未抽薹时的半乳糖、蔗糖、果糖及葡萄糖含量。.3.高水平的内源GA、IAA、ABA和ZR含量，低水平的BR可能促进叶用莴苣抽薹，而MeJA与叶用莴苣抽薹的关系不大。.4.高温处理8 d抽薹和未抽薹植株差异蛋白质组分析共鉴定出1167个蛋白质，在其中有129个蛋白是差异表达蛋白。通过对叶用莴苣差异蛋白进行KEGG pathway和GO分析发现，叶用莴苣差异蛋白质参与的生物过程包括细胞组分组织(26)，代谢过程(84)，信号过程(1)，生物调节过程(10)，生物刺激响应(17)等。其中代谢过程，碳代谢占的比例最大。基因的表达情况表明mRNA和蛋白丰度之间的相关性较低。.5.高温处理32 d抽薹和未抽薹植株差异蛋白质组分析共鉴定出蛋白质 6635 个，其中上调 409 个和下调 349 个。差异蛋白质显著富集的 GO 功能主要为水解酶活性（106）、对刺激的响应（86）、氧化还原（74）等；显著富集到的 KEGG通路为碳代谢（26）、光合作用（15）和色氨酸代谢（4）等。.6. Snakin2、E3UPLALIX7、PSTPKAL 和 PLRLSTPKRIX1 在 Western blot 技术下的蛋白质表达趋势与 iTRAQ 结果一致。基于蛋白质表达量的变化情况，推测 Snakin2 和 PSTPKAL 在抽薹过程中可能起正向调控作用，而 E3UPLALIX7 和 PLRLSTPKRIX1 可能起反向调控作用。.7. 叶用莴苣LsE3、LsTMK1、LsMAPK4基因克隆与表达分析表明，这4个基因与叶用莴苣高温抽薹关系密切。