基于叶绿体蛋白组的荫蔽对大豆光合特性的调控及其机理研究

In China, half of the total grain yield is produced with multiple cropping, intercropping soybean is one of major planting patterns in the southern regions, which plays an important role in increasing planting acreage and grain yield of soybean. Shading decreasing photosynthesis results in yield loss in intercropping soybean production, and chloroplast proteins perform a vital function in the process of photosynthesis. Generally, shading includes the changes of light quantity and light quality, and our studies also found that there are significant relationship of morphological parameters of soybean seedling with R/FR ratio and PAR transmittance in maize–soybean relay intercropping system, especially R/FR ratio of soybean canopy. However, most previous studies concerning some proteins response of soybean photosynthesis to shading have mainly focused on artificial shading or single light quality. Artificial shading with shade nets or screens does not change the components of the spectrum. However, the response mechanisms of soybean photosynthetic characteristics to shading (light quantity and light quality) by analyzing chloroplast proteomics are unclear. Therefore, the experiments of field and indoor pot are set for analyzing the photosynthetic characteristics of soybean under shading conditions. At the same time, the effects of light quantity and R/FR ratio on abundance of the chloroplast proteomics are determined using the iTRAQ technique and then the candidate proteins are obtained. Finally, confirming the key proteins of chloroplast response to shading using real-time quantitative PCR and Western blot technique. These results are intended to reveal the regulatory mechanism of photosynthetic characteristics of intercropping soybean under shading conditions and will provide insight for carrying out functional verification and breeding high light efficiency varieties of intercropping soybean in the future.

南方间套作作为大豆三大优势产区之一，对扩大种植面积，提高大豆产量具有重要意义。在生产中，荫蔽是导致间套作大豆光合作用下降、产量降低的主要因素。我们发现荫蔽下光强和红光/远红光比值与大豆生长密切相关，而叶绿体蛋白在光合作用中执行着重要功能，先前研究仅在弱光或单一光质（未考虑光强）下围绕某一类蛋白进行分析，那么荫蔽如何影响间套作大豆的光合特性，叶绿体中哪些关键蛋白起着关键调控作用，尚不清楚。因此，本研究将通过大田和室内盆栽试验，分析大豆光合特性对荫蔽的响应特征，利用蛋白质组学iTRAQ技术筛选荫蔽中光强和红光/远红光变化下叶绿体差异表达蛋白，获得叶绿体响应荫蔽的候选蛋白；采用实时荧光定量PCR和Western blot技术对候选蛋白进行验证，明确荫蔽调控大豆叶绿体结构和功能的关键蛋白。研究结果旨在揭示荫蔽对间套作大豆光合特性的调控机理及为下一步功能验证，培育高光效间套作大豆品种提供参考。

在间套作生产中，荫蔽是导致间套作大豆光合作用下降、产量降低的主要因素。本研究通过大田和盆栽结合，分析大豆光合特性对荫蔽的响应，应用iTRAQ技术获得大豆叶片叶绿体响应荫蔽的候选蛋白，采用RT-qPCR技术和Western blot技术明确荫蔽调控大豆功能叶片叶绿体的关键蛋白，以期揭示荫蔽对间套作大豆光合特性的调控机理。结果表明：在玉米大豆套作种植中，与净作大豆相比，套作大豆冠层光强降低66.8%左右，红光/远红光比值（R/Fr）为0.7-1.0；套作大豆叶片下表皮的气孔面积和密度下降，而气孔长度、宽度和周长差异不显著；叶绿体数目增加，而大小下降；叶片单位面积光合色素含量、光合速率、非光化学猝灭系数降低。进一步模拟荫蔽下光强和光质变化，设置正常光照，正常光照+远红光，弱光和弱光+远红光4个处理，在相同光强下低R/Fr比值与正常R/Fr比值（1.2）相比，大豆总生物量，叶面积，叶绿素含量，净光合速率和光系统II（PSII）量子效率显著增加；通过不同处理下的大豆叶片蛋白质组学分析，在定量的7834种蛋白质中，有12个与光合作用相关的蛋白丰度大于1.3倍，其中1个与卟啉和叶绿素代谢有关，2个与PSI有关，4个与PSII有关，3个蛋白质参与了光合作用的电子传输以及2个参与淀粉和蔗糖的代谢。与正常光照处理相比，弱光处理通过上调相关蛋白水平以维持光合作用和CO2同化。尽管低R/Fr比值增加了光合碳同化，但正常光照+远红光和弱光+远红光处理下大多数蛋白质表达水平与正常光照处理相比差异不显著。通过对关键蛋白及其基因表达验证，调控大豆光合作用对荫蔽响应的关键蛋白主要为POR， Lhcb1、2、3、4、6，PsaD，PetE，PetF和PetH。因此，荫蔽条件下低R/Fr比值（高丰度远红光）可以通过调控关键蛋白的表达提高光系统的光合效率和碳同化，这些结果为培育高光效优良间套作大豆新品种提供参考依据。