高寒草甸植物叶角质层蜡质的表型可塑性与遗传变异研究

In our previous studies, we have found that great variations of leaf cuticular wax existed among alpine meadow grasses growing under different environmental conditions. However, it is still not clear that such variation is phenotypic plasticity or genotypic variation. Therefore, in current study, common garden experiment, reciprocal experiment and simulated stress experiment will be conducted to elucidate the mechanism of such variation. In reciprocal experiment, four perennial grasses widely distributed in alpine meadow, Poa pratensis, Potentilla nivea, Kobresia humilis and Polygonum viviparum, will be reciprocally planted at two sites with different latitudes. In common garden experiment, P. pratensis seeds sampled from different environments in our previous studies will be planted at two sites, one at alpine meadow and the other at temperate region. In simulated stress experiments, P. pratensis from alpine meadow and low altitude regions will be subjected to drought, low temperature and enhanced ultraviolet radiation. We will analyze the crystal structure, amounts, chemical compositions, and alkane distributions of leaf cuticular waxes, specific leaf area, stomatal characteristics, and cuticular permeability; analyze single nucleotide polymorphism of major cuticular wax genes of P. pratensis, KCS, KCR, CER1and CER4, and DNA methylation of promoter of major cuticular wax genes; analyze the sensitivity of cuticular wax amounts and chemical compositions and major genes to environmental factors. Finally, the project aims to elucidate the variation mechanism of cuticular wax in alpine meadow grasses from the views of environment and genetics, and to discuss the ecological meaning of such variations for alpine meadow grasses.

在前期研究中，我们发现高寒草甸草本植物叶角质层蜡质在异质生境中存在较大变异，但尚不清楚这种变异是可塑性的还是遗传适应的结果。本项目拟，1）选择高寒草甸分布范围相对较广的草地早熟禾、雪白萎陵菜、矮嵩草和珠芽蓼，按纬度设置2个交互移栽实验地；选择前期已采集的不同生境草地早熟禾材料，设置高寒草甸区和温带地区2个同质园实验地；选择草地早熟禾材料，在室内开展模拟逆境胁迫（低温、水分胁迫、紫外线辐射增强）试验；2）分析植物叶角质层蜡质晶体结构、含量及组分、烷烃分布特征、比叶面积、气孔特征、角质层渗透性等；分析草地早熟禾蜡质主效基因（KCS、KCR、CER1及CER4）的SNP多态性及蜡质主效基因启动子的DNA甲基化水平；分析蜡质含量、组分及其主效基因对环境因子的敏感性；3）最终从环境与遗传角度解析高寒草甸草本植物叶角质层蜡质变异的机制，探讨高寒草甸草本植物叶角质层蜡质变异的生态学意义。

角质层蜡质在提高高寒草地植物抵御逆境中发挥着重要的生理生态作用，然而尚不清楚高寒草甸植物叶角质层蜡质在异质环境中产生的差异是环境因素诱导的表型可塑性变化，还是遗传变异的结果。本项目中，我们结合交互移栽、同质园实验和模拟逆境胁迫实验，从表型到分子水上回答了这个问题。1）对6种高寒草甸植物的交互移栽实验发现，生境变化显著影响叶角质层蜡质沉积，移栽植物角质层蜡质总量整体小于本地植物；相关分析结果表明，烷烃含量与叶厚呈显著正相关关系，说明烷烃作为优势组分，在提高高寒草甸植物抗逆性中发挥着重要作用。2）在同质园试验中，32份扁蓿豆材料蜡质组分含量在不同种群间存在显著差异，变异系数达到33.25%-50.89%；然而蜡质组分含量变化与环境因子之间无显著相关关系；对59份羊草的分析发现，群体间蜡质含量和相对丰度差异很大，种内特征变化较明显；冗余分析揭示了气候因素对蜡质特征的影响，其中纬度、干旱指数和6-8月的降水量是影响蜡质沉积、组分含量和相对丰度的重要参数。3）扁蓿豆四个蜡质合成基因（MrFAR3-1、MrFAR3-2、MrCER1和MrKCS1）在不同种群之间的表达量也存在显著差异，其中MrFAR3-2和MrCER1基因的表达量与初级醇含量呈显著正相关关系，MrCER1基因表达量与蜡质总量呈显著正相关关系，说明环境变化从分子水平上影响蜡质沉积。4）扁蓿豆三个蜡质基因符合中性进化模型，说明未受到自然选择作用，而MrCER1与之相反，受到自然选择作用。5）逆境胁迫显著提高了蜡质合成基因的表达水平及蜡质沉积，反映了不同生态区植物叶片角质层蜡质的差异是由环境和遗传变异共同驱动的。长期、稳定的逆境胁迫在诱导植物叶角质层蜡质发生可塑性变化的同时，也将通过遗传变异改变角质层蜡质的沉积规律，以适应外界环境。