楸子脱水素基因家族的干旱适应性进化研究

Variation in climatic conditions is thought to confer strong selective pressure on plant local adaptation, which promotes adaptive phenotypic differentiation between natural populations for fitness related traits. In the previous research on adaptation to drought in wild tomatoes, we found DHN is a ideal candidate gene in plant tolerance adaptation research, because, 1) DHN locus exhibits higher levels of nucleotide diversity than neutral loci; 2) positive selection has been acted on DHN gene in one population of wild tomato, showing a significant haplotype structure and 3) higher genetic differentiation between populations, which is indicative of local adaptation. In recent research we identified 9 members of DHN gene family, they exhibit obviously different expression pattern under drought and low temperature stresses. In this project, we will use Malus prunifolia as the material, firstly, identify and clone DHN gene family in M. prunifolia. Secondly, sequence DHN gene family of 15 populations and try to find which type selection has been acted on this gene family and how population structure changed to adapt to local environment by analyzing nucleotide diversity level, neutrality tests and population difference level. Finally, analysis expression patterns of different gene members to find the relationship between natural selection and gene function. This project will help us to understand the genetic basis of plant adaptation to abiotic stress, also provide theoretical guidance for apple stress resistance breeding.

生态环境的变化给植物生存带来选择压力，进而使得群体表现型发生分化，以适应当地环境。本项目组在前期对野生番茄干旱适应性的研究中发现脱水素基因是研究植物抗旱适应性的理想候选基因：1）各群体在脱水素基因位点上的多态性水平明显高于中性位点；2）正向选择曾作用于野生番茄的一个群体，使脱水素基因呈现出显著的单倍体结构；3）各群体间的分化水平明显增大。项目组还鉴定了9个苹果脱水素基因家族成员，发现各成员响应逆境的表达模式存在明显差异。本项目拟以自然分布广、抗逆性强的15个楸子群体为研究对象，重点研究1）鉴定楸子的脱水素基因家族成员；2）通过对脱水素基因的多态性水平、群体结构及中性检测等进化遗传分析，找出哪些自然选择力曾作用于该基因家族；群体结构发生了哪些变化以适应当地环境。3）结合逆境表达模式探明自然选择力与基因家族成员功能变化的关系。本研究旨在探明植物适应性的遗传学基础，也为苹果抗性育种提供理论基础。

楸子适应性强，抗寒抗旱，是苹果的优良砧木及苹果属重要种质资源。楸子分布范围广，分布区生态环境变化多样，成为研究生态适应性的良好材料。脱水素是一种植物中广泛存在的亲水性蛋白，在低温、干旱、高盐或外源ABA等逆境诱导，提高植物抗逆性。为研究不同生态环境对楸子群体的选择压力，本项目以脱水素基因为候选基因，研究了楸子脱水素基因家族的干旱适应性进化。研究结果如下：（1）首先从楸子中克隆得到脱水素基因家族9个成员（MpDHN1-9），并分析序列特征，她们分别属于SKn、YSKn、YKn、Kn型；（2）用荧光定量PCR技术研究了不同脱水素基因成员分别在盐胁迫、低温胁迫和干旱胁迫下的表达模式，发现其中5个成员在干旱胁迫下表达水平显著上调，3个成员盐胁迫下表达水平显著上调，3个成员在低温胁迫下表达显著上调。（3）进而以在三种胁迫下MRNA表达水平都上调的MpDHN8作为候选基因对楸子个体进行克隆测序，获得66条gDNA序列，并进行序列分析。结果表明各群体内个体间的核苷酸多态性水平较高；而各群体之间的差异不显著；中性检测表明群体SX偏离中性理论，呈正显著，表明存在大量中等频率的等位基因。这可能是由群体瓶颈效应，群体结构，或者平衡选择引起的。