低温预处理诱导紫花苜蓿游离小孢子胚胎发生的机理

Haploids are usually developed by isolated microspore culture technique in many crops. The embryo yield in most alfalfa genotypes is very low, which limits the implementation of this technique in alfalfa breeding. Our previous studies have shown that incubation of isolated microspores at 4℃ had a positive effect on embryogenesis. However, the underlying mechanism of early embryogenesis from alfalfa microspores induced by low temperature stress remains unclear. ‘Baoding’ alfalfa is a highly responsive cultivar in isolated microspore culture and it will be used as experimental material in this study. To gain deep insights into the mechanism, morphological characteristics and developmental pathways of isolated microspores will be observed by fluorescence microscopy. Meanwhile, transcriptional changes in the early stage of low temperature-induced microspore embryogenesis will be characterized by transcriptome sequencing. The differentially expressed genes involved in microspore embryogenesis will be identified, and then the functions, metabolic and regulatory pathways of the genes will be analyzed by bioinformatics methods. Some differentially expressed genes, which are significantly related with embryogenesis, will be cloned from ‘Baoding’ alfalfa. The functions and expression patterns of the genes will be analyzed. The findings in this study will help us to understand the underlying mechanism of embryogenesis under low temperature stress which is contributing to development of an efficient protocol for alfalfa microspore culture.

游离小孢子培养技术是获得单倍体植株的一种重要方式，但是紫花苜蓿小孢子出胚率低严重限制了该技术在其育种中的应用。我们前期研究结果表明，4℃低温预处理能够显著提高紫花苜蓿小孢子的出胚率。为进一步解析4℃低温诱导小孢子早期胚胎发生的机制，本项目拟以出胚率较高的“保定苜蓿”为试验材料，开展以下研究工作：（1）从细胞学水平上研究4℃低温诱导小孢子胚胎发生过程中的细胞形态学变化和小孢子分裂途径；（2）利用转录组测序技术对低温诱导的小孢子早期胚胎发生相关基因的表达情况进行分析，筛选出差异表达基因，利用生物信息学方法对其进行功能、代谢和调控途径分析；（3）筛选出1-2个在小孢子胚胎发生早期发挥关键作用的差异表达基因，分析其表达模式并进行功能验证。预期研究结果将从细胞学水平和分子水平上探明4℃低温诱导紫花苜蓿小孢子早期胚胎发生的机制，为提高紫花苜蓿小孢子的出胚率提供理论依据。

单倍体育种可以显著加快选择进程，缩短育种周期。利用游离小孢子培养技术获得单倍体植株，进而加倍得到纯系已成为现代育种技术中的一个重要手段，紫花苜蓿小孢子成胚率低严重限制了该技术在其育种中的应用。4℃低温预处理能够显著提高“保定苜蓿”小孢子的成胚率，本项目从细胞学水平和分子水平上开展了4℃低温预处理诱导紫花苜蓿小孢子胚胎发生的机理研究，取得了如下结果：通过对4℃低温预处理诱导小孢子胚胎发生过程中的细胞学观察，初步探明紫花苜蓿小孢子胚胎形成过程中的细胞形态学变化特征，推断4℃低温预处理条件下小孢子胚胎发育所经历的分裂途径为B途径。采用Illumina HiSeqTM 2000高通量测序技术，获得紫花苜蓿小孢子4℃低温预处理样本与对照样本的转录组数据26.48 G，明确了4℃低温诱导的紫花苜蓿小孢子早期胚胎发生相关基因的功能、代谢途径和调控途径。参考前人对小孢子胚胎发生相关基因的研究成果，结合转录组测序获得的差异表达基因及其功能分析，筛选出可能在紫花苜蓿小孢子胚胎发生过程中发挥关键作用的差异表达基因2个，MsEDAG1和MsEDAG2。组织特异性表达分析结果显示2个基因均在小孢子中的表达量最高。采用根癌农杆菌介导法将这2个候选基因导入“保定苜蓿”中，转MsEDAG1基因植株和转MsEDAG2基因植株的小孢子出胚率和对照均没有显著性差异。研究结果可为提高紫花苜蓿小孢子的出胚率提供理论依据，对加快紫花苜蓿单倍体育种和种质资源创新具有重要的参考价值。