2020年4月8日，中国农业科学院蔬菜花卉研究所甘蓝青花菜研究团队在期刊peer J（2023IF=2.7） 上发表了题为“Proteomic variations after short-termheat shock treatment reveal differentially expressed proteins involved in early microspore embryo genesis in cabbage (Brassica oleracea)”的研究论文。论文第一作者为苏贺楠博士和陈果硕士（现为在读博士），通讯作者为吕红豪研究员。

小孢子培养是一种广泛应用的单倍体育种方法，可以大大缩短育种周期，为甘蓝、大白菜、油菜等重芸苔属蔬菜的育种提供了一种有效的手段。对于甘蓝而言，短期热激处理在许多情况下都能显著提高小孢子的发育率，但其潜在的作用机制还不清楚。本研究以甘蓝材料(中甘628和87-534)为材料，比较了32◦C处理24 h和25◦C处理24 h的分离小孢子的蛋白质组学差异。经32◦C处理的中甘628的胚产量为19.7个/蕾，而25◦C处理的中甘628和87-534在两种温度下均未观察到胚胎。我们通过无标记蛋白质组学技术鉴定了中甘628和87-534的363和282个差异表达蛋白(DEP)。经32◦C热激处理后，只在中甘628中发现了97个DEPS，而在87-534中没有发现，这可能与热激诱导的体外胚胎发生有关。这些DEPS主要集中在碳代谢过程、蛋白质合成和降解过程以及信号转导过程中。基于蛋白质相互作用和途径浓缩分析，我们认为SGT1同源蛋白A和B(SGT1)、热休克70 kDa蛋白5(HSP70)、细胞分裂控制蛋白48同源A(CDC48)和脂肪酰辅酶A还原酶(FAR)可能在小孢子胚胎发生中起重要作用。这一蛋白质组学研究有助于我们对甘蓝小孢子胚胎发生的分子理解，有助于构建高效的单倍体育种体系。

Figure 2 Principal component analysis at the proteome level. (A1) Zhonggan 628 under 25 ◦C for 24 h. (A2) Zhonggan 628 under 32 ◦C for 24 h. (B1) 87–534 under 25 ◦C for 24 h. (B2) 87–534 under 32 ◦C for
24 h.

Figure 3 Venn diagrams showing numbers of distinct and common proteins in each of the three groups. (Y1) Zhonggan 628 under 32 ◦C vs. 25 ◦C for 24 h. (Y2) 87–534 under 32 ◦C vs. 25 ◦C for 24 h. (Y3) Zhonggan 628 under 32 ◦C for 24 h vs. 87–534 under 32 ◦C for 24 h.

Figure 4 GO function classification of DEPs during embryogenesis caused by high temperature.

Figure 5 KEGG pathway of DEPs during embryogenesis caused by high temperature.

Figure 6 Protein–protein interaction analysis between DEPs. (A) The upregulated proteins. (B) The
downregulated proteins.

Figure 7 mRNA expression level analysis using a qRT-PCR approach on candidate proteins of embryogenesis caused by high temperature. (A) Bo5g021500. (B) Bo8g066630. (C) Bo5g139630. (D) Bo2g165560. (E) Bo1g050780. (F) Bo2g023100. (G) Bo5g138000. (H) Bo3g045210. (I) Bo6g031300 (** P < 0.01).

Figure 8 Abundance patterns of protein species involved in the carbohydrate metabolism. The expression value was log10 transformed. Yellow box (A2/A1): The expression levels of proteins from Zhonggan 628 under 32 ◦C vs. 25 ◦C; Blue box (A2/B2): The expression levels of proteins from Zhonggan 628 vs. 87–534 under 32 ◦C

Figure 9 Abundance patterns of protein species involved in protein synthesis and degradation. The expression value was log10 transformed. Yellow box (A2/A1): The expression levels of proteins from Zhonggan 628 32 ◦C vs. 25 ◦C; Blue box (A2/B2): The expression levels of proteins from Zhonggan 628 vs. 87–534 under 32 ◦C.

Figure 10 Proposed model of microspore embryogenesis induced by heat shock in cabbage.