大豆质核互作雄性不育系NJCMS2A及其保持系的花药蛋白质组比较研究

大豆质核互作雄性不育系NJCMS2A是以栽培大豆组合（N8855 ´ N1628）F₂不育株为母本，以N1628为父本，通过连续回交选育而成的，N1628（或NJCMS2B）为其同型保持系。对NJCMS2A和NJCMS2B的二胞花粉期花药进行蛋白质组比较分析，获得重复性好的双向电泳图谱，在分子量18.4~116.0 kD、等电点4~7线性范围内，检测到约217个蛋白点，其中差异表达蛋白点25个，包括在NJCMS2A 中出现而在NJCMS2B中缺失的蛋白点13个，在NJCMS2A中缺失而在NJCMS2B中出现的蛋白点10个，另有2个蛋白点的表达量在NJCMS2B中比在NJCMS2A中明显增强。利用基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）技术对差异表达蛋白进行分析，获得肽质量指纹图谱，用Mascot软件搜索NCBInr数据库，鉴定出14个差异表达蛋白，其中10个在NJCMS2A中出现而在NJCMS2B中缺失和4个在NJCMS2A中缺失而在NJCMS2B中出现。对热激蛋白22 kD、半胱氨酸蛋白酶、V型H+-ATP酶A亚基、MADS盒蛋白和淀粉分枝酶等主要差异蛋白进行功能分析，推测不育系NJCMS2A雄性不育性可能与能量代谢紊乱、细胞程序化死亡（PCD）、淀粉合成受抑制和花器官发育调节基因作用失控等有关。

A Comparative Study on Anther Proteomics between Cytoplasmic-Nuclear Male-Sterile Line NJCMS2A and Its Maintainer of Soybean

Cytoplasmic-nuclear male sterility is a mojor tool in the hybrid seed production for utilization of heterosis in crops. The soybean cytoplasmic-nuclear male-sterile line NJCMS2A was developed through a consecutive backcross procedure with male-sterile plants of（N8855 ´ N1628）F₂ as donor parent and N1628 (designated as NJCMS2B afterwards) as recurrent parent. The present paper was aimed at the differential expressed proteins of anther at binucleate pollen stage between NJCMS2A and its maintainer NJCMS2B. The developmental stage of the anther was determined by both the external morphological performance of the flower bud and the microscopic observation of the microsporogenesis. Two-dimensional gel electrophoresis (2-DE) technique was used to separate the protein spots and the gels were stained with Coomassie Blue G-250. The obtained 2-DE maps were pretty consistent among replications. The difference between the protein maps of anthers from NJCMS2A and NJCMS2B was analyzed with the PDQuest image software. About 217 protein spots were detected within Mr 18.4–116.0 kD and pH 4–7. Total 25 spots out of 217 were found differentially expressed between NJCMS2A and NJCMS2B. Among these, 13 protein spots were present in the anther protein map of NJCMS2A but absent in that of NJCMS2B, and 10 protein spots present in that of NJCMS2B but absent in that of NJCMS2A, another two protein spots were up-regulated significantly in the map of NJCMS2B in comparison with that of NJCMS2A. The matrix-assisted laser-adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to obtain the peptide mass fingerprinting of the differentially expressed proteins and the MASCOT software was used to search the protein database NCBInr. The results were as follows: 10 proteins were present in NJCMS2A anther at binucleate pollen stage but absent in NJCMS2B. These were heat shock 22 kD protein, AIG1-like protein, oligouridylate binding protein, cysteine proteinase, hypothetical protein MtrDRAFT_AC146570g8v1, vacuolar H+-ATPase A subunit, adenosine/AMP deaminase, translational initiation factor EIF-4A, cullin, and beta-amyrin synthase. Four proteins were absent in NJCMS2A anther but present in NJCMS2B. These were MADS box protein, starch branching enzyme, glutathione-s-transferase, and auxin-induced protein AUX28. According to the literature, the functions, especially those related to the male sterility of the major differentially expressed proteins, including heat shock 22 kD protein, cysteine proteinase, vacuolar H+-ATPase A subunit, MADS box protein, and starch branching enzyme were reviewed and discussed. It was inferred that the male sterility of NJCMS2A might be related to energy metabolism turbulence, the programmed cell death (PCD), starch synthesis suffocation and the disturbed function of the flower developmental gene. But the exact mechanism of the differentially expressed proteins above leading to the male sterility of NJCMS2A are to be further studied.