关键词: 大豆, 质核互作雄性不育, 花药, 蛋白质组, 双向凝胶电泳, 基质辅助激光解吸飞行时间质谱

Abstract:

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.

Key words: Soybean, Cytoplasmic-nuclear male-sterility, Anther, Proteomics, 2-DE, MALDI-TOF-MS