龙眼松散型胚性愈伤组织发生过程中相关蛋白的表达分析

Analysis of Protein Expression during Growth Processes of Friable Embryogenic Callus(FEC) of Longan

以龙眼‘红核子’品种（Dimocarpus longan Lour.cv.Honghezi）松散型胚性愈伤组织（FEC）为材料,采用固相双向凝胶电泳技术和质谱鉴定技术,对其生长过程中相关蛋白的表达变化进行研究。结果表明：（1）龙眼FEC生长过程中,总蛋白点数在400-800之间,蛋白点数变化虽有升降起伏,但总体呈上升趋势;蛋白质pI集中在4-7之间;蛋白质分子量在14-97kD之间,表达量相对较高的是分子量在30-66kD的蛋白质,且30-45kD的蛋白点逐渐增多,45-66kD的逐渐减少。（2）龙眼FEC生长过程中质谱鉴定66个蛋白,其中28.79%为功能未知蛋白,30.30%涉及胁迫应答和抗氧化,10.61%涉及能量和糖代谢,10.61%涉及蛋白代谢和修复,6.06%涉及细胞骨架重构,4.55%蛋白是调控蛋白,3.03%是核酸代谢相关蛋白,而所占比例最少的为信号转导与细胞凋亡相关蛋白、氨基酸代谢相关蛋白、脂类代谢相关蛋白,以及维生素代谢相关蛋白均为1.52%。（3）龙眼FEC生长过程中,胁迫反应/氧化还原相关蛋白（抗坏血酸类过氧化物酶R147、R158和HC21,苄基醚还原酶同系物TH6、谷胱甘肽过氧化酶以及过氧化物酶L26）表达高峰出现在前期10-20d,而过氧化物酶4多在后期40-50d表达;蛋白质合成及修复的相关蛋白,只有蛋白质异形体1在10-30d表达量高,其他蛋白的表达高峰出现在前期10-20d之间,能量与糖代谢相关酶在整个过程中都有较大量的表达。

Using friable embryogenic callus（FEC） of Dirnocarpus longan Lour. cv. Honghezi as the material,we carried out the proteomic analysis of its growth processes by using the immobilized pH gradients two-dimensional electrophoresis technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF/TOF）. The results showed that： （1）The total protein was from 400 to 800 during longan FEC development, and it showed a rising trend in general although its variation trend had in fluctuation. Isoelectric point of all proteins was distributed between 4 and 7. Molecular weight of proteins of FEC during the normal development in longan was within 14--97 kD. The protein expression quantity was at relatively high within 30--66 kD,but within 30--45 kD it showed a rising trend and within 45--66 kD it showed a reverse trend. （2）Sixty-six proteins related to longan FEC development were identified. 28.79% of the proteins were unknown identity or function, 30. 30% of the proteins participated in stress response and anti-oxidation,10.61% of the proteins were involved in energy and carbohydrate me- tabolism,10.61~ of the proteins belonged to protein synthesis related,6.06% of the proteins were struc- tural proteins involved in cytoskeleton remodeling,4.55% of the proteins were regulatory proteins, 3.03 % of the proteins were enzymes involved in nucleic acid metabolism. Additionally, 1.52% of the proteins were associated with signal transduction and PCD, 1.52 % of the proteins were related to amino acid metabolism, 1.52% of the proteins were lipid metabolism-related protein, and 1. 52% of the proteins attached them- selves to vitamin metabolism. （3）In the early stage（10--20 d）,the expression of redox proteins associated with FEC in longan（including ascorbate peroxidase R147,R158 and HC21, phenylcoumaran benzylic ether reductase homolog TH6, glutathione peroxidase and peroxidase L26） was high,while the expression of peroxidase 4 was in late stage（40--50 d）. Protein synthesis-related proteins showed a expression peak within 20 d,besides protein isoform 1 within 30 d. Different energy-associated proteins expressed more abundantly during whole period. There were S-adenosylmethionine synthetase （the enzyme related to precursors of polyamine） and a large number of peroxidase expression at the later stage of longan FEC development. It explained that the polyamine has not only the function like peroxidase for scavenging active oxygen, but enhance the activities of antioxidant enzymes.