白菜型冬油菜质外体抗冻蛋白研究

Study on apoplast anti-freeze proteins in winter turnip rape(Brassica rape L.)

为探明冬油菜抗寒机理,采用大田试验和盆栽试验,对低温胁迫的白菜型冬油菜品种‘陇油6号’叶片和根的质外体蛋白进行抗冻活性检测,通过SDS-PAGE分离质外体蛋白并结合MALDI-TOF/TOF质谱技术对部分高表达蛋白进行了鉴定。结果表明,‘陇油6号’经人工气候室冷驯化后,叶片质外体蛋白质含量显著增加(P<0.05),在第5 d达92.31μg?g?1(FW),比对照增加246.12%。质外体蛋白含量在冷驯化第10 d和第15 d与第5 d相比虽有所降低,但仍显著高于对照(P<0.05)。在冷驯化第20 d和第25 d质外体蛋白含量继续显著增加,第25 d达到最高值(P<0.05)。室温恢复生长10 d后,质外体蛋白含量比之前显著下降(P<0.05),但仍高于对照和冷驯化第10 d。在冷驯化过程中,‘陇油6号’叶片的质外体中出现明显的蛋白质积累,室温恢复生长10 d后,质外体中的蛋白质含量显著降低,可见冷驯化的‘陇油6号’质外体蛋白属低温诱导蛋白。抗冻活性检测发现其具有重结晶抑制活性。质谱鉴定发现多种功能尚不明确的蛋白质,其中β^(-1)-3-葡聚糖酶与冬黑麦中报道的抗冻蛋白一致。对质谱检测到的类葡聚糖酶回收及抗冻活性测试发现其有较弱的冰晶形态修饰作用,表明这个类葡聚糖酶是一种低活性抗冻蛋白。低温胁迫下冬油菜叶片和根质外体中合成分泌了抗冻蛋白,在抵御外界低温中起到积极作用,同时推测超强抗寒性冬油菜质外体中可能存在多种未被发现的抗冻蛋白。

The objective of this paper was to lay the basis for studying cold resistance of winter rapeseed. The anti-freeze activities of apoplast proteins were determined in the ‘Longyou 6’ winter rape leaves and roots under cold vernalization. The apoplast proteins were separated by SDS-PAGE and high expression proteins identified in MALDI-TOF/TOF massspectrometry under field and pot experiments. The results showed that apoplast protein content of ‘Longyou 6’ leaves increased significantly（P 〈 0.05） after cold acclimation in an artificial climate chamber, reaching 92.31 μg ？ g ？ 1（FW） on the fifth day, which represented an increase of 246.12% over CK. Apoplast protein content after 10–15 days of cold acclimation dropped compared with that after 5 days, but was still significantly higher than that of CK（P 〈 0.05）. Apoplast protein content continued to increase with increasing cold acclimation time from 20 to 25 days（P 〈 0.05）. Apoplast protein content decreased significantly with after 10 days of de-acclimation. In the process of cold acclimation, apoplast protein content of ‘Longyou 6’leaves significantly accumulated. However, it decreased significantly after de-acclimation. Obviously, apoplast proteins of‘Longyou 6’ winter rape belonged to low temperature induced proteins. Anti-freeze activity detection analysis suggested that apoplast proteins had re-crystallization inhibition activity. Mass spectrometry identification revealed a variety of proteins with unclear functions along with β-1-3-glucanase consistent anti-freeze proteins reported in winter rye. The class glucanase detected by mass spectrometry was found to have weaker ice crystal forms due to modification effect with reclamation and anti-freeze activity test. The test suggested that this class glucanase was a low activity anti-freeze protein. Many anti-freeze proteins were synthesized and secreted by winter rape in apoplast of leaves and roots under low temperature stress. The proteins likely played a positive role against outside low temperature. Furthermore, it was speculated that there were many undetected anti-freeze apoplast proteins responsible for the strong winter hardiness of winter rapeseed.