【摘要】 通过显微镜观察强抗寒冬油菜叶片几丁质酶蛋白冰晶形态，分析其编码基因家族冷胁迫下的表达模式。克隆几丁质酶基因(BrCTL1),进行亚细胞定位与过表达载体构建，测定过表达株系低温胁迫下生理变化及基因低温表达模式。结果表明，在冷胁迫环境下，从强抗寒白菜型冬油菜叶片中提取的蛋白液，具备显著的重结晶抑制活性，有效抑制冰晶生长，同时含有高活性的抗冻蛋白。SDS-PAGE电泳将该蛋白鉴定为几丁质酶(chitinase, CTL1)。同源克隆油菜BrCTL1(Brapa08T003472.1)基因，其ORF全长963 bp,编码321个氨基酸，BrCTL1蛋白被定位在叶绿体上。低温胁迫下，BrCTL1基因过表达植株形态无明显变化，突变体与野生型植株出现明显冻害症状，过表达拟南芥株系SOD、POD、CAT活性较野生型分别提高20%、17%、48%,CTL1突变体则分别降低65%、32%、67%。低温胁迫下，BrCTL1过表达株系相对电导率和MDA含量较野生型分别降低19%和45%,CTL1突变体分别提高9.4%和5.7%。综上，BrCTL1基因能够显著增强转基因拟南芥植株的抗寒性。更多还原

【Abstract】 This study observed the ice crystal morphology of chitinase protein in the leaves of highly cold-resistant winter rapeseed through a microscope and analyzed the expression patterns of its encoding gene family under cold stress. The chitinase gene(BrCTL1) was cloned, the subcellular localization was carried out, and an overexpression vector was constructed. The physiological changes and gene expression patterns of overexpressing lines under low-temperature stress were determined. The results showed that under cold-stress conditions, the protein solution extracted from the leaves of highly cold-resistant winter rapeseed had significant recrystallization inhibition activity, effectively inhibits ice-crystal growth, and contains highly active antifreeze proteins. The protein was identified as Chitinase(CTL1) by SDS-PAGE electrophoresis. The BrCTL1(Brapa08T003472.1) gene of rapeseed was cloned by the homologous cloning method. Its ORF was 963 bp in full length and encoded 321 amino acids. The BrCTL1 protein was located in the chloroplast. Under low-temperature stress, there were no obvious morphological changes in the BrCTL1 gene overexpression plants, while obvious freezing injury symptoms occurred in the mutant and wild-type plants. The activities of SOD, POD, and CAT in the overexpression Arabidopsis lines increased by 20%, 17%, and 48%, respectively, compared with the wild-type, while those in the CTL1 mutant decreased by 65%, 32%, and 67%, respectively. Under low-temperature stress, the relative conductivity and MDA content in the BrCTL1 overexpression lines decreased by 19% and 45%, respectively, compared with the wild-type, while those in the CTL1 mutant increased by 9.4% and 5.7%, respectively. In conclusion, the BrCTL1 gene can significantly enhance the cold resistance of transgenic Arabidopsis thaliana plants.更多还原