摘要:分析了6种木兰科植物对低温胁迫的生理响应及耐寒相关调控基因HSP90和WRKY33的差异表达，为木兰科植物抗寒机理的研究和抗寒品种的选育提供理论基础。结果表明，6种木兰科植物的低温LT50在-10.64—-22.06℃，从高到低依次为红花深山含笑、峨眉含笑、杂交含笑、阔瓣含笑、六瓣含笑和乐东拟单性木兰；低温过程中，6种木兰科植物叶片可溶性蛋白（SP）、游离脯氨酸（Pro）含量、超氧化物歧化酶（SOD）和过氧化物歧化酶（POD）活性呈先升高后降低的趋势，可溶性糖（SS）和丙二醛含量（MDA）则不断积累；筛选出REC、MDA、SP、SS和Pro作为6种木兰科植物抗寒性评价的关键指标；聚类分析将6种木兰科植物在抗寒性能上分为强、中、弱三类，分别为乐东拟单性木兰和六瓣含笑，阔瓣含笑、杂交含笑和峨眉含笑，以及红花深山含笑。对HSP90、WRKY33基因的差异表达分析表明，2个基因在6种木兰科植物中的相对表达量呈先升高后降低的趋势，在临近各树种LT 50时，2个基因的表达被强烈抑制且后期表达量不可逆。0℃时，2个基因的表达量差异不显著；-5℃时，2个基因开始被激活，表达量增加；-10℃时，HSP90、WRKY33基因在红花深山含笑叶片中的表达量较-5℃时分别下调了0.76倍和0.68倍，而在其他5个树种中的表达被进一步激活；-15℃时，HSP90和WRKY33基因在抗寒性中等的阔瓣含笑、杂交含笑、峨眉含笑中亦被强烈抑制，较-10℃时分别下调了0.38倍、0.33倍、0.32倍和0.71倍、0.72倍、0.74倍，在抗寒性强的乐东拟单性木兰和六瓣含笑中的表达被进一步激活；-20℃以后，2个基因在6个树种中的表达均被强烈抑制，但在抗寒性最强的乐东拟单性木兰中的表达量仍高于其他5个树种。抗寒基因的激活与表达是影响植物抗寒性的重要因素，抗寒性不同的树种对低温的应答机制明显不同。抗寒性越强的树种越能快速启动低温应答机制，激活抗寒相关基因的表达，进而调整生理生化活动以抵御和适应冷应力。不抗寒树种中抗寒基因的表达则受到抑制，降低了其对低温逆境的耐受能力。



Abstract:The physiological response and differential expression of the cold resistance-related genes, Hsp90 and WRKY33, of six varieties of Magnoliaceae under different low temperatures were analyzed in this study, to determine the molecular mechanisms of cold resistance and select cold-resistant Magnoliaceae species. The results showed that the semi-lethal temperature of the six varieties of Magnoliaceae ranged from -22.06 to -10.64℃, ranging from high to low were Michelia maudiae var. rubicunda, M. wilsonii, M. martinii "Tiny" ♀×M. shiluensis, M. platypetala, M. martinii "Tiny", and Parakmeria lotungensis. During low temperatures, the indexes of soluble protein(SP), proline(Pro), superoxide dismutase(SOD), and Peroxidase(POD) initially increased first and then decreased, whereas those of soluble sugar(SS) and Malonaldehyde (MDA) continued accumulating. Relative electrical conductivity(REC), MDA, SP, SS, and Pro could be used as key indicators for the evaluation of cold resistance of six Magnoliaceae varieties. A cluster analysis of the six Magnoliaceae varieties for cold resistance was divided into strong, medium, and weak three categories, and they were P. lotungensis and M. martinii "Tiny", M. platypetala, M. martinii "Tiny" ♀×M. shiluensis and M. wilsonii, and M. maudiae var . rubicunda, respectively. Differential expression analysis revealed that the expression of HSP90 and WRKY33 genes displayed a peak followed by a progressive decline in the six Magnoliaceae varieties. At the semi-lethal temperature of each variety, the expression of both genes was strongly inhibited and irreversible as temperature decreased. There was no significant difference in the expression of both genes between different varieties at 0℃(P>0.05), but they were significantly activated at -5℃. The expression of HSP90 and WRKY33 were downregulated 0.76 and 0.68 times, respectively, than that at -5℃ in M. maudiae var. rubicunda, whereas were further activated in the other five varieties at -10℃. Under low temperature stress at -15℃, HSP90 and WRKY33 expression in the medium cold-tolerant varieties of M. platypetala, M. wilsonii, and M. martinii "Tiny" ♀×M. shiluensis was strongly inhibited, and the expression of both genes was downregulated 0.38, 0.33, 0.32, and 0.71 times, respectively, and 0.72 and 0.74 in P.lotungensis and M. martinii "Tiny", respectively. Under low temperature stress at -20℃, HSP90 and WRKY33 expression in the leaves of the six Magnoliaceae varieties were all strongly inhibited, but the expression was still higher in the strongest cold resistance variety of P. lotungensis than in the other five varieties. The activation and expression of cold-tolerant genes are important factors affecting the cold resistance of plants. Different varieties showed significantly different response mechanisms to low temperature stress. Stronger cold-tolerant varieties activated the response mechanisms more quickly to low temperature stress. Expression of cold tolerance related genes was activated to adjust the physiological and biochemical activities to resist or adapt to cold stress. However, it was inhibited in the cold-intolerant varieties and significantly decreased their adaptation to cold stress.





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