对4 ℃和-30 ℃低温条件下引种自安徽大别山的1 株鹅掌楸[Liriodendron chinense (Hemsl.) Sarg.]植株顶芽总RNA 进行了数字基因表达谱构建和差异表达基因筛选,并对筛选出的抗寒性相关基因进行了实时荧光定量PCR 分析。结果表明:获得的总RNA 样品质量符合实验要求。数字基因表达谱分析结果表明:每个样品的质量控制后序列的总长度均在0. 5 Gbp 以上,碱基错误率为0. 01%,Q20 值超过99%,Q30 值为96. 99% ~97. 23%,GC 含量为44. 97% ~47. 06%,并且每个样品比对成功的序列条数占质量控制后序列总数的百分率均在90%以上。差异表达基因筛选结果显示:共发现9 个与鹅掌楸抗寒性有关的差异表达基因,分别为HSP、MYB、NAC、AP2、Zincfinger、WRKY、FAD、Phospholipase 和β-amylase 基因。实时荧光定量PCR 分析结果表明这9 个基因共有3 种表达模式,其中,HSP 和FAD 基因的相对表达量均随着温度的降低而减少,表现为基因下调表达;AP2、NAC 和Zinc finger基因的相对表达量随着温度的降低而增多,表现为基因上调表达;而β-amylase、WRKY、Phospholipase 和MYB 基因的相对表达量则表现为4 ℃时增加、-30 ℃时减少。从基因的相对表达量来看,最大相对表达量超过5 的基因有NAC 和WRKY,介于2 ~5 之间的基因有AP2、Zinc finger、β-amylase 和MYB,低于2 的基因有Phospholipase、HSP 和FAD。结果显示NAC 和WRKY 基因可能在鹅掌楸的抗寒过程中起主要作用。

Under 4 ℃and -30 ℃low temperature conditions, digital gene expression profile of total RNA from apical buds of one individual of Liriodendron chinense ( Hemsl.) Sarg. introduced from Dabieshan of Anhui was constructed and differential expression genes were selected, and cold resistance related genes selected were analyzed by real-time fluorescence quantitative PCR. The results show that qualities of total RNA samples obtained meet the experimental requirements. Analysis results of digital gene expression profile show that total length of reads after quality control of each sample is above 0. 5 Gbp, error rate of base is 0. 01%, Q20 value is more than 99%, Q30 value is 96. 99% -97. 23%, GC content is 44. 97% -47. 06%, and percentage of successfully mapped read number to total read number after quality control of each sample is above 90%. Selection result of differential expression genes shows that nine differential expression genes related to cold resistance of L. chinense are founded totally, they are HSP, MYB, NAC, AP2, Zinc finger, WRKY, FAD, Phospholipase and β-amylase genes, respectively. Real-time fluorescence quantitative PCR analysis result shows that these nine genes have three expression modes, in which, relative expressions of HSP and FAD genes both decrease with decreasing of temperature, appearing gene down-regulated expression; those of AP2, NAC and Zinc finger genes increase with decreasing of temperature, appearing gene up-regulated expression; while those of β-amylase, WRKY, Phospholipase and MYB genes appear increasing at 4 ℃and decreasing at -30 ℃. On the view of gene relative expression, genes with maximum relative expression over 5 are NAC and WRKY, genes with that between 2-5 are AP2, Zinc finger, β-amylase and MYB, and genes with that under 2 are Phospholipase, HSP and FAD. It is suggested that NAC and WRKY genes may play a major role in the process of cold resistance of L. chinense.