采用DDRT-PCR 技术,对低温(4℃)胁迫处理不同时间(0、8、12、24 和48 h)后茶树[Camellia sinensis(Linn.) O. Ktze.]抗寒品种‘紫阳圆叶’(‘Ziyangyuanye’)叶片中差异表达的基因进行分离和测序,并采用半定量RT-PCR 对差异表达基因的表达特性进行了比较。结果表明:有12 个引物对扩增出有明显差异的cDNA 片段,其中3 个片段是与抗寒性相关的差异片段,分别被命名为Csgsf、Cscaf1 和Cscaf2,碱基数分别为217、316 和232 bp。比对结果显示:Csgsf 与茶树品种‘安吉白茶’(‘Anjibaicha’)和‘龙井43’(‘Longjing 43’)的谷氨酰胺合成酶基因的同源性分别为96%和91%,与菜豆(Phaseolus vulgaris Linn.)、蒺藜苜蓿(Medicago truncatula Gaertn.)、油棕(Elaeis guineensis Jacq.)、西洋参(Panax quinquefolius Linn.)和水稻(Oryza sativa Linn.)等植物的谷氨酰胺合成酶的基因序列同源性均达到90%以上,因此,Csgsf 应为茶树谷氨酰胺合成酶基因片段;Cscaf2 与干旱胁迫条件下茶树表达的cDNA 的同源性为100%,为茶树应答干旱和低温胁迫的基因片段;Cscaf1 未检索出同源序列,推测其为与冷胁迫相关的未知基因片段。半定量RT-PCR 分析结果表明:Csgsf 和Cscaf1 在低温胁迫的初始期即开始表达且表达量随低温胁迫时间延长逐渐下调;而Cscaf2 的表达量随低温胁迫时间延长逐渐上调并在胁迫48 h 后达到最大,3 个片段 的表达特性均与差异扩增结果相符。

Using DDRT-PCR technique, differential expression genes in leaf of cold-resistance cultivar ‘Ziyangyuanye’of Camellia sinensis (Linn.) O. Ktze. under low temperature (4 ℃) stress for different times (0, 8, 12, 24 and 48 h) were separated and sequenced. And their expression characteristics were compared by semi-quantitative RT-PCR. The results show that twelve primer pairs can amplify cDNA segments with obvious difference, in which, three segments are differential segments related to cold-resistance and are named Csgsf, Cscaf1 and Cscaf2 with base number of 217, 316 and 232 bp, respectively. The alignment results reveal that the homology of Csgsf with glutamine synthetase gene of C. sinensis cultivars ‘Anjibaicha’ and  ‘Longjing 43’is 96% and 91%, respectively, and the homology of Csgsf with gene sequence of glutamine synthetase of Phaseolus vulgaris Linn., Medicago truncatula Gaertn., Elaeis guineensis Jacq., Panax quinquefolius Linn. and Oryza sativa Linn. is more than 90%, therefore, Csgsf should be glutamine synthetase gene segment of C. sinensis. The homology of Cscaf2 with cDNA of C. sinensis expressed under drought stress condition is 100%, so Cscaf2 is an responded gene segment of C. sinensis to drought and low temperature stresses. No homology sequence with Cscaf1 is detected, and it is conjectured that Cscaf1 is an unknown gene segment related to cold stress. The analysis result of semi-quantitative RT-PCR shows that Csgsf and Cscaf1 begin to express at initial period of low temperature stress and their expression amount is down-regulated with prolonging of low temperature stress time, while expression amount of Cscaf2 is up-regulated with prolonging of low temperature stress time and reaches to the highest after 48 h of low temperature stress. The expression characteristics of three segments are consistent with the result of their differential amplification.