地黄中响应连作基因的时空表达与分析

Spatiotemporal expression and analysis of responding consecutive monoculture genes in Rehmannia glutinosa

目的:前期作者利用抑制性消减杂交技术(SSH)构建了连作地黄的正反消减cDNA文库。该研究从两库中各选5个可能引起地黄连作障碍的基因做时空表达分析,以探讨其在地黄连作障碍中的作用。方法:应用实时荧光定量PCR技术检测这10个基因在正茬、重茬地黄中不同时期不同组织的相对表达量。结果:正库中筛选的5个编码基因(钙依赖性蛋白激酶、SAM合成酶、ACC氧酶、甲基转移酶、钙蛋白酶)在重茬地黄中有较高的表达量,在正茬地黄中几乎无表达;反库中筛选的5个编码基因(RNA依赖的RNA聚合酶、RNA复制酶、依赖于DNA的RNA聚合酶IIa、细胞周期蛋白D、RNA结合蛋白)在正茬地黄中均得到较高表达,而在重茬中却被下调或关闭。结论:参与核心生命过程的关键调控基因(如细胞周期蛋白D、依赖于DNA的RNA聚合酶IIa)在重茬地黄中被抑制或关闭,扰乱了植株正常的基因表达程序,通过钙信号传导和乙烯合成信号,干扰了地黄生长发育的重要代谢过程,从而导致连作地黄植株矮小,发育不良,引起地黄的连作障碍现象。

Objective： Based on previous study, authors used the suppression subtractive hybridization （SSH） technique to construct the forward and reverse subtractive cDNA libraries of consecutive monouhure Rehmannia glutinosa. Five genes related with consecutive mouoculture problem of R. glutinosa were chosen from the each of two subtraetive libraries. And their spatiotemporal ex- pression was measured in order to explore the functions in consecutive monoculture problem of R. glutinosa. Method： Using the real- time quantitative PCR, we tested the relative expression values of the genes in different development stages and tissues of normal growth （ one-year culturing） and consecutive monoculture （two-year culturing） R. glutinosa. Result： The five genes （calcium-dependent pro- tein kinase, s-adenosyl-methionine synthetase, Aminocyclopropane-l-carboxylate oxidase, mcthyhransferase, calpain）, which were chosen from the forward libraP/had high expression in consecutive monocuhure R. glutinosa, especially in root, and were hardly ex- pression in normal growth R. glutinosa. On the contrary, the other five genes （ RNA-dependent RNA polymerase, RNA replicase, DNA-directed RNA polymerase lla, cyclin D, RNA binding protein） chosen from the reverse library had high expression in one-year R. glutinosa, but were down regulated or shut down in consecutive monoeuhure R. glutinosa. Conclusion： The key genes, which reg- ulate inessential metabolism parthway （such as cyclin D, DNA-directed RNA polymerase IIa） , were restrained or shut down in consec- utive monoculture R. glutinosa. Calcium and ethylene signaling might played key roles in the formation of consecutive monoculture problem, resulting in disturbing normal metabolic process and syndrome of disease in R. glutinosa appeared in turn.