烟草类胡萝卜素降解关键基因CCD1的克隆与表达分析

Cloning and expression of gene CCD1 critical to cleavage of tobacco carotenoid

为深入探索CCD1的功能，利用RACE方法获得了烟草品种K326类胡萝卜素降解关键基因CCD1的2个cDNA全长序列。序列分析表明，CCD1-1与CCD1-2各包含一个1635和1647bp的开放读码框（ORF），编码544和548个氨基酸。分别与林烟草和绒毛状烟草CCD1存在一个碱基的差异。CCD1-1与CCD1-2基因序列GC含量为43.24%和42.87％。预测的蛋白质分子量均为61 kDa,理论等电点（pI）6.51和6.55。与番茄和辣椒等植物的类胡萝卜素CCD1的同源性达到80％以上。跨膜区预测表明分别在138～158和142~162位氨基酸间有1个跨膜螺旋区。蛋白质二级结构分析表明，均以β折叠和无规则卷曲为主。蛋白三维结构预测分析表明CCD1-1与CCD1-2空间结构极为相似。组织表达分析表明，CCD1-1与CCD1-2在花中的表达最强，根中的表达最弱，茎和叶次之。这些结果为进一步研究类胡萝卜素降解基因的功能提供了依据。

Tobacco carotenoids and their apocarotenoid derivatives play essential physiological and developmental roles and provide plants tolerance to a variety of stresses. Using RACE method, two copies （named CCD1-1 and CCD1-2） of the full-length cDNA sequence of tobacco carotenoid cleavage oxygenases1 were cloned from K326. Sequences analysis showed that CCD1-1 and CCD1-2 contained of open reading frame （ORF ） 1635-bp and 1647-bp and coded 544 and 548 amino acid residues with the theoretical isoelectric point （pI） of 6.51 and 6.55, respectively. The calculated molecular masses were both 157 kDa. The GC contents of two copied gene were 43.24%and 42.78%. The similarity of CCD1 with tomato, pepper and other plants reached to more than 80%. Protein hydropho bicity and transmembrane region’s prediction analysis indicated that there was one transmembrane helical region from 138 to 158 amino acids in CCD1-1 and from 142 to 162 in CCD1-2. The secondary structure prediction results showed that they were mainly made up by beta turns and random coil. The tertiary structure prediction results displayed that CCD1-1 showed high similarity with CCD1-2 except in random coil. Expression analysis showed that the expression level was highest in flower, and lowest in root. These results provide some basis for the function analysis to CCD1.