烟草MADS-box家族基因保守片段的克隆与序列分析

摘要

MADS-box家族基因在植物的生长发育过程中具有重要功能.在烟草生产中出现的早花现象与MADS-box家族基因相关,为了能从烟草中克隆出与开花时间相关的基因,根据不同植物MADS-box家族基因的保守区序列,设计简并引物,采用RT-PCR技术从烟草品种NC82茎尖中成功分离出27条cDNA版段,长度在122～146 bp之间.序列分析表明,其中有16条具有MADS_MEF2保守结构域,其推导的氨基酸序列与拟南芥等MADS-box家族基因具有同源性,相似性平均能达到76%,并具有15个保守的氨基酸位点,其中7个位点参与蛋白与DNA的相互作用,4个位点具有蛋白二聚化功能.系统发育分析结果表明,这些保守片段分别和拟南芥9个不同的MADS-box家族基因亚类最近似,其中许多亚家族与成花相关,说明NC82中存在具有调控开花的MADS-box家族基因表达,并可能 NC82低温敏感,易早花特性相关.%MADS-box family genes play an important role as transcription factors in plant developments, especially in the development of floral organ, and florescence. Early flowering in tobacco agricultural production is associated with MADS-box family genes. In order to isolate new MADS-box family genes from tobacco, degenerate primers were designed according to conserved domain of various plant MADS-box family genes, and 27 cDNA fragments were amplified from the young shoot tip of tobacco variety NC82 using RT-PCR technology, fragment lengths were between 122 bp and 146 bp. By using Blast in NCBI, 16 among these 27 cDNA fragments' deduced amino acid sequences contained MADS_MEF2_like region. Multiple sequence alignment analysis indicated that these 16 deduced amino acid sequences had high homology with MADS-box family genes of Arabidopsis and other species, the average identity could reach 76％. Meanwhile, the 16 fragments contained 15 conserved amino acid motifs, of which 7 motifs involved in the interaction between protein and DNA, 4 motifs with protein dimerization function. Phylogenetic analysis shows these conserved MADS-box family gene fragments could be classified under different MADS-box subfamilies (FLC,STMADS11, TM3, AGL15, AG, AGL2, AGL6, DEF/GLO, and AGL12). FLC, STMADS11, TM3 and AGL6 were associated with flowering. These results indicated that there might be several MADS-box family genes involved in low temperature response and early flowering of cold-sensitive tobacco NC82.