Russinova E, Slater A, Atanassov A I, Elliott M C
Norman Borlaug Institute for Plant Science Research, De Montfort University, Scraptoft, Leicester, UK.
Cell Mol Biol (Noisy-le-grand). 1995 Jul;41(5):703-14.
Cyclins are a complex group of proteins involved in regulation of the eukaryotic cell division cycle via their interaction with cyclin dependent kinases (Cdks). Cyclin gene sequences have been cloned from a number of plant species, including alfalfa, but the diversity of these genes suggests that there are many plant cyclins which have yet to be characterized. A RACE-PCR strategy has been adopted for cloning cyclin gene sequences expressed during direct somatic embryogenesis in alfalfa. RT-PCR with nested degenerate primers was used to amplify the highly conserved "cyclin box" region of a novel A-like cyclin mRNA sequence expressed after induction of somatic embryogenesis. The sequence of this PCR product was used to design primers for 5'- and 3'-RACE protocols. 5'-RACE using a modified SLIC (single strand ligation to single stranded cDNA) procedure revealed considerable sequence heterogeneity in the N-terminal region of the coding sequence with several closely related sequences apparent. Conventional 3'-RACE generated a single cyclin sequence. The complete coding sequence of one member of this A-like cyclin subgroup has been obtained by this RACE strategy and confirmed by PCR amplification and sequencing of alfalfa genomic DNA.
细胞周期蛋白是一类复杂的蛋白质，它们通过与细胞周期蛋白依赖性激酶（Cdks）相互作用，参与真核细胞分裂周期的调控。细胞周期蛋白基因序列已从包括苜蓿在内的多种植物物种中克隆出来，但这些基因的多样性表明，仍有许多植物细胞周期蛋白有待鉴定。已采用一种RACE-PCR策略来克隆苜蓿直接体细胞胚胎发生过程中表达的细胞周期蛋白基因序列。使用巢式简并引物进行RT-PCR，以扩增体细胞胚胎发生诱导后表达的一种新型A类细胞周期蛋白mRNA序列的高度保守的“细胞周期蛋白框”区域。该PCR产物的序列用于设计5'-和3'-RACE方案的引物。使用改良的SLIC（单链连接到单链cDNA）方法进行5'-RACE，发现在编码序列的N端区域存在相当大的序列异质性，有几个密切相关的序列很明显。传统的3'-RACE产生了一个单一的细胞周期蛋白序列。通过这种RACE策略获得了该A类细胞周期蛋白亚组一个成员的完整编码序列，并通过苜蓿基因组DNA的PCR扩增和测序得到了证实。
