彩叶草叶片衰老相关新基因Cbcbs的分子特征和功能分析

 叶片衰老是观叶植物观赏性降低的重要因素之一.为研究彩叶草叶片衰老变化的分子机理，在构建彩叶草衰老叶片cDNA文库及小型EST库的基础上，以1条新的具有胱硫醚 β 合酶(cystathionine beta synthase, CBS)结构域的EST序列为探针，通过RACE与文库结合的 方法，克隆了1个具有1对完整CBS结构域的全长cDNA，Cbcbs.Cbcbs cDNA 全长859 bp，包含1个609 bp的ORF框，编码202个氨基酸.其5′UTR区含有1个终止子TAA，3′UTR区含有推测的加尾信号AATAAA和ATTTA元件.CbCBS N端含有线粒体转运肽，具有2个保守的CBS结构域，4个酪蛋白激酶Ⅱ(casein kinase Ⅱ，CKⅡ)磷酸化位点，3个蛋白激酶C(protein kinase c，PKC)磷酸化位点和1个酪氨酸硫化(tyrosine sulfation，TS)位点.序列比较和进化分析表明，CbCBS是与衰老或应急相关的蛋白.二级结构和三级结构预测表明，CbCBS的功能主要由CBS结构域决定.RT-PCR分析表明，该基因在叶的各个时期均有表达，但随叶片衰老进程的加快而表达增加，是一个叶衰老相关基因（SAG），推测在线粒体中成熟的CbCBS可能作为细胞能量传感器，在叶衰老引起的能量应急中参与细胞能量水平的调节.

Molecular Characterization and Functional Prediction of a Novel Leaf SAG Encoding a CBS-domain-containing Protein from Coleus blumei

Leaf senescence is considered as one of important factors to decrease ornamental values of foliage plants. In the attempt to study and understand the molecular mechanism of leaf senescence, a senescent leaf cDNA library of Coleus blumei was constructed and a small EST library was obtained. According to the sequence of an EST fragment with a cystathionine beta synthase (CBS) domain, a novel leaf senescenceassociated gene (SAG) full-length cDNA encoding a CBS-domain-containing protein, denoted Cbcbs, was rapidly cloned using a strategy of RACE combined with cDNA library. The full length of the Cbcbs gene was 859 bp long (accession No. EF076754) and contained a 609 bp open reading frame (ORF) encoding a 202amino acid protein. One stop codon (TAA) was found in 5' UTR and one possible polyadenylation signal,AATAAA, and a pentanucleotide motif, ATTTA, were found in 3' UTR. The CbCBS contained a predicted mitochondrial targeting peptide in the N-terminal region, two conserved and intact CBS domains, four casein kinase Ⅱ (CK Ⅱ) phosphorylation sites, three protein kinase c (PKC) phosphorylation sites and one tyrosine sulfation (TS) site. Sequence comparisons and phylogenetic analysis showed that CbCBS was a novel senescence or stress-associated protein. The prediction analysis of secondary structure and three dimensional structure of CbCBS suggested that the chief function of the protein was decided by the CBS domain pair. The expression pattern of Cbcbs in leaves was analyzed by RT-PCR. It was demonstrated that Cbcbs gene was a senescence-associated gene (SAG) and expressed in all leaf stages, young stage (Y) being the lowest and terminal senescence stage (S3) being the highest, and was upregulated along with the leaf senescence.Function analysis showed that the mature CbCBS maybe acts as a sensor of cellular energy status and directly or indirectly regulates cellular energy levels to increase ATP content in mitochondria during periods of metabolic stress of senescent leaves.