CAJ | 학술논문

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609bp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,CK Ⅱ)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.
협편쇠로시관협식물관상성강저적중요인소지일.위연구채협초협편쇠로변화적분자궤리,재구건채협초쇠로협편cDNA문고급소형EST고적기출상,이1조신적구유광류미-β-합매(cystathionine beta synthase,CBS)결구역적EST서렬위탐침,통과RACE여문고결합적방법,극륭료1개구유1대완정CBS결구역적전장cDNA,Cbcbs.Cbcbs cDNA전장859 bp,포함1개609bp적ORF광,편마202개안기산.기5'UTR구함유1개종지자TAA,3'UTR구함유추측적가미신호AATAAA화ATTTA원건.CbCBS N단함유선립체전운태,구유2개보수적CBS결구역,4개락단백격매 Ⅱ(casein kinase Ⅱ,CK Ⅱ)린산화위점,3개단백격매C(protein kinase c,PKC)린산화위점화1개락안산류화(tyrosine sulfation,TS)위점.서렬비교화진화분석표명,CbCBS시여쇠로혹응급상관적단백.이급결구화삼급결구예측표명,CbCBS적공능주요유CBS결구역결정.RT-PCR분석표명,해기인재협적각개시기균유표체,단수협편쇠로진정적가쾌이표체증가,시일개협쇠로상관기인(SAG),추측재선립체중성숙적CbCBS가능작위세포능량전감기,재협쇠로인기적능량응급중삼여세포능량수평적조절.
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.