芹菜核酸内切酶CELI基因的克隆及其表达分析

Cloning and expression pattern analysis of CELI gene in celery

CELI是一种单链特异性核酸酶(singlestrand specific nuclease),主要应用于清除DNA或RNA双链分子存在的单链。测定不同品种芹菜中CELI基因非生物胁迫诱导表达情况,以进一步研究芹菜中核酸内切酶的功能及应用。以芹菜(Apium graveolens L.)为试验材料,分别从‘六合黄心芹’和‘文图拉’中克隆出核酸内切酶CELI基因序列。通过生物信息学的方法对所得序列进行分析,通过实时定量PCR方法进行该基因在芹菜中表达分析。结果表明:来源于2种芹菜的CELI基因核苷酸序列高度保守,基因全长均为891 bp,分别编码296个氨基酸。2种芹菜的CELI基因核苷酸序列之间共有20个位点不同,导致3个氨基酸位点发生改变。2种芹菜该酶的蛋白质相对分子质量分别为33.88×103和33.92×103,pI值分别为6.51和6.37。进化分析显示,芹菜中的CELI与同属于伞形科的欧芹进化关系最近,伞形科CELI进化上更接近于菊科。实时定量PCR分析表明,芹菜中CELI基因在根、茎、叶、花不同组织及不同品种之间的表达量有明显差异。对2种芹菜分别进行4℃、38℃、0.2 mol·L-1NaCl、200 g·L-1PEG处理2 h,表达分析显示,4种处理条件下芹菜中CELI基因表达量有明显差异,其中PEG处理表达量呈明显下降趋势。结论:通过逆境处理后的基因表达分析发现,芹菜中CELI基因对非生物胁迫有响应,‘六合黄心芹’中CELI基因的诱导表达量大于‘文图拉’。

CELI was the first mismatch endonuclease,which was identified from the celery（Apium graveolens L.）. CELI is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion.This study measured the different varieties of celery CELI gene expressions under abiotic stress,in order to further study the function and application of endonuclease.The cDNAs encoding the CELI were cloned from two celery variaties of‘Liuhehuangxinqin＇and‘Ventura＇,respectively.The sequences were analysed and used for quantitative real-time PCR to detecte the expression profiles under different abiotic stress treatments.CELI sequences of various types of plants in this study were from the NCBI database.The amino acid composition and physicochemical properties were analysed by the Expasy related software.MEGA 5 test and editor of the evolutionary tree by DNAMAN6.0 was used to generate report graphics.The results showed that the lengths of CELI genes were 891 bp,encoding 296 amino acids.There were 20 different nucleotide sites,and 3 amino acid residues were changed between the two celery cultivars.There was highly nucleotide identity between the CELI genes from the two celery cultivars.Multiple sequence alignment display showed 77.42% similarity.Analysis of amino acid composition and physicochemical properties showed that the predicted molecular weights of the CELI were 33.88×103and 33.92×103,and the pI values were 6.51 and 6.37,respectively.Phylogenetic analysis illuminated that the CELI of celery was close to Petroselinum crispum,an Apiaceae plant.The CELI of Apiaceae plant was close to the plant of Compositae.The expression profiles of the CELI genes of organizations in two-month-old mature plants were detected by quantitative real-time PCR.The expression of each tissue descended as flowers,leaves,roots,stems in‘Liuhehuangxinqin＇,but stems,flowers,roots,leaves in‘Ventura＇.Quantitative real-time PCR analysis showed that the CELI genes were tissue-specific expressed in the two celery cultivars.The expression profiles of the CELI genes exposed to various treatments（4 ℃,38 ℃,0.2 mol·L-1NaCl and 200 g·L-1PEG） for 2 h were detected by quantitative real-time PCR.The gene expression levels were significantly different in the two celery cultivars,and the level significantly decreasedafter treated by 200 g·L-1PEG.Conclusions： Under the treatments of high temperature,low temperature,drought,high salt,the celery plants of‘Ventura＇resistance performance is better.In this study,through analysis of gene expression after these abiotic stress treatments,it was found that the expression level of CELI gene induced in‘Liuhehuangxinqin＇was greater than in‘Ventura＇.