甘蓝型油菜ADC基因片段的克隆及序列分析  

Cloning and sequence analysis of ADC gene fragments in Brassica napus L.

作　　者：洪林[1,2] 闫晓红[1] 李晨[1] 刘正富[2] 魏文辉[1] 

机构地区：[1]中国农业科学院油料作物研究所/农业部油料作物生物学重点实验室,武汉430062 [2]重庆市农业科学院果树研究所,重庆402260

出　　处：《南方农业学报》2012年第2期135-140,共6页Journal of Southern Agriculture

基　　金：国家自然科学基金面上项目(30671312);农业部油料作物生物学重点实验室开放课题项目(201004)

摘　　要：【目的】克隆甘蓝型油菜精氨酸脱羧酶(ADC)基因的cDNA片段,为进一步获得ADC基因全长及研究其的生物学功能奠定基础。【方法】以甘蓝型油菜(Brassica napus L.)品种中双6号的花蕾为材料,设计简并引物,采用同源克隆法扩增ADC基因的cDNA片段。【结果】成功扩增获得8个不同的ADC基因拷贝,按长度将其分为1011、999、981bp3类,8个拷贝核苷酸序列之间的同源性为81%～99%,其推导氨基酸序列之间的同源性为86%～99%。1011bp片段包含3种不同的序列S1～S3,序列间存在9个单核苷酸多态性(SNP)位点。999bp序列包含S4和S5,具有3个SNP位点,两个区段出现连续碱基缺失。981bp序列包含S6～S8,发现14个SNP位点,且第67位核苷酸出现无义突变,4个区段发生碱基缺失。序列S4～S8共同缺失第632～634、642～650位的核苷酸小片段。氨基酸同源比对和系统进化分析结果表明,8个不同ADC基因拷贝与芥菜(Brassica juncea L.)、拟南芥(Arabidopsis thaliana L.)、盐芥(Thellungiella halophila L.)等物种的ADC基因同源性高,亲缘关系近。【结论】所获得的8个基因拷贝即为来自于甘蓝型油菜ADC基因的片段。[Objective]The present experiment was conducted to isolate the cDNA fragment of arginine decarboxylase（ADC） gene from Brassica napus L.in order to provide information for obtaining its full length gene and understanding its biological functions.[Method]The cDNA fragments of ADC were isolated from floral buds of Brassica napus L.variety Zhongshuang 6 using degenerated primers and homologous cloning method.[Result]Eight different sequences of ADC gene which were divided into three groups,viz.,1011,999 and 981 bp were obtained successfully.These 8 DNA sequences shared 81-99% homology in nucleotides and 86-99% homology in deduced amino acid sequences.All of the 8 sequences were named as S1-S3 of 1011 bp,S4-S5 of 999 bp,S6-S8 of 981 bp with 9,3 and 14 SNP sites in three groups,respectively.Compared with S1-S3,there were 2 sections base deletion in S4 and S5,and 4 sections base deletion in S6-S8,and the nucleotide sequence in sites 632-634 and 642-650 were absent in both S4-S5 and S6-S8.Furthermore,a nonsense mutation located in the nucleotide sequence in site 67 of S8 was identified.Amino acids homology and evolutionary analysis results indicated that the 8 sequences had high homology with those of other reported plants including Brassica juncea L.,Arabidopsis thaliana L.,Thellungiella halophila L.and so on.[Conclusion]The studies clearly demonstrated that these 8 sequences were derived from ADC gene of Brassica napus L.variety Zhongshuang 6.

关 键 词：甘蓝型油菜 精氨酸脱羧酶(ADC)基因 同源克隆 序列分析 

分 类 号：S635[农业科学—蔬菜学]