水稻精细胞差异表达基因RSG6的克隆和抗体制备  

CLONING OF A DIFFERENTIALLY EXPRESSED GENE IN RICE SPERM CELLS AND PREPARATION OF ANTISERUM AGAINST EXPRESSED PRODUCT

作　　者：白彧[1] 苟小平[1] 徐莺[1] 颜钫[1] 唐琳[1] 陈放[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《应用与环境生物学报》2002年第4期378-382,共5页Chinese Journal of Applied and Environmental Biology

基　　金：欧盟国际合作项目(ERBIC1 8CT960 0 72 );教育部博士点基金;教育部骨干教师计划专项资金资助项目~~

摘　　要：选用在水稻精细胞和二细胞花粉的差减文库中获得的在精细胞中表达量显著增强且可能代表新基因的EST之一 (BF4 75 2 0 7)为探针 ,筛选水稻精细胞cDNA文库 ,得到一全长为 1176bp的序列 ,其开放读码框编码 2 81个氨基酸 ,与已知蛋白质无明显同源性 ,属于一新发现的基因 ,GenBank登录号为AF4 4 2 4 90 .这是第 1次从高等植物精细胞中分离到的全长基因 .RT PCR结果证明该基因在根、叶、二细胞花粉、成熟花粉、授粉子房和精细胞中均有表达 ,但在精细胞中的表达量要高得多 ,是精细胞差异表达基因 .将此基因命名为RSG6 (ricespermgene6 ) .将RSG6的编码区克隆到表达载体pQE30上 ,构建重组质粒 .经IPTG诱导后 ,在E .coliM15 (pREP4 )中表达出了N端融合了 6×His的融合蛋白 .用纯化的融合蛋白免疫家兔 ,制得高效价。Subtracted clone BF475207 had a higher expression in sperm cells than in two cell stage pollen. The inserted fragment of this clone was amplified via PCR method and labeled as a probe to screen the cDNA library of rice sperm cells. One 1 176 bp gene containing an open reading frame of 281 amino acids was cloned and named as RSG 6 (accession number in GenBank: AF442490). It is the first gene isolated directly from sperm cells of higher plants with complete sequence. Sequence of RSG 6 and the deduced amino acid sequence did not reveal remarkable similarity to any known sequences in GenBank, showing that it was a new gene. Reverse transcriptase polymerase chain reaction analysis showed that RSG 6 gene expressed in all rice organs or cell types (including roots, leaves, two cell stage pollen, mature pollen and sperm cells), but the level of expression in sperm cells was particularly higher, which suggested that RSG 6 be a specially abundant expressed gene of rice sperm cells. The open reading frame of RSG 6 gene was cloned into expression vector pQE30. After induction with IPTG, RSG 6 gene was expressed in E.coli M15 (pREP4) consisting of 6×His fused to the N terminal. The purified protein as antigen was used to immunize rabbits and high specific and high titer antibody was obtained.

关 键 词：水稻 精细胞 差异表达基因RSG6 克隆 抗体制备 大肠杆菌 表达 融合蛋白 抗血清 

分 类 号：Q943.2[生物学—植物学]