摘要
A gene with complete sequence of 1 176 bp containing an open reading frame (ORF) of 281 amino acids, RSG6 (accession number in GenBank: AF442490) was cloned from rice (Oryza sativa L.) sperm cell cDNA library by using sperm cell higher expression clone BF475207 as probe. It is the first gene isolated directly from sperm cells of higher plants. The sequences of RSG6 gene and its deduced amino acid did not reveal remarkable similarity to that of any known ones in GenBank. Southern hybridization suggested that there may be introns. in this gene. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) showed that the RSG6 gene was expressed in all nice organs or cell types examined (including roots, leaves, two-cell stage pollen, mature pollen, pollinated ovary and sperm cells), but the level of expression in sperm cells was particularly higher than that in all the other organs or cells. The RSG6 gene was cloned into a vector pQE30 and expressed in Escherichia coli M15 (pREP4). High specific antibody with high titer was obtained from rabbits.
用水稻 (OryzasativaL .)精细胞优势表达克隆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。Southern杂交显示该基因可能含有内含子。RT_PCR结果显示该基因在根、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 (ricespermgene 6 )。将RSG6的编码区克隆到表达载体pQE30上 ,构建重组质粒。在大肠杆菌M15中表达出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。
基金
欧盟国际合作项目 (ERBIC18CT960 0 72 )
教育部博士点基金 ( 980 610 3 0 )
教育部骨干教师计划专项 ( 2 0 0 0 65 15 )
四川省重点科研项目 ( 1997JB0 0 2 )~~
