摘要
本文采用RT-PCR方法,从籽粒苋(Amaranthus hypochondriacus)"千穗谷1号"的幼嫩种子克隆出AmA1基因的完整开放阅读框序列(ORF),其长度为915bp,编码305个氨基酸。经Blast同源性分析,结果表明该序列与GenBank登录的籽粒苋AmA1基因的核苷酸序列基本一致,只在起始密码子下游51bp处由G变成C,但不影响氨基酸的编码。将该基因插入原核表达载体pET28a(+),并转化到大肠杆菌BL21中,经过IPTG诱导,能正确表达出37kD的融合蛋白。同时构建了AmA1基因双T-DNA双标记植物表达载体PCDMAR-AmA1-dsRed2-hpt,该载体含有2个独立的T-DNA区,其中一个T-DNA区含有选择标记基因hpt和可视标记基因DsRed2(红色荧光蛋白基因),另一个T-DNA区含有由水稻胚乳特异性表达启动子pGt1驱动的AmA1基因,在AmA1基因的两侧连有两段正向重复的烟草Rb7MARs,以增强AmA1基因的表达。实验结果为下一步规模化培育稻米氨基酸组成平衡无选择标记的转基因水稻奠定了基础。
The whole open reading fragment （ORF） ofgene encodind protein with well-balanced amino acid composition was cloned from A maranthus hypochondriacus ＂Qiansuigu No. 1 ＂using RT-PCR. The length ofA mA 1 gene is 915 bp, which encoding a peptide with 305 amino acid residues. The homology analysis results showed that A- mA 1 gene share high similarity with A maranthus hypochondriacus AmA 1 protein gene which had been registered in GenBank.Comparing to original AmA1 gene, the base of AreA1 gene change from G to C at 51 bp downstream of the start condon, but it does not result in change of amino acid encoding. AmA 1 gene was cloned into PET-28a（＋） vector and then transformed into E. coli BL21. SDS-PAGE analysis showed that a band with about 35 kD in molec ular mass was found after inducation with IPTG. A dual marker double T-DNA vector PCDMAR-A mA 1-dsRed2- hpt was constructed, which was composed of two transfering DNA region, one of T-DNA regions include selecting marker gene hpt and red fuorescent protein gene DsRed2 as a visual marker, the other T-DNA region includes AreA 1 gene driven by rice endosperm-specific promoter pGtl. Two piece of directrepeat tabacoo Rb7 MAR sequence flanked the A mA 1 gene to increase target gene stable expression. This result provide experimental base for the large-scale breeding of marker-free transgenic rice with well-balanced amino acid composition.
出处
《分子植物育种》
CAS
CSCD
2009年第4期793-800,共8页
Molecular Plant Breeding
基金
国家转基因生物新品种培育重大专项(2009ZX08001-032B
2008ZX08001-006)
福建省科技创新平台建设项目(2007S1001)
福建省科技厅重大专项专题项目(2008NZ0001-4)共同资助
关键词
籽粒苋
AmA1
基因克隆
原核表达
载体构建
A maranthus hypochondriaeus, AmA 1, Gene cloning, Prokaryotic expression, Vector construction
