摘要
根据灯盏花转录组所注释的CHI unigene片段,设计了RACE相关引物,克隆到灯盏花CHI的cDNA全长序列,序列全长717bp,编码238个氨基酸.该氨基酸序列与我们前期所克隆的灯盏花CHI cDNA序列相比,在345、351位点发生了突变,碱基均由C突变为T,两个位点的突变均属于同义突变,编码的氨基酸分别为115、117位异亮氨酸、酪氨酸.同时将CHI基因片段插入到pDM 18-T,构建pDM 18-T-eBCHI中间载体.经测序验证后,根据pBI121-EGFP图谱,设计带有SalI、SpeI酶切位点的引物,以pDM 18-T-eBCHI中间载体为模板,扩增带酶切位点的CHI序列,扩增产物经回收、纯化后与双酶切的pBI121-EGFP链接,构建了重组表达载体pBI121-EGFP-CHI,CHI基因插入植物表达载体pBI121-EGFP的6xHis组氨酸标签与GFP基因间.经SalI、SpeI双酶切和PCR验证重组质粒,均能得到约750bp的目的片段,通过进一步测序证明,CHI基因已成功地连接到pBI121-EGFP-eBCHI中.采用电击转化法将重组质粒导入根癌农杆菌GV3101,用叶盘法转化灯盏花叶片,筛选出具有卡那霉素抗性的愈伤组织,经PCR扩增和叶绿素荧光成像检测证明,重组基因已成功地转化到灯盏花愈伤组织中.为利用转基因技术研究CHI基因的表达及其亚细胞定位打下基础.
In order to study subcellular location of CHI encoding protein derived from Erigeron breviscapus and its effecte on phenolis phenotype under overexpression of CHI. The full length of eDNA of CHI was cloned via RACE technique according to a pair of primers designed from se quence annotated CHI unigene of transcriptome data under low nitrogen in Erigeron breviscapus. After sequencing,the results showed that the cDNA 238 amino acids. Comparing to the amino acid sequence which we port,two point mutation occurred at 345 and 351 in the new CHI is mutated to T. The mutation of two sites belong to synonymous are isoleucine and tyrosine, at position 115 cloned to pI）M 18 T,a intermediate vector, of CHI was 717 bp,encoding had cloned in our previous re eDNA sequence by the base C mutations,encoded amino acids and ll7,respectively. And the eDNA of CHI was pDM 18-eBCHI. After sequencing of pDM 18-eBCHI, according to the map of pBI121 EGFP, a pair of primers was design with SalI, SpeI restriction sites,and the CHI sequnence was amplified using the intermediate vector,pDM 18 eBCHI,as tern plate,after recovered and purified,the PCR products of CHI was linked with pBI121-EGFP which were double digested with SalI and SpeI to construct the recombinant expression vector,pBI121- EGFP eBCHI which with CaMV35S as promoter. CHI gene was inserted into a plant expression vector pBI121 EGFP between the histidine tag and 6xHis GFP gene. After SalI, SpeI digested of plasmid and PCR validation,a approximately 750 bp fragment can be obtained. Further sequencing of pBI121-EGFP-eBCHI showed that the CHI gene has been successfully inserted into the pBI121-EGFP eBCHI. Electroporation method was utilized to introduce the recombinant plasmid into Agrobacterium tumeflaciens GV3101,and then transfered into Erigeron breviscapus by leaf disc method with leaf as explants. The kanamycin resistant transformants were obtained by kanamycin screening. And the resistant transgenic calluses of Erigeron [reviscapus were detected by PCR and the observation of
出处
《云南师范大学学报（自然科学版）》
2016年第3期53-60,共8页
Journal of Yunnan Normal University:Natural Sciences Edition
基金
国家自然科学基金资助项目(30660075
31360263)
云南省科技厅社会发展科技计划基础研究面上资助项目(2009CD052)
云南省应用基础研究计划资助项目(2011FA016)
关键词
灯盏花
查尔酮异构酶
绿色荧光蛋白
表达载体
Erigeron breviscapus（Vant.）Hand.-Mazz
Chalcone isomerase
Green fluorescence protein
Expression vector
