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根据本实验室已建立的杜仲转录组数据库提供的序列信息设计引物,以杜仲含胶组织叶和皮为材料提取总m RNA,采用RACE技术克隆了5’端700 bp和3’端600 bp c DNA片段,连接至p GEM-T载体,经测序拼接后获得全长1 075 bp的杜仲橡胶延长因子基因(Eucommia ulmoides rubber elongation factor 1,Eu REF 1)c DNA序列,其中开放阅读框678 bp,共编码225个氨基酸残基。在NCBI中blastp比对,Eu REF 1与葡萄、蓖麻REF序列同源性分别为42%和41%;预测的蛋白质Eu REF 1氨基酸序列经blastp比对,显示该预测蛋白质序列属于REF超家族。本研究首次克隆杜仲的橡胶延长因子基因c DNA序列,构建了表达载体p SH-35S-Eu REF 1,通过农杆菌介导法对烟草长脖黄进行遗传转化,经PCR检测和GUS组织染色,获得转基因烟草43株,移栽生长四个月的转基因烟草在表型上与野生型在叶片长宽比上有明显差异。
Primers were designed based on the sequence information provided by the database of Eucommia ulmoides transcriptome laboratory. The total m RNA was extracted from the leaves and husk of the eucommia ulmoides tissues. The 700 bp 5’-end and 600 bp c DNA fragment was ligated to p GEM-T vector. After sequencing, the DNA fragment of Eucommia ulmoides rubber elongation factor 1 (Eu REF 1) cDNA with a length of 1 075 bp was obtained. The open reading frame (ORF) Encoding a total of 225 amino acid residues. In NCBI, blastp alignment showed that the EU REF 1 homology was 42% and 41%, respectively. The predicted amino acid sequence of Eu REF 1 was blastp-aligned and showed that the predicted protein sequence belonged to REF superfamily . In this study, the c DNA sequence of rubber elongation factor gene was first cloned and the expression vector p SH-35S-Eu REF 1 was constructed. 43 transgenic tobacco plants were obtained, and the transgenic tobacco with four-month transplanting showed significant differences in aspect ratio and length-width ratio of the leaves.