肥城桃果实乙烯受体ETR1蛋白重组条件优化及NO对ETR1基因表达的影响

Optimization of Recombination Proteins of Ethylene Receptor ETR1 from Feicheng Peach Fruit and Effect of Nitric Oxide on Expression of ETR1

克隆与肥城桃成熟有关的乙烯受体ETR1基因片段,优化ETR1的表达条件,获得ETR1重组蛋白,为进一步研究ETR1功能并改善肥城桃贮运性能奠定基础。以肥城桃成熟果实总RNA反转录cDNA为模板,经PCR扩增得到特异片段,将该片段连接到质粒,经双酶切后,连接至质粒并转染至表达载体,IPTG诱导,优化表达条件。并以cD-NA为模板,进行实时荧光定量PCR,测定外源NO对ETR1相对表达量的影响。序列分析结果表明,该序列与Gen-Bank中的AF124527的cDNA序列同源性为99%,氨基酸序列同源性为99%。优化蛋白表达条件为:IPTG最佳浓度为0.5 mmol/L,最适温度为30℃,最适诱导时间为8 h。经优化后,ETR1重组蛋白成功表达,为TETR1的重组生产和对ETR1功能的体外研究提供了条件。此外,外源NO对体外表达的ETR1基因有抑制作用。

In this study a ripening related ethylene receptor ETR1 gene in Feicheng peach fruits was cloned and the recombination protein of ETR1 was obtained for the further researches on the functions of ETR1 and improving the storage and transport quality of Feicheng peach fruits.Using cDNA reverse-transcribed from total RNA in mature Feicheng peach fruits as template,the specific PCR product of ETR1 was obtained,then it was ligated to pEASY-T1 vector.The sequencing data showed that the PCR product was about 2 500 bp.After digested by BamHⅠ and NdeⅠ,the sequence was ligated to pET15b and transformed to E.coli.Recombinant ETR1 was induced by IPTG,and the expression of ETR1 was optimized.And with the cDNA as templates,perform real-time quantitative fluorescence PCR,determine the influence of exogenous NO on expression of ETR1.Comparison with the cDNA,sequence of AF124527 indicated the homology was 99%,and amino acid identity was 99%.The proper conditions for recombinant ETR1 protein were that the mutant host strains E.coli was C43（DE3）.The final concentration of IPTG was 0.5 mmol/L.The proper temperature was 30℃ and the induction time was 8 h.Recombinant ETR1 was highly expressed under the optimized conditions,which was helpful for studying the functions of ETR1 in vitro.And,the inhibition by NO on the expression of ETR1 depended on the dose of NO.