梨茎尖中PpKO基因表达量的实时荧光定量PCR分析

Expression Analysis of PpKOGene in Pear Shoots Apical Tissue by Real-time Fluorescent Quantitative PCR

以黄金梨不同发育时间的新梢茎尖为试材,应用实时荧光定量PCR技术,对18S rRNA、Actin、GAPDH和S19这4个常用内参基因在梨茎尖组织中的表达稳定性进行了评估。GeNorm软件分析显示,它们表达稳定度的平均值(M)依次是0.365,0.392,0.457和0.517,即表达稳定性从高到低的排序是:Actin>18S rRNA>GADPH>S19。选择Actin作为校正内参基因,对处于不同发育时期的新梢茎尖组织中贝壳杉烯氧化酶基因PpKO的表达量进行研究,发现该基因在新梢生长过程中出现2次表达高峰,基本与春梢、秋梢的快速生长期吻合,且秋梢中的表达量远高于春梢。

The expression stability of 4 normal used reference genes,18S rRNA,Actin,GAPDH and S19,in pear shoot apical tissue at different developmental period was evaluated by real-time fluorescent quantitative PCR technique.GeNorm software analysis indicated that the value of average expression stability（M） was 0.365,0.392,0.457 and 0.517,respectively,which meant the sequence of expression stability from higher to lower was Actin〉18S rRNA〉GADPH〉S19.Finally,Actin was selected as the reference gene for assaying ent-kaurene oxidase gene PpKO expression in the apical tissue of pear shoots.The result showed that there were two expression peaks in the procedure of shoots development under growing season coinciding with the shoots primary and secondary rapid growth period.Moreover,the expression was much higher in the secondary shoots than the primary shoots.