烟草叶片愈伤组织形成过程中的细胞程序性死亡(英文)

Programmed Cell Death in the Process of Nicotiana benthamianav Leaves Callus Inducement

以烟草叶片在MS培养基上诱导的10个不同时期的愈伤组织为材料,通过荧光染色显微观察到细胞核在形态上发生了缩缢变形,DNA Ladder检测也出现了标志细胞程序性死亡特征的DNA片断,表明了愈伤组织形成过程中发生了细胞程序性死亡(programmed cell death,PCD),而在诱导的第六个时期(13 d)DNA的脱尾现象最为明显,表明这一时期是PCD发生较为活跃的时期。利用RT-PCR法在诱导的前九个时期,即诱导的第3、5、7、9、11、13、15、17、19 d的叶片的愈伤组织中均扩增出了细胞程序性死亡相关Beclin1基因的cDNA片段(678 bp),Northen杂交结果显示,在诱导的第13 d,类Beclin1基因表达较强。通过酶联免疫法测定诱导前14 d叶片中内源激素ABA的含量变化,在诱导的第六时期ABA含量相对达到了峰值,结果表明:在烟草叶片愈伤组织诱导过程中伴随PCD的发生,在此过程中内源激素ABA与Beclin1基因起到了调节作用。

We induced the ten different stages callus from the leaves of Nicotiana benthamiana （tobacco） cultured in the MS medium. The morphologic varieties of nucleolus was observed through the fluorescence microscope and DNA Ladder was tested during ten different stages of the induced callus tissue of tobacco leaves, which correlated with PCD by RT-PCR method in the nine different stages, leaf segments cultured at 3,5,7,9,11,13,15,17,19 days. Northern blotting result showed that the expression of Beclinl -like gene at stage six, the leaf segments cultured 13 days was showed Programmed cell death （PCD） happened in the process of callus inducement, and stage six, the leaf segments cultured 13 days was the active period of PCD occurred. Then we cloned fragment of Beclinl gene （678 bp） cDNA which was higher than those of the other eight stages, Furthermore the content of endogenous hormones ABA in leaf segments was measured by ELISA method, ABA reach to peak at stage six, All these results proved that PCD certainly happened during the tobacco leaves callus induced, ABA and Beclinl gene had regulated function in this process.