PBU诱导尾叶桉愈伤分化过程中SOD活性及基因表达的变化

Changes of SOD Activity and Gene Expression during Callus Differentiation of Eucalyptus urophylla Induced by PBU

为了探讨脲类细胞分裂素PBU在尾叶桉(Eucalyptus urophylla)愈伤分化过程的作用机理,将下胚轴接种于含0.57μmol/L IAA和细胞分裂素(分别为4.56μmol/L PBU,4.56μmol/L 6-BA和3.99μmol/L PBU+0.57μmol/L 6-BA)的SPCa培养基中,诱导愈伤组织形成,检测尾叶桉不同时期愈伤组织的SOD酶活性、SOD基因表达水平及其他指标。结果表明:PBU诱导的2周龄愈伤组织的SOD酶活性显著高于6-BA或PBU+6-BA。PBU诱导的6周龄愈伤组织的超氧阴离子产生速率和过氧化氢质量摩尔浓度显著低于其他激素组合。检测PBU诱导组不同阶段的愈伤组织SOD酶活性,前6周酶活性无明显区别,8周龄白色愈伤组织SOD酶活性下降,仅为50.67 U/mg,但10周龄的再生型愈伤组织SOD酶活性高达968.70 U/mg。设2周龄愈伤组织基因相对表达量为1,SOD1、SOD2、SOD3、SOD4、SOD5表达均在2~6周表现出不断升高的趋势,并在10周龄的绿色愈伤组织中最高,分别是24.45、8.18、11.96、14.96、7.22。SOD6第6周表达最高,为5.51,但随后下降,10周绿色愈伤组织中的相对表达量仅为1.03。结合基因编码蛋白的细胞定位及酶类型分析:PBU诱导定位于叶绿体和线粒体的SOD高效表达,提高不同类型SOD酶活性,维持ROS代谢平衡,减轻褐化,促进再生型愈伤组织分化。

In order to explore the mechanism of urea cytokinin PBU in callus differentiation of Eucalyptus urophylla,the hypocotyls of E.urophylla were inoculated on SPCa medium containing 0.57 μmol/L IAA,supplemented with 4.56 μmol/L PBU,4.56 μmol/L 6-BA or 3.99 μmol/L PBU and 0.57 μmol/L 6-BA,respectively.The SOD activity of two-week-old calli induced by PBU was 183.67 U/mg protein,which was significantly higher than that induced by 6-BA or PBU and 6-BA.The generation rate of superoxide anion and the content of hydrogen peroxide of six-week-old calli induced by PBU were significantly lower than that induced by 6-BA or PBU and 6-BA.There was no obvious difference in SOD activity of calli induced by PBU from 2 nd week to 6 th week.The SOD activity of white calli at 8 th week declined,only 50.67 U/mg protein.However,the organogenic calli at 10 th week was as high as 968.70 U/mg protein.The relative transcription levels of six SOD genes in calli at different stages were detected by qPCR with the transcription level of the relative gene in two-week-old calli normalized to one.The gene expression of SOD1,SOD2,SOD3,SOD4 and SOD5 all showed a rising trend from 2 nd week to 6 th week.The transcription levels of above genes were the highest in 10-week-old organogeniccalli,which were 24.45,8.18,11.96,14.96 and 7.22,respectively.For the SOD6 gene,the transcription level,5.51,was the highest at 6 th week.Then,it declined and the transcription level in the organogeniccalli was only 1.03.The results indicated that PBU induced the high-efficiency expression of SOD gene that located in chloroplast and mitochondria,improved the activity of different types of SOD,maintained the balance of ROS metabolism,reduced browning and promoted the organogeniccalli formation.The study might provide a reference for exploring the mechanism of urea cytokinins and the establishment of an efficient regeneration system of eucalyptus.