外源NAA对甜瓜嫁接愈合关键期保护性酶活性及相关基因表达的影响

Effects of exogenous NAA on the protective enzymes activities and related genes expression at the key stages of melon graft healing

通过分析外源施用植物激素NAA(萘乙酸)条件下甜瓜嫁接愈合3个关键时期(隔离层形成期、愈伤组织形成期、维管束桥形成期)保护性酶活性及相关基因表达的变化,探析外源施用NAA对甜瓜愈合过程中生理及其基因表达特性的影响。以蘸取清水的嫁接苗为对照,外源施用40 mg·L-1NAA的嫁接苗为处理,薄皮甜瓜‘银泉一号’为接穗,白籽南瓜‘圣砧一号’为砧木,采用顶端贴接法嫁接。利用石蜡切片筛选嫁接愈合关键时期,测定嫁接愈合过程中4种关键代谢酶(SOD、POD、PPO和PAL)活性,并利用实时荧光定量(qRT-PCR)测定其相关基因表达量。通过石蜡切片观察发现,外源NAA处理后,愈伤组织形成期和维管束桥形成期较对照提前1 d。SOD、POD酶活性呈先升高后降低的趋势,在愈伤组织形成期达到最高值,且酶活性显著高于对照,PAL酶活性在愈合过程中均高于对照,且在维管束桥形成时期达到最高值。PPO酶活性在隔离层形成期显著高于对照,随后其活性略低于对照,但差异不显著。除CmPOD外,外源NAA处理能够显著提高CmFe-SOD、CmCu-Zn-SOD、CmMn-SOD、CmPPO相对表达量,且其变化趋势与酶活性基本一致。综合分析表明,外源施用NAA后,缩短了甜瓜嫁接苗的愈合期,并且能够诱导影响嫁接愈合的关键酶基因表达,提高其相关酶活性,进而促进甜瓜嫁接苗的愈合进程。

The protective enzyme activities and related gene expression were analyzed to investigate the effects of exogenous NAA(naphthalene acetic acid) application on the physiological and related genes expression characteristics at three key stages of melon graft healing process(isolated layer formation, callus formation, vascular bundle formation). In this experiment, the grafted seedlings that graft union dipped 40 mg×L^-1 NAA were used as the treatment, and those dipped water were used as the control. We used the oriental melon‘Yinquan No. 1'as the scion, and the white-seed pumpkin'Shengzhen No.1' as the rootstock. Splice approach grafting was used as the grafting method. The paraffin sections screened out the key stages of graft healing process. The activities of four key metabolic enzymes(SOD, POD, PPO and PAL) during graft healing were determined, and the expression of related genes was determined by real-time quantitative quantification(qRT-PCR). It was observed by paraffin sections that the formation of the callus and vascular bundle were1 day earlier than the control by exogenous NAA treatment. The activities of SOD and POD increased first and then decreased, reached the highest value at the stage of callus formation, and the enzyme activities were significantly higher than that of control. The activities of PAL were higher than that of control and reached the highest value at the formation of vascular bundle. The activities of PPO were significantly higher than that of control at the formation of the isolated layer and indistinctively lower than the control with the grafting seedlings growth. Exogenous NAA treatment significantly increased the relative expression of CmFe-SOD, CmCu-Zn-SOD, CmMn-SOD and CmPPO except CmPOD, and the trend of changes is basically consistent with the enzyme activities changes. The comprehensive analysis showed that the exogenous NAA application shortened the time of grafted healing, induced the expression of key enzyme genes that involved in graft union and increased the related enzyme activities, which promoted the graft healing process of grafting melon seedlings.