桃芽中PpMPK3介导的ABA调控休眠的分子机制研究

Dormancy has emerged as the focus of concern under the background of global climate change, and it is seen as the crucial link to determine the ripening date of fruit tree grown within agricultural facilities. Our previous study found that ABA played a key role in controlling the dormancy, and the mechanism of dormancy was evolutionarily conservative between bud and seed tissues. Furthermore, the SSH cDNA library correlated with dormancy has been constructed and it suggested that MAPK gene family was likely involved in controlling dormancy. The phylogenetic relationships within members of the gene family have been analyzed, and the expression patterns of the gene family have been examined during dormancy. As a result, ABA mediates inhibition of seed germination and the expression level of MAPK is controlled by ABA. But the signal pathway of ABA mediated by PpMPK3 controlling bud dormancy is still to be elaborated. So the PpMPK3 was selected as the likely candidate gene involved in dormancy. To characterize the function of PpMPK3 gene, over-expression of this gene will be individually performed in the wild-type and mutant of Arabidopsis thaliana and poplar lines. As we expect, the function of PpMPK3 will be further characterized, and the molecular mechanism of ABA mediated by PpMPK3 regulating bud dormancy will be preliminarily revealed. This study will provide a theoretical basis for manual regulating the bud dormancy of fruit tree.

休眠是全球气候变化中人们关注的热点领域，亦是果树设施栽培熟期调控的科学问题。本课题组前期研究发现ABA在休眠中发挥至关重要的作用，且芽休眠与种子休眠有一致性。利用构建的休眠相关SSH文库发现MAPK基因家族与休眠相关，进一步对该基因家族进行筛选比对和荧光定量检测发现PpMPK3参与调控休眠过程。已有研究证明：ABA是抑制种子萌发的激素信号，MAPK在转录水平受ABA调控，而在PpMPK3介导下ABA调控桃芽休眠过程的信号通路尚无报道。本研究拟构建PpMPK3的表达载体，转化拟南芥和杨树进行生物学功能鉴定。研究结果可明确PpMPK3基因的功能，阐明PpMPK3介导ABA调控桃芽休眠的分子机制，为桃树休眠的人工调控奠定理论基础。

以‘中油四号’油桃为材料，筛选了桃树基因组中调控 ABA 代谢的PpMPK3 关键基因，检测 ABA 、GA处理下PpMPK3 基因在种子休眠以及芽休眠过程中的表达模式、组织表达特性及种子发育期表达；测定了油桃自然休眠期间花芽中内源 ABA、H2O2 的含量变化；研究了 H2O2、PD98059 处理对休眠期间桃芽 MAPK 相关基因、H2O2 及相关酶活性的影响。主要结果如下：1.通过亚细胞定位发现，PpMPK3定位于细胞核。qT-PCR结果表明， PpMPK3的表达模式与休眠解除过程相一致。推测PpMPK3能够响应GA信号，促进休眠解除。油桃芽内 ABA 含量在整个休眠期间呈先升后降的变化趋势，深休眠期 ABA 一直保持在较高水平，随着休眠解除期的临近 ABA 开始迅速下降，并在休眠解除后维持在较低水平；芽内 H2O2 含量在自然休眠期间同样呈先升后降的变化趋势，与 ABA 含量变化基本一致，在深休眠期保持在较高水平，休眠中后期至休眠解除呈稳步下降的趋势；油桃中存在 15 个 MAPK基因和 8 个 MAPKK 基因，为 A、B、C、D 四个组，在各组织中均有表达，其中有6 个 MAPK 和 4 个 MAPKK 在花芽休眠解除过程中呈现表达量下降趋势，与花芽休眠解除过程中 ABA、H2O2 水平的下降趋势相吻合，说明在休眠解除过程中 MAPK 相关基因很可能参与 ABA、H2O2 的信号转导，从而解除休眠；休眠解除期 H2O2 处理对芽内H2O2 含量与 CAT 活性无显著影响，提高了5 个 MAPK基因的表达水平；休眠中期 PD98059处理对芽内 H2O2 含量与 CAT 活性无显著影响，降低了 Pp MPK12、Pp MPK13、Pp MKK4 的表达水平。