苹果矮化砧木M9根系IPT5b基因表达调控及其致矮机理的研究

发布时间：2018-03-24 19:50

  本文选题：苹果　切入点：矮化　出处：《中国农业大学》2017年博士论文

【摘要】：砧木对果树树体的生长发育、果实产量和品质有着重要的影响,目前矮化砧木在世界主要苹果生产国中得到了广泛的应用。遗憾的是,矮化砧木致矮的分子机理国内外虽有研究,但至今尚不明确。本研究以不同苹果砧木为材料,检测其根系玉米素含量,发现矮化砧木根系玉米素含量显著低于乔化砧木,对苹果砧木根系细胞分裂素代谢通路的基因家族成员进行筛选,发现IPT5b基因在不同乔化、矮化砧木间存在表达差异。进一步研究发现,矮化砧木M9和乔化砧木八棱海棠的IPT5b基因启动子序列上存在着丰富的SNP和InDel差异,分别构建两种材料的启动子序列的分段删除载体和定点突变载体,利用拟南芥原生质体转化体系,最终确定了 IPT5b基因启动子区上游距离ATG起始密码1172bp处的Deletion变异是导致基因表达差异的主要原因。基于Deletion变异在苹果砧木中检测方法的开发,这一变异与致矮性的关系在苹果砧木种质资源和八棱海棠×M9杂交群体中得到了验证。IPT5b基因异源转化试验证明了 Deletion变异影响根系玉米素合成,这说明Deletion变异可能通过调控根系玉米素含量,影响砧木矮化特性。此外,本研究还发现,M9砧木IPT5b基因启动子区域CpG2岛和CpG4岛上甲基化水平显著高于八棱海棠,对M9砧木根系外施DNA甲基转移酶抑制剂5-氮胞苷后,M9砧木根系IPT5b基因表达明显上调,但尚未达到乔化砧木的平均水平,说明M9砧木IPT5b基因启动子区甲基化的维持对基因表达有一定影响。鉴于拟南芥根系中AUX/IAA家族基因对AtIPT5基因具有调控作用,AUX/IAA家族基因编码区Domain Ⅱ的变异通过影响AUX/IAA蛋白的泛素化降解途径而抑制AtIPT5基因表达。本研究克隆了 M9和八棱海棠中AUX/IAA基因家族成员编码区,并对Domain Ⅱ的序列进行比较后发现,M9砧木中AUX/IAA114基因的Domain Ⅱ存在两个氨基酸的缺失,并初步探讨了IPT5b基因的上游调控机理。
[Abstract]:Rootstocks play an important role in the growth, development, yield and quality of fruit trees. At present, dwarf rootstocks are widely used in major apple producing countries in the world. Unfortunately, Although the molecular mechanism of dwarf rootstocks has been studied at home and abroad, it is still unclear. In this study, different apple rootstocks were used as materials to detect the content of zeatin in root system, and it was found that the content of zeatin in dwarf rootstocks was significantly lower than that in arborization rootstocks. The gene family members of cytokinin metabolism pathway in apple rootstocks were screened, and the expression of IPT5b gene was found to be different among different arborescence and dwarf rootstocks. The IPT5b gene promoter sequence of dwarf rootstock M9 and Qiaohua rootstock was different in SNP and InDel, respectively. The fragment deletion vector and the site-directed mutant vector of the promoter sequence of the two materials were constructed, respectively. By using the protoplast transformation system of Arabidopsis thaliana, it was finally determined that the Deletion variation at the upstream of the promoter of the IPT5b gene was the main cause of the difference in gene expression. Based on the development of the detection method of Deletion mutation in apple rootstock, the main reason for the difference in gene expression was found in the upstream of the promoter region of the IPT5b gene. The relationship between this variation and dwarfism was verified in apple rootstock germplasm resources and hybrid population of Begonia octagnoides 脳 M9. The results of heterogenous transformation of Deletion gene showed that Deletion variation affected root zeatin synthesis. The results suggested that Deletion mutation might affect the dwarfing characteristics of rootstocks by regulating root zeatin content. In addition, the methylation levels of CpG2 island and CpG4 island in IPT5b gene promoter region of M9 rootstock were significantly higher than those of Begonia sinensis. After applying DNA methyltransferase inhibitor 5-azacytidine to root system of M 9 rootstock, the expression of IPT5b gene in root of M 9 rootstock was upregulated obviously, but it did not reach the average level of arborization rootstock. The results indicate that the maintenance of promoter methylation of IPT5b gene in M 9 rootstock has a certain effect on gene expression. In view of the regulation of AtIPT5 gene by AUX/IAA family genes in Arabidopsis thaliana root system, the variation of Domain 鈪，

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