發(fā)布時(shí)間：2018-03-24 19:50

  本文選題：蘋果　切入點(diǎn)：矮化　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：砧木對(duì)果樹樹體的生長發(fā)育、果實(shí)產(chǎn)量和品質(zhì)有著重要的影響,目前矮化砧木在世界主要蘋果生產(chǎn)國中得到了廣泛的應(yīng)用。遺憾的是,矮化砧木致矮的分子機(jī)理國內(nèi)外雖有研究,但至今尚不明確。本研究以不同蘋果砧木為材料,檢測其根系玉米素含量,發(fā)現(xiàn)矮化砧木根系玉米素含量顯著低于喬化砧木,對(duì)蘋果砧木根系細(xì)胞分裂素代謝通路的基因家族成員進(jìn)行篩選,發(fā)現(xiàn)IPT5b基因在不同喬化、矮化砧木間存在表達(dá)差異。進(jìn)一步研究發(fā)現(xiàn),矮化砧木M9和喬化砧木八棱海棠的IPT5b基因啟動(dòng)子序列上存在著豐富的SNP和InDel差異,分別構(gòu)建兩種材料的啟動(dòng)子序列的分段刪除載體和定點(diǎn)突變載體,利用擬南芥原生質(zhì)體轉(zhuǎn)化體系,最終確定了 IPT5b基因啟動(dòng)子區(qū)上游距離ATG起始密碼1172bp處的Deletion變異是導(dǎo)致基因表達(dá)差異的主要原因。基于Deletion變異在蘋果砧木中檢測方法的開發(fā),這一變異與致矮性的關(guān)系在蘋果砧木種質(zhì)資源和八棱海棠×M9雜交群體中得到了驗(yàn)證。IPT5b基因異源轉(zhuǎn)化試驗(yàn)證明了 Deletion變異影響根系玉米素合成,這說明Deletion變異可能通過調(diào)控根系玉米素含量,影響砧木矮化特性。此外,本研究還發(fā)現(xiàn),M9砧木IPT5b基因啟動(dòng)子區(qū)域CpG2島和CpG4島上甲基化水平顯著高于八棱海棠,對(duì)M9砧木根系外施DNA甲基轉(zhuǎn)移酶抑制劑5-氮胞苷后,M9砧木根系IPT5b基因表達(dá)明顯上調(diào),但尚未達(dá)到喬化砧木的平均水平,說明M9砧木IPT5b基因啟動(dòng)子區(qū)甲基化的維持對(duì)基因表達(dá)有一定影響。鑒于擬南芥根系中AUX/IAA家族基因?qū)tIPT5基因具有調(diào)控作用,AUX/IAA家族基因編碼區(qū)Domain Ⅱ的變異通過影響AUX/IAA蛋白的泛素化降解途徑而抑制AtIPT5基因表達(dá)。本研究克隆了 M9和八棱海棠中AUX/IAA基因家族成員編碼區(qū),并對(duì)Domain Ⅱ的序列進(jìn)行比較后發(fā)現(xiàn),M9砧木中AUX/IAA114基因的Domain Ⅱ存在兩個(gè)氨基酸的缺失,并初步探討了IPT5b基因的上游調(diào)控機(jī)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，

本文編號(hào)：1659781