苹果生长素合成基因MdYUCCA8通过调控IAA和ABA合成影响侧根发育的分子机制研究

Auxin plays an important role in lateral root development. But the mechanism of the crosstalk between auxin and ABA affects lateral root development has not yet been fully elucidated. At earlier stage, Ectopic overexpression of MdYUCCA8 gene in Arabidopsis increased lateral roots numbers and lateral root length. Auxin content was significantly increased, but ABA content was significantly reduced in the transgentic lines. Based on the previous research results, this project intends to obtain transgenetic apple material to observe the root phenotype of transgenic plants and determination IAA, ABA and other hormone content. Transgenic apple plants were treated with auxin, ABA and their inhibitors to determine the role of auxin and ABA in root development. The expression of MdWRKY40 were detected to determined whether auxin treatment and overexpression of MdYUCCA8 promoted MdWRKY40 expression. The coding region of MdWRKY40 was cloned and transformed into apple rootstock M26. The contents of ABA, MdNCED and other related genes were determined in MdWRKY40 over-expressing transgenic plants to determine whether the MdWRKY40 inhibit ABA synthesis. Using yeast one hybridization, dual luciferase assay, EMSA techniques to analyze the interaction of MdWRKY40 protein and MdNCED3b, MdNCED5b promoter. At the end, elucidate the role of "MdYUCCA8-auxin-MDWRKY40-MdNCED-ABA" network in regulation of apple lateral root development.

生长素在侧根发育中的作用已经有了深入的研究。生长素和ABA相互作用调控侧根发育的机制尚未完全阐明。前期在拟南芥中异位表达苹果生长素合成基因MdYUCCA8导致植株侧根数量和侧根长度显著高于野生型，生长素含量增加，ABA含量降低。在此基础上，本项目拟获得过表达MdYUCCA8的苹果转基因材料，观察其根系表型，测定生长素、ABA等激素含量。用生长素、ABA及其抑制剂处理转基因苹果苗，确定生长素和ABA在MdYUCCA8促进根系发育中的作用。检测生长素处理和过表达MdYUCCA8是否促进MdWRKY40的表达。获得过表达MdWRKY40的苹果材料，测定ABA含量、ABA合成基因MdNCED及其它相关基因的表达量。分析MdWRKY40与MdNCED3b、MdNCED5b的启动子互作情况。从而阐明“MdYUCCA8-生长素-MdWRKY40-MdNCED-ABA”调控苹果侧根发育的机制。

生长素在侧根发育中的作用已经有了深入的研究，但生长素和ABA相互作用调控侧根发育的机制尚未完全阐明。在本研究中，在拟南芥中异位表达苹果生长素合成基因MdYUCCA8导致植株侧根数量和侧根长度显著高于野生型，生长素含量增加，ABA含量降低。对YUCCA8过表达拟南芥的转录组分析结果显示，过表达YUCCA8可能通过降低ABA合成基因NCED来降低ABA含量。进一步分析了差异的转录因子，结果显示WRKY40可能是调控ABA合成的关键差异表达转录因子。对苹果根系施加外源生长素类似物NAA，结果显示MdWRKY40a、MdWRKY40b、MdNCED3b和MdNCED5b都受生长素的诱导表达。酵母单杂交和双荧光素酶的结果显示，MdWRKY40a、MdWRKY40b分别与MdNCED3b、MdNCED5b启动子存在相互作用。基于上面的结果，提出了一个“MdYUCCA8-生长素-MdWRKY40-MdNCED-ABA-侧根发育”的模式图：生长素合成基因YUCCA8过量表达，生长素含量增加，上调了WRKY40基因表达，WRK40结合在NCED启动子上，抑制NCED表达，导致ABA含量降低，升高的生长素和降低的ABA促进侧根生长。