基于转录组测序筛选的MdCoLBDs和MdKS基因功能分析及其调控苹果柱型机理研究

Co (Columnar) gene of Malus×domestica Borkh is a valuable genetic resource, due to its regulating columnar architecture formation with dwarf and less branches tree, which is easy to pruning and suitable for high density planting. Co is located in the linkage group 10 (LG10) based on molecular markers on the linkage map, but the molecular mechanism of columnar traits genetically regulated by the Co gene is unclear. Using the RNA-Seq and positional screening, we have screened the LBD transcription factors (MdCoLBD1, MdCoLBD2) and gibberellin synthesis key enzyme MdKS as putative Co candidate genes. Based on these preliminary research, we proposed a sytematic study on apple Co candidate genes with three specific objectives: 1) function and expression characteristics analyzing of Co candidate genes by transforming 'Gala' apple and columnar recovery mutant 'M-95-121' with binary vectors, in situ hybridization, and investigating expression differences of Co candidates genes between the columnar and standard apple trees using Real-Time quantatative PCR (RT-qPCR); 2) identifying significant differences genes on metabolic pathways using RNA-Seq analysis under candidate genes overexpressed; 3) expression pattern analyzing of key genes of AUX/IAA GA20ox, KS, IPT and LBDs on hormone metabolic pathways after IAA, GA3, 6-BA and inhibitor treating transformated plants, and to explore how the candidate genes regulate the columnar formation through the relationship with IAA, GA, CTK. Based on the above study, the molecular regulation mechanism of columnar growing habits will be characterized through candidate genes expression analyzing, biological function, and how to affect the columnar formation by hormone metabolism and interactions. This study will provide a theoretical basis for genetic engineering to improve the apple tree's architecture suitable for high density planting.

苹果柱型基因Co调控树体柱型发育，使其矮生、分枝少，易于修剪并适宜密植栽培，是树型育种重要基因资源。遗传图谱将Co定位在LG10连锁群，但Co及其调控机理尚不清楚。课题组已通过转录组测序结合定位筛选将LBD转录因子（MdCoLBD1、MdCoLBD2）和赤霉素合成关键酶MdKS初定为候选基因。本项目在此基础上拟通过转化'嘎啦' 苹果和柱型恢复突变体、荧光定量PCR、原位杂交分析其功能和表达特性；RNA-Seq探寻转基因后代谢途径上显著差异基因变化规律；用IAA、GA3、6-BA及抑制剂处理转基因植株，分析激素代谢及关键基因AUX/IAA、GA20ox、KS、IPT 和LBDs表达规律，结合转录因子结合位点分析，探讨候选基因与IAA、GA、CTK作用关系及其对柱型的调控。综上从基因表达特性、生物学功能、与激素作用关系影响柱型形成三层面揭示柱型调控机理，以期为基因工程选育矮化密植树型提供参考。

苹果柱型基因Co调控树体柱型发育，使其矮生、分枝少，易于修剪并适宜密植栽培，是树型育种重要基因资源。在前期研究的基础上，本项目构建了Co候选基因MdCoL（MdCoLBD）和MdKS双元表达载体，获得转基因嘎啦苹果株系5个、愈伤组织株系9个、转基因烟草株系17个，利用不同激素处理柱型苹果以及转基因株系，测定了其激素含量，荧光定量PCR分析了相关基因表达，利用酵母双杂交技术及双分子荧光试验筛选了与Co候选基因MdCoL互作的蛋白。研究结果表明：Co候选基因MdCoL和MdKS基因均定位于细胞膜和细胞核上。MdCoL在柱型苹果芽和新梢中表达量都明显高于普通型苹果。MdKS基因在普通型和柱型苹果根、春梢、夏梢、秋梢、叶、花、果实均有表达，表达量普通型高于同期相应器官的柱型苹果。转MdCoL基因株系植株矮小，粗壮，节间短、长分枝少，表现出明显的柱型性状，说明MdCoL基因在调控植株的株型上起重要作用。对转基因材料进行RNA-Seq，获得了142个上、下调与株型形成相关的Unigene，进一步筛选了与株型显著相关21个Unigene。酵母双杂交文库筛选获得与Co候选基因MdCoL互作的蛋白20个。通过激素测定发现柱型苹果茎尖以及转基因株系ABA的含量是显著高于普通型苹果和未转基因对照，而TZR和GA3的含量均低于普通型苹果和未转基因植株。赤霉素生物合成途径中的关键基因MdGA2oxI、MdGA3oxI、 MdKs和MdKAO 明显低于普通型苹果。转MdCoL基因烟草GA2oxI和GA3oxI基因显著下调表达；赤霉素受体相关基因（GID1a）和DELLA蛋白合成相关基因与未转基因对照相比表达量明显上升。综合以上结果，初步得出Co候选基因调控苹果柱型性状形成的分子机理：柱型性状不仅是由于体内低的GA和TZR决定，同时受较高ABA含量影响；Co候选关键基因MdCoL可能通过调控DELLA蛋白相关基因的表达使植株体内GA含量控制在较低的范围，同时MdCoL基因通过与MdDREB2互作，参与调控ABA合成中关键基因NCED（MdNCED3、MdNCED6、MdNCED9）的表达量提高，以合成更多的ABA，从而控制植株的柱型性状。以上试验证明了Co候选关键基因MdCoL在调控苹果柱型中的作用，为今后通过基因工程选育矮化密植树型提供了理论和基因方面的参考。