苹果不同砧穗互作的抗旱分子机理研究

Here, we will investigate the drought resistance of two-year-old apple rootstocks and grafting seedlings grown in control, mild, moderate, and severe drought stress treatments. To investigate molecular responses in apple rootstocks, the transcriptomes from roots and leaves of M. prunifolia seedlings treated with well-watered and drought stress treatments will be built and sequenced using Illumina Solexa technology, respectively. Bioinformatics will be used to identify the differentially expressed genes based on transcriptome sequencing library of M. prunifolia, and real-time quantitative PCR will be performed to analyze the relative expression levels of the differentially expressed genes in different organs and tissues of M. prunifolia under different stresses. cDNA-AFLP technique will be used to identify differentially expressed transcript-derived fragments (TDFs) in the roots and leaves of rootstock–scion interactions under well-watered and drought stress treatments, and the TDFs will be validated, cloned, sequenced and the analyzed. A comparative study with differences in proteins from the roots and leaves of rootstock–scion interactions will be carried out by using two-dimensional gel electrophoresis and mass spectrometry during drought and normal processing conditions, respectively. Together, this study will strikingly elucidate the molecular responses in apple induced by drought stress, which will ultimately provide us a theoretical foundation for cultivation adaptability and molecular breeding of apple in China.

本研究以2年生苹果砧木及不同嫁接苗作为研究对象，从分子水平揭示苹果砧木及其嫁接苗的抗旱机理。采用RNA-seq及高通量测序技术构建干旱处理下楸子根系和叶片的转录组测序文库，借助生物信息学分析差异表达基因，用实时荧光定量PCR技术验证其组织特异性及时空表达模式。利用cDNA-AFLP技术筛选不同砧穗组合根系和叶片中的差异表达转录衍生片段（TDFs），并对其进行验证、克隆、测序和生物信息学分析。利用双向电泳技术结合质谱检测技术对不同砧穗组合在干旱胁迫与正常条件下根系和叶片表达差异蛋白质组进行对比研究。结果将阐明苹果对干旱胁迫的分子应答机制，为我国苹果适应性栽培及分子育种提供理论依据。

栽培苹果主要通过嫁接进行繁育，嫁接砧木影响着苹果耐旱性等园艺性状。目前人们对苹果砧穗互作抗旱分子机制的认识尚不清楚。本研究以2年生嘎拉/楸子与嘎拉/平邑甜茶嫁接苗及楸子实生苗为研究对象，首先比较分析了干旱胁迫下2种苹果嫁接苗形态结构、光合生理及气孔反应的变化，明确了2种苹果砧穗组合对干旱胁迫的适应能力；接着利用RNA-seq技术构建了干旱胁迫下2种苹果嫁接苗及楸子实生苗叶片及根系的转录组文库，利用生物信息学等方法比较分析不同砧穗组合及嫁接砧木应答干旱胁迫的差异表达基因（DEGs）及转录因子，挖掘出参与砧穗互作的干旱相关基因，如生长素响应蛋白IAA10、乙烯受体ETR2、同源盒亮氨酸拉链蛋白ATHB-7、NAC72、bHLH130、MYB62、WRKY61等基因。基于iTRAQ及质谱技术构建了楸子叶片和根系应答干旱胁迫的差异蛋白质组，从转录组和蛋白质组水平挖掘了干旱胁迫下楸子不同组织的DEGs与差异表达蛋白（DEPs），利用生物信息学等方法对苹果砧木抗旱关键基因及代谢途径进行了鉴定分析；基于转录组与蛋白质组联合分析，鉴定出苹果嫁接苗参与抗旱反应的关联基因与蛋白，明确了干旱胁迫下苹果关联蛋白与基因的表达模式。利用RT-PCR技术从楸子叶片中克隆出2个苹果（Malus）新基因MpRZ1和MPGR-RBP2，结合转录组分析数据对苹果GR-RBPs基因家族的RRM保守结构域、进化关系、基因表达谱等进行了分析。此外，从解剖结构与生理生化2个方面对干旱及ABA处理下2种苹果砧木（楸子与平邑甜茶）的旱生性结构及生理反应进行了比较研究，明确了2种苹果砧木应答干旱胁迫的抗性生理机制。上述结果主要阐明了苹果不同砧穗组合及苹果砧木对干旱胁迫的生理及分子应答机制，这为我国苹果适应性栽培及分子育种提供了理论支撑。