梨果实糖酸性状形成的分子机制及重要功能基因的挖掘

Bad quality resulting from poor flavor of the fruit is one of the most dominant problems for pear production at present. Basing on the results of identification of genetic diversity of pear gerplasm resources, phenotype analysis of fruit sugars & organic acids, whole genome sequencing and resquencing, we want to uncover the molecular mechanism(s) of sugars & organic acids formation in pear fruit, and provide genetic resources for directional quality improvement in this project. In detail, the SNP clip is going to be developed for screening the genes encoding sugars & organic acids and their location in charomosomal. The differentially expressed genes in transcriptional level among the cultivars will be screened by using the methods of high-flux RNA-Seq sequencing. While iTRAQ and LC?MS/MS techniques will be employed to analyze the differential protein profiles among the cultivars. Analysis on relationship between transcriptome and proteome, as well as gene annotation, expression pattern and related metabolism pathway will be performed to confirm differentially expressed genes and their involvement in the potential metabolic pathway, to screen critical genes encoding sugars and organic acids, and further validation of their functions by transgenic method. Besides, up-regulated and down-regulated genes associated with sugars & organic acids traits, as well as the transcription factors, small RNA, and transposon involved in sugars & organic acids metabolism will also be investigated. Taken together, results from above research will be expected to provide an important scientific basis for regulating fruit quality as well as provide gene resource for realization of directional improvement quality of pear.

针对梨生产中普遍存在果实风味品质较差的突出问题。在已开展梨种质资源遗传多样性鉴定、果实糖酸性状表型分析及梨全基因组测序及重测序工作的基础上；利用基因组重测序结果，开发SNP基因芯片，进行糖、酸性状基因的筛选及其染色体定位；采用基于高通量测序的RNA-Seq数字表达谱分析技术，筛选果实糖、酸性状转录水平表达差异基因；同时应用TRAQ及LC-MS/MS分析技术比较不同品种糖、酸性状蛋白差异表达谱；通过转录组及蛋白质组差异表达谱的关联性分析、基因注释、表达模式和相关代谢通路分析，明确差异表达基因及其参与调控的代谢途径，筛选出控制果实糖、酸性状关键基因，并转基因验证其功能；探讨果实糖、酸性状的上调或下调表达基因、参与糖、酸代谢调控的重要转录因子、small RNA、以及转座子等，构建果实糖酸性状基因的调控网络；本研究将全面揭示梨果实糖、酸性状形成的分子及调控机制，为梨果实品质定向改良提供基因资源。

针对梨生产中普遍存在果实风味品质较差的突出问题，我们开展了以下研究：1）采用梨基因组重测序结果筛选基因组内均匀分布的SNP位点，构建梨果实糖、酸性状的SNP基因芯片，实现利用基因芯片进行梨品种资源及杂种后代果实糖、酸性状的基因分型；2）建立利用转录组、蛋白质组分析及全基因组关联分析等多种手段筛选梨果实糖、酸性状重要候选基因的技术体系，完善转基因番茄验证重要候选基因功能的技术，挖掘出梨果实糖、酸性状基因（基因家族）；3）明确梨果实糖、酸性状相关的重要转录因子、转座子和small RNA等，揭示果实糖、酸性状形成的‘基因-基因’互作关系及基因调控网络，阐明梨果实糖、酸性状形成的分子及调控机制。研究结果主要包括：首先，从梨中克隆了梨果实糖转运蛋白PbSUT、PbHT、PbSOT、PbTMT基因，己糖激酶基因PbHXK、PbFRK和山梨醇代谢相关基因 PbS6PDH、PbSDH。分析了这些基因家族的结构和进化特征，证明了PbSUT2促进转基因番茄成熟果实中蔗糖含量的提高，而果糖、葡萄糖和可溶性总糖含量下降；PbHT1促进转基因番茄成熟果实中己糖和可溶性总糖含量的积累，但对蔗糖含量无显著影响；PbSOT2的表达、NAD-SDH与NADP-SDH 的活性和己糖的积累呈正相关。PbHXK1和PbFRK1负调控果实糖积累，并影响植株的生长发育。其次，对酸积累相关基因PbALMT9、PbTDT1和PbVHA-c4研究，发现5 个ALMT家族基因与苹果酸代谢相关，其中PbALMT9不仅转录表达水平高于其他基因，且表达模式与梨果实整个发育时期的苹果酸含量变化呈正相关；PbTDT1有助于果实发育过程中有机酸的积累，超表达转基因番茄成熟果实中的苹果酸、延胡索酸含量高于野生型和 RNAi转基因株系，而柠檬酸含量低于野生型和 RNAi转基因株系；PbVHA-c4促进梨果实中柠檬酸的积累，其超表达转基因株系的番茄果实中柠檬酸含量明显高于野生型。研究结果不仅为梨果实品质定向改良奠定理论基础和提供基因资源，也为分子辅助育种提供有效方法。