玉米杂种优势转录组新转录本和特异差异表达基因及其调控代谢功能的研究

Hybrid vigor, or heterosis, is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic superior phenotypes and stress tolerance relative to their parents and widely exploited in applied plant breeding. However, the responsible molecular mechanisms have not been determined. Understanding the novel transcripts(NTs）and differential expression genes (DEGs）is essential for studying the complexity of molecular mechanisms of hybrid vigor. Here, we present the first dynamic transcriptomes to switch on heterosis in cultivated maize. Using high-throughput paired-end RNA-seq, we have unambiguously detected 3 dynamic transcriptomes of maize hybrid F1 and both parents for the reaction after treated by NaCl. We sequenced 7,224Gb RNA that 3.14-fold coverage of the classic maize genome, and analyzed 71,364 cDNAs and compared 121 metabolic pathways to study the heterosis. We conducted a comparative analysis of hybrids and parental inbred lines, corresponding a rigorous algorithm showing only 865 DEGs and 257 NTs compared with its parental lines. DEGs and NTs with more significance will be carried out into functional analysis. The NTs and DEGs will be validated by using RT-PCR (reverse transcription-PCR). Furthermore, the genes expression will be detected via the real- time PCR experiments. The metabolic networks will be conducted by HPLC and Gene Ontology to exhibit distinct features for hybrid line compared with its parental lines. Moreover, more important genes will be characterized by gene transformation to detect related function for the phenomenon of heterosis. A gene pool which is involed in switching on heterosis in maize will be created for prediction of hybrid performance and improvement of the efficiency in maize hybrid breeding programs.

杂种优势的分子机理依然没有完全揭开。了解杂种优势的新转录本(NTs）和特异差异表达基因(DEGs）,不仅对全面揭开杂种优势分子机理有创新性的贡献,还对品质的预测、种质的创新、杂优育种效率的提高有重要意义和应用前景。与国际上其他报道相比(Nature Genet. 2012；2010；Science. 2009等)，我们分析了F1与亲本对环境反应的一系列超亲现象,首次做了杂种优势启动模式的3个转录组测序, 测序长度相当于覆盖经典玉米全基因组3.14倍。本项目对首次获得的257个F1 NTs,865个杂优特异DEGs进行研究, 利用RT-PCR、Real-time PCR、HPLC和Gene ontology等，研究最少有多少NTs和DEGs是杂种优势所必须的基因；它们有什么特殊功能；及对代谢组的优化作用；并通过转基因研究重要基因对优势性状的多重贡献, 最后，创建玉米杂种优势启动基因库。

玉米商业杂交种天塔5具有高产稳产抗逆等优良性状。本研究利用高通量测序技术检测了萌发45天玉米天塔5及其父母本在盐胁迫情况下的3个转录组，研究了257个新转录本（NTs）和865个特异差异表达基因（DEGs）对杂种优势的贡献。DEGs的代谢通路富集分析发现天塔5（F1）对父母本均富集的pathway主要包括phenylpropanoid biosynthesis, flavonoid biosynthesis, alpha-Linolenic acid metabolism, plant-pathogen interaction, nitrogen metabolism and starch and sucrose metabolism, etc. 对比HRGD（Heterosis Related Gene Database）发现本研究中有 648个杂种优势相关基因被检测到，这将有助于理解杂种优势在不同时期具有不同表现的遗传机理。进一步比较转录组分析发现，一系列转录因子 (TFs) 在植物盐胁迫反应中发挥了重要的作用，包括 NAC, WRKY, ERF, bZIP, HSF, bHLH and MYB superfamily。qRT-PCR验证了不同转录因子家族/亚家族的表达模式，表明了其对杂种优势的耐盐表现具有不同贡献。天塔5启动了抗性机能提高和毒性物质抑制增强的保护机制保证了植物的生长发育，这可能与杂种优势的形成有一定的关系，如氰化物(cyanide)对生物有机体有剧毒，F1玉米细胞氰化物解毒途径上的重要基因β-CAS 和NIT上调，使F1的根长显著长于亲本。进一步，我们检测并克隆了杂种优势直接相关的基因，包括光合色素、激素合成、产量、抗逆等基因，并重组于含有Bar基因的工程载体中，（为了避免在玉米中基因沉默，本研究在枸杞和拟南芥中进行基因克隆，通过转基因转入烟草、玉米中），获得的转基因植物抗逆性明显提高，且有些转基因玉米表现出高产性状等。本项目还构建了玉米杂种优势相关基因的数据库，为后续研究提供数据基础。.本项目完成了预期的研究计划，发表了15篇论文，其中9篇SCI论文，5篇EI论文，其他被北大中文核心期刊论文收录，申请专利2项，获得专利1项，培养人才7名。