蒙古沙冬青4个寒旱诱导表达DREB基因的功能研究

Environmental stresses, such as cold and drought, are important infactors that greatly affect plant growth and development. It is important to investigate the molecular mechanisms of plant resistance to such stresses and to mine the key stress-resistant genes. Substantial evidence has demonstrated that transcription factors play pivotal roles in plant stress resistance by regulating the expression of many stress-responsive genes.Among them,DREBs have been identified functioning mainly on plant resistance to cold and/or drought,and for some DREB genes also to salt or heat stress. DREBs activate specifically the expression of the stress-responsive genes with DRE/CRT element in their promotor region.Over the past 20 years,large numbers of the DREB genes,especially the genes for DREB2s, have been cloned and characterized.However, majority of the known DREB genes were isolated from atypical stress-resistant or even stress-sensitive plants, such as Arabidopsis and rice, and the researches focused largely on the DREB1s/CBFs.Therefore it is necessary to clone and characterize more DREB genes, especially the DREB2s, from strong-resistant plants..Ammopiptanthus mongolicus is the unique evergreen broadleaf shrub endemic mainly to the west desert of Inner Mongolia and has evolved extreme resistance to harsh environments, especially to freezing/cold and drought. It is recognized as an ideal model species for elucidating the mechanisms of plant resistance to cold and drought stresses, as well as for mining the key stress-resistant genes. In the previous study,we have performed de novo transcriptome assembly and transcriptome profiling analysis of A.mongolicus under cold and drought stresses using RNA-Seq approach. Totally 16 Unigenes for DREBs have been obtained, and 11 of them were induced rapidly and significantly by cold and drought stresses. We have cloned the complete coding region cDNAs of the 4 stress-induced genes, namely AmDREB2, AmDREB3, AmDREB1F and AmDREB2C (designated according to the most homologous genes in other plants). We have laso obtained transgenic Arabidopsis seeds of AmDREB3 and finished the preliminary experiment for the 4 genes`expression profile analysis during cold stress using real time quantitive PCR method..In this project, we will analyze the 4 genes`introns; detect their expression profiles under different abiotic stress treatments in laboratory and in natural low temperature conditions in the wild in autumn, winter and spring; characterize their roles in resistance to cold,drought,salt and heat stresses and analyze their regulatory mechanisms by using their transgenic plants. We aim at 1) to provide evidence for elucidating the stress-resistant function and regulatory mechanisms of these genes in A.mongolicus,2)to gain new knowledge for futher understanding the resistant mechanisms of plant to cold and drought stresses,3) to provide useful genes for improving plant stress resistance by genetic engineering.

低温和干旱等非生物逆境是影响植物生长发育的重要因素。DREBs是目前所知最为重要的一类植物抗逆转录因子，可通过诱导许多低温和干旱应答基因的表达而显著提高植物的抗寒和抗旱等抗逆性能。前人已克隆和鉴定了许多DREB基因，但其中绝大多数来自拟南芥和水稻等非典型抗逆或不抗逆植物。从强抗逆植物克隆此类基因是当前的重大课题。蒙古沙冬青是内蒙古西部荒漠区唯一的常绿阔叶植物，具有极强的抗寒、抗旱等抗逆特性。我们已从其转录组分析中获得一批寒旱诱导型DREB基因，本项目将克隆其中4个并分析其内含子结构，研究其在室内不同胁迫处理和室外秋冬春季自然冷冻条件下的表达特性，鉴定其在转基因植物抗寒和抗旱等抗逆性中的功能并分析其靶基因。旨在为阐明这些基因在蒙古沙冬青抵抗寒冷和干旱等逆境中的功能及其调节机理提供依据，为进一步揭示植物抗寒、抗旱性的分子机理提供新知识,为通过基因工程提高植物的抗寒抗旱等抗逆性能提供基因资源。

DREB蛋白是目前所知最为重要的一类植物抗逆转录因子，可通过诱导许多胁迫应答基因的表达而提高植物对低温、干旱、高盐和热胁迫的抗性。来自不同植物的DREB基因对抗逆性的作用存在差异，因此研究强抗逆植物的DREB基因很有必要。蒙古沙冬青（亦称沙冬青）是中国西北荒漠区唯一的常绿阔叶植物，具有极强的抗寒和抗旱等抗逆特性。本项目从中克隆到四个DREB基因（AmDREB1F/2/2C/3），对其基本结构、蛋白质亚细胞分布、胁迫诱导表达和器官表达模式、转基因拟南芥抗逆性变化及其调节的胁迫应答基因等进行了研究。.在四个基因的编码区内均无内含子，其中AmDREB1F、AmDREB2C和AmDREB3的编码蛋白已被证实定位于细胞核内。.在室内正常培养条件下，四个基因在沙冬青幼苗中仅有少量表达或不表达，低温和干燥胁迫可诱导其表达量上调；除AmDREB1F外其余三个基因也受热胁迫诱导；AmDREB2和AmDREB2C对这些胁迫的响应可能与ABA信号途径有关。在野外生长的沙冬青嫩叶中，四个基因主要在寒冷的冬季和晚秋或早春时节大量表达。其器官表达模式为：AmDREB1F和AmDREB3主要在根中表达，AmDREB2C只在嫩叶和未成熟果荚中高表达，AmDREB2在不同器官中均高丰度表达。 .将AmDREB2C在拟南芥中超表达可以激活RD29A和COR47等多个胁迫诱导基因转录，并可以显著提高转基因株系的抗冻、耐热和耐旱性。超表达该基因还可以提高拟南芥种子和叶片中三烯酸含量，后一功能在DREBs中未见报道。将AmDREB3超表达可以增强拟南芥的耐旱、耐热和耐盐性，但其调节的胁迫应答基因与其他DREBs有所不同。该基因还可能增加高盐和干旱胁迫下花青素类的积累，类似的功能在DREBs中未见报道。AmDREB2主要在耐受水分胁迫中起一定作用。此外，四个基因中只有AmDREB1F在超表达时可引起生长延滞，将其诱导表达可提高对水分胁迫和盐胁迫的耐性。.本项目首次发现DREBs可以提高转基因植物叶片中不饱和脂肪酸或花青素类含量，从而为揭示DREBs在植物抵抗非生物逆境中的功能及其调节机理提供了新知识。此外还筛选出两个重要抗逆基因（AmDREB2C和AmDREB3），为作物耐逆性分子改良提供了基因资源。