银杏开花调控关键基因鉴定及分子机理研究

Research of Herbaceous plant flowering mechanism has made important progress. The function of the key flowering regulation genes from Arabidopsis thaliana such as FT, TFL1, SOC1, FLC, SPL and AP1 was identified. Molecular mechanism of flowering regulation from photoperiod pathway, vernalization pathway, GA pathway of Arabidopsis thaliana and one new age pathway of the perennial herb, is clear. However, the molecular mechanism of flowering about Ginkgo of juvenile stage for up to 20 years, which is an ancient Gymnosperm and unique economic tree in China, is unclear..In the project we shall clone 6 flowering regulation candidate genes GbFT,GbTFL1,GbSOC1,GbFLC, GbSPL and GbAP1 homologous to Arabidopsis thaliana by using PCR、RT-PCR, based on Ginkgo cDNA and EST library. Furthermore, the function confirmation of these genes will implemented by building sense and anti-sense expression vectors, transferring them to the cells of Arabidopsis thaliana and poplar.The mRNA expression of candidate genes would be determined by using the fluorescence quantitative reverse transcription PCR (qRT-PCR) technology, and the temporal and spatial expression characterization would be analised, in order to identify the key flowering regulation genes,flowering pathway and molecular mechanism in Ginkgo. .We shall firstly identify the key flowering regulation genes in Ginkgo, discover their temporal and spatial expression characterization, and explain molecular mechanism of flowering regulation in Ginkgo, which is of important theoretic and practical value to molecular breeding and flowering regulation in Ginkgo, and provide the basis for studying Gymnosperm flowering molecular mechanism.

草本植物开花机理研究已取得了重要进展,拟南芥开花调控关键基因如FT、TFL1、SOC1、FLC、SPL、AP1的功能已鉴定，开花主要途径——光周期途径、春化途径和年龄途径等的分子机理已较清楚。而银杏是古老的木本裸子植物、我国特产经济树种，幼年期长达20年才开花，其开花调控分子机制尚不清楚。本项目基于转录组学方法，采用cDNA 文库、EST 技术、PCR和qRT-PCR技术等对银杏开花调控关键基因进行分离、鉴定与表达，阐明银杏开花调控的途径及分子机理：（1）银杏开花调控候选关键基因（GbFT、GbTFL1、GbSOC1、GbFLC、GbSPL、GbAP1）克隆；（2）银杏开花调控候选关键基因转拟南芥和杨树功能鉴定；（3）银杏开花调控途径基因表达验证及分子机理分析。项目完成将首次揭示银杏开花调控的关键基因及分子机理，阐明与草本植物开花调控机理的异同，为银杏分子育种与生产栽培提供理论指导。

草本植物拟南芥开花调控途径及分子机理研究已取得重要成就，但木本裸子植物开花调控机理尚不清楚。银杏是我国特产的重要经济树种，最古老的木本裸子植物，在生物进化上位于种子植物的源头，其开花分子机制应具有保守性与特异性。本项目主要通过转录组学分析银杏开花相关基因的差异表达与功能，探索银杏开花调控的关键基因及分子机理，揭示其与草本植物开花调控的异同。.本项目的主要研究内容：（1）银杏不同花芽分化期和不同生长发育期的基因表达差异研究；（2）银杏开花调控关键基因筛选、克隆与功能验证；（3）银杏开花调控分子机理分析；（4）银杏细胞培养及遗传转化。.本项目的重要结果、关键数据：（1）完成了银杏花、芽、叶等11个样品的转录组测序，共获得93.07Gb 表达基因数据库，Unigenes基因35610条，有功能注释基因35,179条，新基因431条；大量的基因归为分子功能、细胞组分、生物过程3个大类50个小类，其中代谢过程、催化活性和细胞过程等富集程度较高，与银杏花芽分化有关。（2）对银杏差异基因表达模式分析，结合荧光定量基因表达验证，筛选鉴定克隆出与开花调控相关的10个关键基因和4个关键miRNA：GbFT、GbSOC1（GBM6）、GbLFY、GbFUL、GbSVP、GbAG（GBM5）、GbCOL、GbGI、GbSPL3、GbFLC；gbi-miRNA159、gbi-miRNA 319、gbi-miR156、gbi-miRNA172。（3）初步揭示了银杏开花调控的分子机理，构建了银杏开花途径基因调控网络模式图：随着银杏年龄的增长，银杏茎端gbi-miRNA159、gbi-miRNA 319表达增加，其靶基因MYBs表达下调，从而引起开花决定基因GbLFY、GbSOC1解除部分抑制，茎端完成从幼年期到成熟期转变；同时银杏叶片在长日照条件下，GbGI表达促使GbCOL表达，进而促使GbFT基因表达，GbFT蛋白运输到茎端进一步解除GbSOC1等抑制，从而诱导银杏开花；银杏中没有发现开花决定关键基因AP1和开花抑制基因TFL1。.本项目发表核心期刊论文7篇、会议论文12篇（优秀会议论文7篇），申请国家发明专利1件，培养研究生7名（已毕业4人），项目研究成果为银杏分子育种与生产栽培提供了理论与实践指导，为其它木本裸子植物开花分子机理研究提供了借鉴。