【作者】 潘衍有；

【导师】 张宪省； 赵翔宇；

【作者基本信息】 山东农业大学 ， 发育生物学， 2011， 硕士

【摘要】 开花是高等植物完成生活周期的必需过程,光周期途径是一条重要的开花调控途径,与昼夜节律钟密切相关。我们实验室从同源六倍体小麦中克隆了拟南芥昼夜节律钟重要基因AtTOC1的三个同源基因TaTOC1-A、TaTOC1-B和TaTOC1-D。我们首先将它们转化模式植物拟南芥,结果表明它们的过表达在短日照条件下能够延迟拟南芥开花,而在长日照条件下对拟南芥开花没有显著影响。并且能够恢复拟南芥toc1-1突变体的早花表型。我们对开花关键基因GI、CO、FT和SOC1在拟南芥toc1-1突变体中的表达模式进行了分析,发现较野生型对照,GI、CO和FT基因在突变体中的表达量升高,SOC1基因的变化不明显。为了进一步研究TaTOC1-A、TaTOC1-B和TaTOC1-D在小麦中的生物学功能,我们利用它们的同源区段构建了RNAi表达载体,利用农杆菌侵染幼胚的小麦遗传转化技术将其转化至小麦CB037中,得到1053株再生植株,经过筛选最终获得了13株阳性转基因植株。转基因植株比对照抽穗时间提早5-7天。半定量RT-PCR(semi-quantitative reverse transcription PCR)以及实时定量PCR(real-time quantitative PCR)结果显示,转基因植株中TaTOC1-A、TaTOC1-B和TaTOC1-D的表达量均有不同程度的降低。为了进一步了解TaTOC1基因在小麦抽穗过程中的作用机制,我们对抽穗关键基因TaGI1、TaHd1、TaFT和TaSOC1在转基因小麦中的表达模式进行了分析。实时定量PCR结果显示,较对照小麦,长日照的转基因小麦中,TaGI1、TaHd1和TaFT基因的表达水平有所提高,TaSOC1基因表达变化不明显;短日照的转基因小麦中,TaGI1和TaFT的表达峰值高于对照,其他基因的表达变化不明显。我们推测,TaTOC1-A、TaTOC1-B和TaTOC1-D基因是通过调控下游TaGI1、TaHd1和TaFT等基因的表达来调节小麦的成花转变。同时,对TaTOC1-A、TaTOC1-B和TaTOC1-D基因进行的组织特异性表达发现,三个部分同源基因在被检测的小麦组织和器官中的表达量不同,在叶片组织中有表达优势。而在叶中,我们原位杂交结果显示,TaTOC1 mRNA被定位于叶片维管束组织中,在韧皮部的杂交信号最明显。更多还原

【Abstract】 The timing of the change from vegetative to reproductive growth is critical for higher plant. Photoperiodic pathway is an important flowering regulatory pathway that is one of flowering regulatory pathways. In previous studies, three nearly full-length cDNA of homolog genes TaTOC1-A, TaTOC1-B and TaTOC1-D were isolated from homologous hexaploid wheat. Here, to investigate their function, we constructed overexpression vectors and transformated them into Arabidopsis. Under short-day conditions, the transgenic plant overexprssing TaTOC1 genes delayed flowering. But it had no visible effect on flowering under long-day conditions. The results of genetic complementation experiments indicated that the early flowering phenotype of toc1-1 were recovered by overexprssion of TaTOC1 genes. Then we analyzed the expression patterns of key regulate flowering genes such as GI, CO, FT and SOC1 in toc1-1 mutant. Compared with the wild-type control, we found that the expression of GI, CO and FT genes increased in toc1-1 mutant, while SOC1 gene’s expression was not significantly changed. To further study their function in wheat (Triticum astivum L.), an RNAi vector was constructed to knock down there genes including TaTOC1-A, TaTOC1-B and TaTOC1-D. Then it was transformed into the wheat (cv. CB037) mediated Agrobacterium tumefaciens. And we obtained 1053 regenerate seedings. After molecular identification, we selected 13 positive transgenic plants from the resistant seedlings. And the heading time of transgenic plants is 5-7 days earlier than that of control. Semi-quantitative reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction results showed that the expression of TaTOC1-A, TaTOC1-B and TaTOC1-D genes were reduced in transgenic plants. This suggests that the reduction of TaTOC1-A, TaTOC1-B and TaTOC1-D gene in transcript levels promote heading earlier in wheat. Further, we analyzed the expression patterns of key regulate flowering genes such as TaGI1, TaHd1, TaFT and TaSOC1. Compared with the wild-type wheat, we found that the expression of TaGI1, TaHd1 and TaFT genes increased in transgenic wheat under long-days. TaGI1 and TaFT genes’ expression had a higher peak in transgenic wheat under short-day conditions. And the other gene’s expression was not significantly changed. These genes’ expressions were changed by reducing the expression of TaTOC1 gene. Meanwhile, semi-quantitative reverse transcription PCR and real-time quantitative PCR results showed that TaTOC1-A, TaTOC1-B and TaTOC1-D gene had different expression in detected tissues and organs. And their expression had an higher levels in the leaves. In the leaves, TaTOC1 mRNA was localized in the leaf vascular bundle tissue and the most obvious hybridization signals was detected in the phloem.更多还原