【作者】 郭志爱；

【导师】 任正隆； 贾继增；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 光周期反应是植物通过感受昼夜长短变化而控制开花的一种生理现象。普通小麦(Triticumaestivum L.)是典型的长日照作物,光周期反应对其生育期、适应性以及产量均具有重要影响。近年来模式植物拟南芥、水稻光周期途径的分子生物学研究已取得很大的进展,但作为主要粮食作物的小麦,其光周期反应研究则相对滞后。本研究主要针对小麦光周期基因多样性变异和与其同家族的生物钟基因TaPRR1展开研究,取得如下主要研究进展:1.根据光周期基因Ppd-D1的5个等位变异位点,开发新的分子标记,同时结合已报道的标记检测来源于世界不同地理生态环境的495份普通小麦品种以及25份人工合成种和30份粗山羊草(Ae.tauschii),对Ppd-D1基因单倍型种类、分布特点、表达规律及其功能进行了分析,研究结果发现:(1)Ppd-D1基因共存在6种单倍型,分别命名为单倍型Ⅰ一Ⅵ;(2)单倍型Ⅱ、Ⅴ和Ⅵ存在于合成种和粗山羊草中,是比较原始的类型,并在小麦驯化进程中单倍型Ⅴ和Ⅵ被丢失,只有Ⅱ被保留下来。(3)不同单倍型在短日照条件下具有相似的表达模式,但其最高表达量存在显著差异。单倍型Ⅰ的表达量最高,Ⅲ的表达量最低,其余呈现一系列过渡类型:(4)相关分析发现Ppd-D1单倍型在短日照条件下其表达量与抽穗期呈负相关,单倍型间的千粒重、株高等农艺性状均存在显著差异;(5)不同单倍型的地理分布呈现明显的地域特征,单倍型Ⅰ主要分布在亚洲、大洋洲和北美洲低纬度国家,单倍型Ⅱ主要集中在亚洲,单倍型Ⅲ主要分布在欧洲和北美洲的高纬度国家,单倍型Ⅳ分布较为广泛,几乎每个小麦种植区域都普遍存在。2.研究光周期基因Ppd-B1在转录水平上的表达特点,结果发现:(1)Ppd-B1是选择性剪切基因,存在3个可选择剪切位点,分别以外显子增加、5’剪切位点改变和内含子保留形式分布在5’UTR、第5外显子和第6内含子,其中前两者不同剪切方式没有引起蛋白保守结构域的改变,后者不同剪切方式会导致基因移码突变:(2)Ppd-B1选择性剪切可产生8种不同形式的转录本,其中4种含有完整的编码序列,表达丰度较高,能够翻译成功能蛋白;另外4种表达丰度较低,不翻译或翻译时被提前终止;(3)Ppd-B1不同转录本的相对表达量受到材料光周期反应特性和外界光周期环境的影响而变化。对Ppd-B1启动子区与编码区进行多样性分析,结果发现:(1)启动子区存在6种序列,编码区存在4种序列类型,包括10个SNP变异位点,这些SNP在基因启动子、外显子、内含子各个区域均有分布;(2)每种材料都含有一种相同的启动子序列类型,此外有的材料还存在另外一种启动子序列类型;(3)序列多样性证明该基因在有些材料中至少存在两个拷贝,而且初步推测认为基因序列类型的多样性与材料的光周期反应存在联系。3.对小麦生物钟基因TaPRR1(即拟南芥TOC1同源基因)进行了如下研究:(1)采用同源克隆的方法,克隆了普通小麦的三个同源基因TaPRR1-A1,TaPRR1-B1,TaPRR1-D1;(2)基因表达发现TaPRR1在种子萌发后即能响应短日照光周期迅速表达,并在第六天表达量快速增加:(3)TaPRR1的表达随昼夜节律交替循环而变化,但在长短日照下的表达模式不同。在短日照条件下。其表达模式为单峰,与拟南芥TOC1和水稻OsPRR1的表达很相似;而在长日照条件下TaPRR1的表达则呈现双峰:(4)三个TaPRR1同源基因在长短日照下的表达均有差异,TaPRR1-D1的表达节律最强,TaPRR1-A1的表达节律最弱,TaPRR1-B1介于前两者之间:(5)将TaPRR1定位于小麦染色体第6部分同源群,并将TaPRR-D1定位于小麦6D染色体RFLP标记Xbcd1716和Xcfd76之间,遗传距离分别为4.2 cM和6.3 cM。更多还原

【Abstract】 Photoperiod response of plant is an important physiological phenomenon which control flowering time by receiveing day length.Wheat(Triticum aestivum L.) is a typical long-day plant and the photoperiod response greatly affects wheat growth period,adaptability and yield.The molecular mechanism of photoperiod pathway has made a great progress in the model species including Arabidopsis and rice in recent years,but it is less clear in wheat,the main food crop in the world.The present study focused on the diversity of Ppd-D1,Ppd-B1,the main photoperiod genes,and the circadian clock gene TaPRRl belonged to the same gene family with Ppds.The progresses were showed as follows:1.Three new diagnostic molecular markers combined with previously reported markers identified five polymorphisms in the Ppd-Dl gene in 495 wheat varieties originating from diverse geographic locations,25 synthetic varieties,and 30 Aegilops tauschii accessions.The discovery,distribution, expression and function of Ppd-D1 haplotypes were investigated.The major results showed as follows. (1) There are six haplotypes of Ppd-D1,named for haplotypeⅠtoⅥ.(2) HaplotypeⅡ,ⅤandⅥwere ancient types because they were found in synthetic wheat and Ae.tauschii.Among them,haplotypeⅤandⅥwere lost whereasⅡwas remained in common wheat during the wheat domestication.(3) HaplotypesⅠ-Ⅴhad a similar expression pattern and all the expression peaks were at the same time,but their levels were different from one to another.The values of expression peaks of haplotypeⅠandⅢwere the highest and the lowest,respectively.The others were intermediate.(4) Their expression abundance in the morning under the short day photoperiod condition were negatively correlated with the heading time and the haplotypes were associated with main agronomic traits such as thousand grain weight and plant height.(5) HaplotypeⅠvarieties distributed mainly in lower latitudes regions, especially Asia,Oceania and Mexico;haplotypeⅡvarieties were most frequently found in Asia; haploytpeⅢwere most commonly found in wheat at higher latitudes of Europe and North America. HaplotypeⅣwas widely found in the wheat grown in much larger areas of different continents.2.The transcripts of Ppd-B1 were analyzed in present study,and the main results were as follows:(1) Ppd-B1 is an alternative splicing gene.There were three alternative splicing loci in the forms of exon inclusion,alternative 5’ splice site and intron retention,which locate in 5’UTR,exon 5 and intron 6, respectively.The formers two did not change the conserved domains of PRR protein,while the last resulted in a frameshift mutation.(2) Ppd-B1 produces eight different transcripts through alternative splicing.Four of them including complete open reading frame could translate to be functional proteins. The others encoded truncated proteins for translation stopped ahead and they expressed at very low levels.(3) The alternative spliced transcripts of Ppd-B1 were differentially expressed,which were affected by photoperiod response of varieties and photoperiod conditions.The diversity of Ppd-B1 nucleotide acid sequences were detected in present studies.(1) There were six sequences types found in Ppd-B1 promoter region and four types in the gene coding region that were comprised 10 SNP ploymorphisms located on 5’UTR,exons and introns.(2) Each variety has a same promoter sequence and there were also another one sequences in some varieties.(3) There were at least two copies of Ppd-B1 sequences found in some varieties.The preliminary results implied that the diversity of Ppd-B1 sequences probably associated with the gene function for photoperiod response.3.The study on the circadian clock gene TaPRR1(the homolog to TOC1 from Arabidopsis) was carried out.The results were showed.(1) The orthologous gene TaPRR1-A1,TaPRR1-B1,TaPRR1-D1 from A,B and D genomes of hexaploid wheat(Triticum aestivum L.),respectively,were isolated by using homologous cloning combined with cDNA library screening methods.(2) TaPRR1 expressed quickly in the leaves of wheat seedling after germination in short day photoperiod condition,and their expression values increased quickly in the 6th day.(3) The expression of TaPRR1 varied with the cycles of circadian rhythms,but the expression patterns between long day and short day photoperiod conditions were different.TaPRR1 showed a single expression peak in short day period,which was similar to the.expression of TOC1 and OsPRR1 in Arabidopsis and rice,respectively.Under a long day period,TaPRR1 showed two peaks expression pattern.(4) The expressions of TaPRR1-A1,TaPRR1-B1 and TaPRR1-D1 showed some differences under both long day and short day photoperiod.Among them, TaPRR1-D1 expressed with the well circadian rhythm,the poor rhythm for TaPRR1-A1 and intermediate for TaPRR1-B1.(5) The TaPRR1 were located on wheat homoeologous chromosome 6 and TaPRR1-D1 was mapped between RFLP marker Xbcd1716 and Xcfd76 of wheat chromosome 6D with the genetic distances of 4.2 cM and 6.3 cM,respectively.更多还原