【作者】 王丹；

【导师】 王明波；

【作者基本信息】 哈尔滨师范大学 ， 遗传学， 2018， 博士

【摘要】 杀配子染色体可以通过引起染色体断裂来杀死那些不含有他们的配子,并保证自身的优先传递,即杀配子染色体同时具有“断裂”和“保护”的双重功能。由于杀配子染色体可以引起染色体断裂并进一步引起染色体重组,因此,可以用于创制易位系及缺失系,从而将外源有益基因导入到普通小麦。但是,杀配子染色体作用的分子机制目前仍不清楚。本研究构建了普通小麦“中国春”(CS)及“中国春”-杀配子染色体3C单体附加系(CS-3C)的小RNA文库,对其进行了测序分析,筛选出在杀配子作用过程中关键的miRNAs及24 nt siRNAs,以研究这两类small RNAs是否参与小麦杀配子作用。主要研究结果如下:1.关键miRNAs的鉴定与分析本研究共鉴定出239个保守的及72个假定的新miRNAs。在所鉴定出的311个miRNAs中,共有104个保守的及31个假定的新miRNAs在这两种植物材料间差异表达,其中74个miRNAs在CS-3C中上调表达,61个miRNAs在CS-3C中下调表达。差异表达miRNAs的靶基因预测及靶基因功能注释分析结果表明,许多差异表达miRNAs的靶基因其功能可能与杀配子作用相关。2.差异表达miRNAs及其靶基因的验证随机挑选4个差异表达的保守miRNAs进行TaqMan荧光定量PCR分析,以验证测序分析结果的准确性。sRNAs测序数据与TaqMan qPCR数据显示出了高度的一致性,表明高通量测序数据的结果是可信的。通过qRT-PCR技术分析了上述4个miRNAs的6个靶基因的表达模式,结果表明靶基因与其相应miRNAs之间呈现负调控关系。通过5′RACE实验,进一步验证了靶基因的断裂位点。3.Tae-miR9657b-3p对水稻的遗传转化及其功能验证克隆得到包含有tae-miR9657b-3p前体结构的序列,并获得tae-miR9657b-3p过表达水稻植株。选取表达量较高的3个株系用于后续分析。靶基因TC492017的qRT-PCR结果显示,该基因在转基因水稻中下调表达,即miR9657b-3p与其靶基因呈现负调控关系。与野生型相比,转基因水稻育性下降,花粉萌发率降低,说明tae-miR9657b-3p的过度积累会降低植物育性。6个育性相关基因,SPX1,GEN-L,UDT1,PAIR1,PAIR2以及REC8,在转基因水稻中都下调表达,说明miR9657b-3p的过度积累影响了这些育性相关基因的表达,进而引起转基因水稻育性下降。以上结果说明,tae-miR9657b-3p参与植物育性的调控,说明taemiR9657b-3p可能在小麦杀配子现象中具有重要作用。4.24 nt siRNAs的鉴定与差异表达分析分析了CS-3C与CS小麦品系间24 nt siRNAs在相应染色体上的分布及密度差异。注释分析结果表明,大部分位于差异表达滑动窗口中的siRNAs都比对到染色体的重复序列上,而这部分重复序列中包含了一些已知类型的转座子。与CS相比,在CS-3C中,24 nt siRNAs的表达水平通常都较低。对位于差异表达滑动窗口中的siRNAs在蛋白质编码基因(包括转录区域及其上下游3 kb区域)中的分布趋势进行分析。结果表明,无论是在CS还是在CS-3C中,24 nt siRNAs的最大密度峰都位于转录区域上下游1 kb区域内。那些能够比对到转录区域及其上游1 kb区域中的差异表达siRNAs所对应基因的GO分析结果表明,这些基因主要富集在“催化”和“结合”活性的分子功能上。5.24 nt siRNAs相关转座子的重亚硫酸盐测序分析对四个LINE/L1反转录转座子的CHH甲基化水平进行了测序分析。结果表明,与CS相比,在CS-3C小麦品系中这些转座子的CHH甲基化水平降低。24 nt重复序列相关siRNAs的差异积累可能是与一些转座子序列中差异的CHH甲基化相关。在CS-3C小麦品系中,重复序列相关24 nt siRNAs的减少及相应CHH甲基化的减少可能引起了基因组的不稳定,并因此参与小麦杀配子作用。更多还原

【Abstract】 Gametocidal(Gc)chromosomes can kill the gametes without themselves by causing chromosomal breakage and ensure their preferential transmission.That is,Gc genes have the dual functions,one of which gives “breaking” function and another “protecting” function.As the Gc chromosome can induce chromosomal breakage and further cause chromosomal recombination,it has been used for creating translocation and deletion lines to transfer the beneficial alien genes to common wheat.However,the molecular mechanism of Gc action remains unclear so far.In this study,small RNA libraries from Triticum aestivum cv.Chinese Spring(CS)and Chinese Spring-Gc 3C chromosome monosomic addition line(CS-3C)were constructed and sequenced,and the key miRNAs and 24 nt siRNAs in Gc action were identified to investigate whether these two categories of small RNAs can participate in Gc action of wheat.The main research results are as follows:1.Identification and analysis of the key miRNAs:This study identified 239 conserved and 72 putative novel miRNAs.Among these 311 miRNAs,104 conserved and 31 putative novel miRNAs were differentially expressed between these two plant materials,of which 74 miRNAs were up-regulated in CS-3C,and 61 were down-regulated.The results of target genes prediction of differentially expressed miRNAs and GO analysis of the targets revealed that the functions of many target genes of these differentially expressed miRNAs maybe relevant to the Gc action.2.Verification of the differentially expressed miRNAs and their target genes:To verify the accuracy of the sequencing analysis results,4 differentially expressed conserved miRNAs were randomly selected to perform the TaqMan qRT-PCR.The high degree of consistency between sRNA sequencing data and TaqMan qPCR data indicated that the result of high-throughput sequencing data is credible.The expression patterns of 6 target genes of the 4 miRNAs above were analyzed by qRT-PCR and the results indicated that the expression profiles of target genes were negative correlated with their corresponding miRNAs.Finally,5′RACE were performed to further verify the cleavage site of target gene.3.Genetic transformation and functional analysis of tae-miR9657b-3p in rice:Cloned the sequence which contains pre-tae-miR9657b-3p and obtained the tae-miR9657b-3p over-expressed rice plants.Three lines which exhibited high levels of miR9657b-3p expression were used for subsequent analysis.QRT-PCR result of the target gene TC492017 indicated that it was down-regulated in transgenic rice,that is,miR9657b-3p was negative correlate with its target gene.Compared with wild-type lines,the fertility and pollen germination rate of the transgenic rice was reduced,indicating that over-accumulation of tae-miR9657b-3p can reduce plant fertility.Six fertility-related genes SPX1,GEN-L,UDT1,PAIR1,PAIR2 and REC8 were down-regulated in transgenic rice lines,which suggested that over-accumulation of miR9657b-3p affected the expression of these fertility-related genes,and further caused the decline in the fertility of transgenic rice.These results above indicated that tae-miR9657b-3p can participate in the regulation of plant fertility and may play an important role in the gametocidal phenomenon in wheat.4.Identification and differentially expressed analysis of 24 nt siRNAsAnalyzed the differences in distribution and density of 24 nt siRNAs on the corresponding chromosomes between CS-3C and CS wheat lines.The results of annotation analysis indicated that most of the siRNAs in the differential sliding windows corresponded to repeat sequences of the chromosomes,including some known types of TEs.Compared with CS,the expression level of 24 nt siRNAs was general lower in CS-3C.The distribution of siRNAs within the differential sliding windows in protein-coding genes,including both transcribed and 3 kb upstream and downstream flanking regions was examined.The results indicated that the highest density of 24 nt siRNAs occurred within 1 kb upstream and downstream of the transcribed regions in both CS and CS-3C.The result of the gene ontology(GO)analysis which was performed on genes associated with differentially expressed siRNAs within the transcribed and 1 kb upstream regions showed that these genes were enriched for the molecular functions “catalytic” and “binding” activities.5.Bisulfite sequencing analysis of 24 nt siRNA-associated transposonThe CHH methylation levels of four LINE/L1 retrotransposon were analyzed by bisulfite sequencing.The results indicated that CHH methylation levels were reduced in these TEs in CS-3C wheat line compared with those in CS.The differential accumulation of repeat-associated 24 nt siRNAs maybe associated with differential CHH methylation at the some of the TE sequences.The decrease of repeat-associated 24 nt siRNAs and the associated reduction in CHH methylation in the CS-3C line may cause genome instability and hence contribute to Gc action in wheat.更多还原