【作者】 王宁；

【导师】 高新起； 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2020， 硕士

【摘要】 冬小麦在秋天种植,经历一段长时间的低温(春化过程),在第二年春季开花结实。春化不仅是冬小麦从营养生长向生殖生长转变的必要过程,对小麦后续的生长发育以及最终产量也起到重要的调节作用。亩穗数、每穗粒数和千粒重是小麦产量构成的三个重要因素。其中千粒重和穗粒数都受到穗型影响。小穗数是决定小麦穗型的重要因子。为了研究不同长度的春化时间对小麦小穗数的影响,本研究首先确认了济麦22在4℃春化条件下所需的最低春化时长,我们发现春化26天是济麦22完成正常生长发育所需的最低春化时长。随后我们以济麦22为实验材料设置了26天、33天和40天共3个春化时间。统计发现济麦22主分蘖的小穗数随春化时长的延长呈先下降后平缓的趋势,春化26天的植株产生的小穗数最多。观察茎尖分生组织的发育过程发现,随春化时间的延长,茎尖分生组织伸长期至二棱期各阶段的发育时间逐渐变短,这可能是造成小麦小穗数的减少的原因。我们进一步对不同春化时长下济麦22的茎尖分生组织取材进行了转录组分析。结果显示3个春化时长下小麦茎尖分生组织中有3142个基因的表达发生了变化。并且这些基因在核小体装配及染色体组装等过程中富集。其中共计有733个差异表达基因(包括192个上调表达基因和541个下调表达基因)的表达变化趋势与小穗数的变化趋势一致。GO以及KEGG富集分析显示这些差异表达基因与转录调控以及茉莉酸代谢和信号途径密切相关。这些与茉莉酸代谢和信号途径相关的差异表达基因的启动子上都含有春化相关转录因子VRN1的结合序列CArG box motif。我们推测茉莉酸途径与春化影响小麦小穗数相关。将差异表达基因与现有的与产量相关的QTL进行了对比分析,筛选出了65个春化影响小麦小穗数的候选基因。并且选取了一部分基因构建了遗传转化载体,将来观察其在小穗数调控过程中的功能。另外,我们还对差异表达基因中的TaTFL1-4A基因进行了克隆及功能分析。该基因是拟南芥开花相关基因TERMINAL FLOWER 1(TFL1)在小麦中的同源基因。荧光定量PCR显示该基因在小穗中表达量最高,原位杂交结果显示该基因在单棱期和二棱期的茎尖分生组织、小穗原基以及小花分化时期均特异表达。同时我们构建了该基因的过表达载体转化拟南芥tfl1突变体,发现可以部分互补tfl1突变体顶端花的表型。由此我们推测该基因可能拥有与拟南芥TFL1基因相似的延长植物营养生长阶段并维持花序的无限生长状态的功能。更多还原

【Abstract】 Winter wheat is planted in autumn,undergo a long period of low temperature stress(Vernalization),and blossom and bear fruit in the following spring.Vernalization is a necessary process for the transition from vegetative growth to reproductive growth of winter wheat,but also plays an important role in the subsequent growth and development of wheat as well as the final yield.Thousand kernel weight,spike number per square meter and kernel number per spike are three important factors affecting yield.Thousand kernel weight and kernel number per spike were affected by the spike architecture.The number of spikelet is an important factor to determine the spike architecture in wheat.In order to investigate the effect of vernalization duration on the spikelet number of common wheat(Triticum aestivum L.),we firstly confirmed the minimum vernalization time requirement for Jimai 22 under 4 °C.We found that 26-day vernalization was the minimum requirment for Jimai 22 to complete its growth and development.Statistic analysis revealed that the number of spikelet in the main tillers of Jimai 22 was significantly decreased at first and then gradual with the elongation of vernalization duration.Plants under 26-day vernalization produced the largest number of spikelet.Observing the development of the SAM,we found that the duration time from the elongation stage to the double ridge stage gradually became shorter with the elongation of vernalization duration,which could be the reason for the decrease in the number of spikelet in wheat.We performed RNA-seq analysis on the shoot apical meristem(SAM)of seedlings under three vernalization duration(26d,33 d,and 40d).RNA-seq analysis revealed that the expression of 3142 genes in the SAM was significantly changed.These differentially expressed genes(DEGs)are enriched in the process of nucleosome assembly and chromosome assembly.A total of 733 DEGs(including 192 up-regulated expression genes and 541 down-regulated expression genes)showed a similar expression changes with those of spikelet numbers.GO and KEGG enrichment analysis showed that these DEGs were closely related to transcriptional regulation,jasmonic acid metabolism and signaling pathways.Thepromoters of these DEGs related to jasmonic acid metabolism and signaling pathways contain the binding sequence of the vernalization-related transcription factor VRN1,CArG box motif.Therefore,we suggest that the jasmonic acid pathway may be related to the vernalization-regulated spikelet number of wheat.We compared the DEGs with the identified yield-related QTL,and 65 candidate genes were identified that may affect the spikelet number of wheat.Furthermore,a subset of DEG genes was selected to construct genetic transformation vectors to explore their functions in spikelet development of wheat.In addition,we cloned and analyzed TFL1-4A,a homologous gene of Arabidopsis thaliana TERMINAL FLOWER 1(TFL1)in wheat.qRT-PCR analysis showed that the gene has the highest expression in spikelets,and in situ hybridization showed that the gene was specifically expressed in the single ridge stage,double ridge stage,spikelet primordium stage and floret differentiation stages of SAM.We constructed an overexpression vector of TFL1-4A gene and transformed it into the Arabidopsis tfl1 mutant,and found that the phenotype of the apex flower of tfl1 could be partially recovered.Therefore,we suggest that TFL1-4A is similar to the AtTFL1 that extends vegetative growth stage and maintains the infinite growth of inflorescence in wheat.更多还原