Identification Of Wheat Mirna In Different Growth And Development Stages And Prelimary Functional Study Of The Two Spike/grain Highly Expressed Mirnas In Common Wheat (Triticum Aestivum L.)

Common wheat?Triticum aestivum L.?is one of the most important staple crops for human diet.With the increasing global population,the shortage of foods supply will become more and more serious.Therefore,better understand of genes that control wheat growth and development is essential for future wheat breeding.MicroRNAs?miRNA?are a class of small non-coding RNAs that act as powerful endogenous regulators in plant growth,development and stress response.Therefore,deep understanding of the involvement of miRNAs in wheat development and yield is crucial for wheat improvement.In the past decade,a vast majority of miRNAs has been discovered by small RNA deep sequencing.This growth represents a significant challenge to the overall quality of the miRNA sequence data set.In order to ensure the quality and credibility of plant miRNAs identified,two international research groups proposed new criteria for high confidence miRNA identification.Since 2007,there have been many studies to identify wheat miRNA based on deep sequencing,most of which have been completed before the new criteria.Therefore,it is necessary to re-identify previously reported wheat miRNA based on new criteria to ensure high confidence.In this study,we systematically identify more high confidence wheat miRNAs involved in regulating wheat growth and development by reanalyzing sets of deep sequencing data from different wheat tissues and developmental stages using the new high confidence criteria.The expression profiles of identified miRNA were analyzed.Target genes of miRNAs identified were predicted.The regulatory network of miRNAs and their target genes was also analyzed to identify key genes during wheat growth and development.In addition,in order to quickly and effectively identify the function of wheat miRNA,we also try to develop BSMV-induced miRNA silencing system.By using this system,combined with BSMV-VIGS system,we preliminary study the function of tae-miR319 and tae-miR9677 in this study.The main topics of thesis are as follows:1.To obtain high confidence miRNAs involved in wheat growth and development,we systematically re-analyzed 12 sRNA libraries?including 7-day-old leaves,flag leaves after 12 days anthesis,spikes with anther length of approximately 1.0mm,1.5mm,2.2mm,3.0mm,spikelets,five-leaf-stage seedlings,flag leaves at heading stage and immature seeds at 5 days-post-anthesis,10 days-post-anthesis,and 20 days-post-anthesis?from different wheat tissues and developmental stages using the new high confidence criteria.As a result,we annotated 128 high confidence miRNAs?including 102 previously reported and 26 novel miRNAs?belonging to 107 families.Conservation analysis showed that 102 high confidence miRNAs were sorted into three categories: 15 miRNA from 9 families are highly conserved,17 miRNAs from 13 families are moderately conserved and the rest 70 miRNAs from 63 families are wheat-specific miRNAs.Location analysis showed miRNA loci in A,B and D sub-genomes was uneven and a large number miRNAs were preferentially located in the B sub-genome.2.Cluster analysis of 128 miRNAs in spatiotemporal expression patterns in 12 different tissue/organ showed that most miRNAs were tissue-specific or-biased expression in wheat.Further analysis showed about 30 miRNAs are differently expressed in wheat sipkes,seeds and vegetative tissues.Among these miRNAs,8 showed to be grain-biased including tae-miR4081 b,tae-miR9654 a,tae-miR9655,tae-miR9657 b,tae-miR9659,tae-miR9678,tae-13 and tae-17,suggesting these miRNAs may related to grain development.Six miRNAs showed to be sipke-biased including miR319 f,tae-miR9666 a,tae-miR9677,tae-2,tae-4 and tae-7,suggesting these miRNAs may related to spike development.3.Putative targets of the 128 identified miRNAs were also predicted.Among 102 known miRNAs,a total of 581 unique genes were predicted as targets for 93 miRNAs.However,the rest 9 miRNAs?tae-miR1847,tae-miR3022 a,tae-miR4048,tae-miR9656,tae-miR9658,tae-miR9665,tae-miR9662 b,tae-miR9669 and tae-miR9671?have no target genes.Among 26 novel miRNA,24 unique target genes for 13 novel miRNA were identified.And the rest 13 novel miRNA have no target genes under current condition.GO annotation analysis revealed that these target genes mainly play important roles in biological process,cellular process,primary metabolic process and organic substance process.miRNA-target gene regulatory network revealed that four key genes?Traes₇BL₈C101A09C?Traes₇DL₁8F2A8567?Traes₅AS₄6DF79652 and Traes₄DS_D9999817B?are in the centre position of network,and further study showed that these genes are involved in cell signal transduction,glucose metabolism and stress response,suggesting these key genes play core roles in wheat growth and development.4.We demonstrated that a BSMV-derived vector can be used to silence endogenous target miRNAs in wheat.By utilizing the BSMV systems,we successfully knocked down the levels of endogenous miR156 and miR166.Target genes of miR156 and miR166 are predicted to be Squamosa promoter-binding protein2-like?TaSPL2?and Homeobox-leucine zipper protein HOX33-like?TaHD-ZIP?respectively.And silencing miR156 resulted in a great increase in the abundance of its target mRNA,and led to a leaf-curl phenotype and earlier flower time in wheat,suggesting that miR156 plays important roles in leaf development by regulating its target gene expression in wheat seedlings.While silencing miR166 led to a large increase in the m RNA level of its target gene TaHD-ZIP,and generated a leaf-twist phenotype in wheat seedlings,indicating that miR166 functions in wheat growth and leaf status.5.Grains and spikes biased tae-miR319 and tae-miR9677 were further studied in this study.Silencing tae-miR319 during grain development stage can reduced grain size.Silencing TaMYB,target gene of tae-miR319 can enlarged grain size in wheat,suggesting that tae-miR319 positive regulates grain size.However,no obvious distinguished symptoms were found when silenced tae-miR9677.In brief,our study first identified 128 high confidence miRNAs from 12 sRNA from different wheat tissues and developmental stages using the new high confidence criteria.The spatial and temporal expression profiles were analyzed to identify spike / grain biased miRNA in wheat.Target genes and miRNA-gene regulatary network were also performed in this study.In addtion,BSMV-induced miRNA silencing system was constructed in this study.By utilized used this system,we quickly studied function of miR156 and miR166 in wheat.We further performed BSMV induced miRNA silencing and overexpressing system to silence and overexpress tae-miR319,and also silenced TaMYB,target genes of tae-miR319 by BSMV-VIGS.The results suggested tae-miR319 positive regulates seed size in wheat.