| Vernalization is an important regulatory process of growth and development in wheat,and the prolonged exposure to cold can effectively accelerate inflorescence initiation and development,which directly affected the frost tolerance of wheat seedlings,as well as the final grain yield and flour quality.Therefore,the research work focused on the molecular mechanisms of vernalization is crucial important to the molecular breeding for frost tolerance and high yield.In this work,the vernalization-responsive transcriptomes were systematically analysed via Illumina high-throughput sequencing technology,and their differential expression patterns during the vernalization process were profiled.Additionally,several vernalization-responsive candidate genes were functionally identified via VIGS technology.The main results were summarized as follows: 1.Vernalization-responsive transcriptome and their differential expression profiles Two wheat varieties Liaochun 10(spring)and Jing 841(winter)were used as the experimental materials and the transcriptomes were analyzed via high-throughput Illumina sequencing technology.69,751,192 raw reads and 5,787,106,380(5.78Gb)nucleotide were obtained and 138,062 sequences can be divided into 61 feature sets based on sequence homology,56,868 assembly sequence were assigned to 25 COG clusters.Eight digital gene expression profiles were constructed using the materials of vernalized-and nonvernalizedbuds,vernalized-and nonvernalized-leaves at double ridge stage in Liaochun 10 and Jing 841,respectively,and a set of differentially expressed genes were obtained.The expression patterns of some differentially expressed candidate genes were validated by using q RT-PCR,which including 9 transcription factors,7 methylation-related genes,2 flowering regulation-related genes,2 vernalization-related genes,1 cold-responsive gene,1 dehydrin,1 ice recrystallization inhibition proteins,and 1 ABA related gene.The results showed that the datas of digital gene expressions from high-throughput sequencing technology were generally accurate and reliable.2.vernalization-responsive candidate genes and their expression patterns The expression profiling indicated that 29498 genes were specifically expressed in Jing841 via comparisons of J-NV&J-V and LC-NV&LC-V.639 vernalization response genes belonged to 8 categories were screened according to the function annotation of differentially expressed gene.A total of 17 genes were selected from the 8 categories respectively on the basis of the higher differential expression,and the expression patterns during the vernalization were analyzed.Including 6 transcription factors,3 methylation-related genes,2 flowering regulation-related genes,2 vernalization-related genes,1 cold-responsive gene,1 dehydrin,1 ice recrystallization inhibition proteins,and 1 ABA related gene.6 from the 17 genes,CL8746.Contig1(TF:B-box transcription factor),CL18846.Contig2(TF : zinc finger protein),Unigene10196(TF),CL12788.Contig3(TF:zinc finger protein),CL14010.Contig2(vernalization),Unigene16777(methylation)were selected with differentially expressed in multiples over 10 times and longer than 1000 bp,and so did the CL176.Contig1(CDT1-like protein)from differentially expressed gene of J-NV&J-V.These 7 genes were cloned and sequence analysed in Jing841,involved in signal transduction,cell cycle,senescence,and defense-related,DNA methylation and mitosis.Expression patterns analysis of 7 genes showed that,the overall trends were down regulated,CL8746.Contig1 up-regulated in 6th day,and then down-regulated;Unigene16777 and CL176.Contig1 down-regulated with a fluctuation;and the rest showed a continuing downward trend.The above results indicated that they played important roles during the vernalization response processes and multiple gene regulatory pathways were involved.3.Functionally identification of several crucial candidate genes Using VIGS technology and taking the Barley stripe mosaic virus as a carrier to establish technology system of VIGS gene function verification,which took Jing841 as experimental material.Function of 7 genes from vernalization response transcriptome were verified based on the above system.The results indicated that the wheat infected by BSMV recombinant vector which constructed in this paper,process of young spike development were influenced differently.Differences among blank,positive and negative control were not significant,but there were significant differences between control and the target genes.There were 85.0 days for blank control from germination to double ridge stage,but the longest and shortest days for plants infected by BSMV recombinant vector were 66.5 and 59.8 respectively.Development progress from germination to double ridge stage were greatly shorten.The study showed that,the 7 candidate genes with flowering inhibitory effect in flowering regulation process in wheat,however,the specific biological functions in wheat vernalization process remain to be further studied.4.Cloning and expression characterization of VRN1 and VER2 Two candidate genes,CL19305.Contig2 and CL14010.Contig2,was highly identical to the vernalizaiton genes of VRN1 and VER2,respectively.Here,complete ORFs of these two genes were cloned and analyzed in 8 wheat varieties,which representing typical different vernalization phenotypes.The results showed that sequences of these two genes in eight species were highly similar.Expression patterns during vernalization were systematically analyzed.The expression level of VRN1 in winter varieties were very low in the vernalization early,and increased as vernalization prolonged.The time required for VRN1 expression up-regulated consistent with the vernalization time required for different wheat varieties,perhaps,the expression level of up-regulated VRN1 promoted the development process of winter varieties,to be heading in normal.In the spring of varieties,VRN1 began to express in the initial stage of vernalization,increased with the extension of time.The expression patterns of VER2 during vernalization were different in 8 varieties,that is up-regulated in spring varieties,while down-regulated in winter varieties.The VER2 was down regulated in vernalized-and nonvernalized-treatment in Liaochun 10 and Jing 841. |
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