Preliminary Identification Of Long Non-coding Rna In Wheat Powdery Mildew Resistance And Preliminary Analysis Of Its Regulatory Mechanism

Bread wheat(Triticum aestivum L.)is one of the most important food crops in China.During it is production,Powdery mildew(Blumeria graminis f.sp.Tritici,Bgt)is an important worldwide fungal foliar disease responsible for severe losses in yield.The development of resistance genes and dissection of the resistance mechanism will therefore be beneficial in controlling disease and minimizing crop losses.With the emergence of new virulent varieties and the simplification of resistance genes caused by the wide spread of resistant varieties,resistance breeding has always been in a passive situation.Therefore,it is of great significance to understand the regulatory mechanism of wheat powdery mildew resistance response and to explore resistance genes.Long chain non coding RNA(lnc RNAs),as a kind of nucleic acid sequence encoding non functional proteins,plays an important regulatory role in plant development and stress response.In this study,N9134,a powdery mildew resistant germplasm,was used to mine the lncrna related to the disease resistance response through the transcriptome data of er09 infection,and its regulatory mechanism was preliminarily analyzed.The research methods and results are as following:1.The transcriptome of the hexaploid wheat line N9134 inoculated with the Chinese Pst race CYR31 and Bgt race E09 at 1,2,and 3 days post-inoculation was recapitulated to detect the linc RNAs.Here,283 differential expressed linc RNAs were identified from 58218 putative linc RNAs,which account for 31.2% of transcriptome.Of which,254 DE-Linc RNAs responded to the Bgt stress.Among them,1328 sn RNP motifs(sm sites)were detected and showed RRU4-11 RR sm site element and consensus RRU1-9VU1-7RR sn RNP motifs,where the total number of uridine was more than 3 but less than 11.Additionally,101 DE-linc RNAs were predicted as targets of mi RNA by ps RNATarget,while 5 target mimics were identified using target mimicry search in TAPIR.2.A total of 461 functional genes close to 249 DE-lnc RNAs we identified by alignment of the DE-lnc RNA sequences with the reference sequences of Chinese Spring.Furthermore,we evaluated the possible role of lnc RNAs in regulating functional genes in wheat responding to Bgt pathogen,using genome-wide transcriptome analysis and quantitative RT-PCR.Our results demonstrated that both long intron nc RNAs(linnc RNA)and long intergenic nc RNAs(linc RNAs)play roles in regulating allele-specific genes,including transcription factors,both positively and negatively.The correlation of expression between linc RNAs and flanking functional genes increased as the intervening distance decreased.Co-expression of micro RNAs,their target lnc RNA and target functional genes showed that linc RNA interacted competitively with functional genes via mi RNA regulation.3.Here,by RNA sequencing,we identified three co-expressed gene modules using pairwise comparisons and weighted gene co-expression network analysis during wheat–Bgt interactions compared with mock-infected plants.Hub genes of stress-specific modules were significantly enriched in spliceosomes,phagosomes,the m RNA surveillance pathway,protein processing in the endoplasmic reticulum,and endocytosis.Induced module genes located on chromosome 5BL were selected to construct a protein–protein interaction network.Several proteins were predicted as the key hub node,including Hsp70,DEAD/DEAH box RNA helicase PRH75,elongation factor EF-2,cell division cycle 5,ARF guanine-nucleotide exchange factor GNOM-like,and protein phosphatase 2C 70 protein,which interacted with several disease resistance proteins such as RLP37,RPP13,and RPS2 analogs.Gene Ontology enrichment results showed that wheat could activate binding functional genes via an m RNA transcription mechanism in response to Bgt stress.4.Combining the genetic map fragment fo Pm AS84 in N9134 with these detected nodes in WGCNA net,the results showed that GNOM-like,PP2 C isoform X1,and transmembrane 9 superfamily member 9 were mapped onto the genetic fragment of Pm AS846 with a distance of 4.8 Mb.This means that they maybe candidate genes of the resistance gene Pm AS846.Taken together,our findings indicate that the linc RNA of wheat responded to Bgt stress and played important roles in splicesome and inter-regulating with mi RNA.The sm site of wheat showed a more complex construction than that in mammal and model plant.The mass sequence data generated in this study provide a cue for future functional and molecular research on wheat–fungus interactions.These results will be beneficial for further dissecting molecular mechanisms of lnc RNAs functions at the transcriptional and post-transcriptional levels in wheat,as well as for understanding the outline of resistance mechanism and cloning the resistance gene Pm AS846.