| Forests are the essential composition for eco-environment system,which have crucial economic and ecological values.In recent years,climate change has increased the frequency of extreme high temperature in the world.Heat stress is one of the main environmental stresses,which affects forest productivity and limits plant growth,metabolism and yield.To study the transcriptional regulation mechanism of forest trees in response to heat stress and analyze the molecular mechanism is the key scientific problem to cultivate new resistant varieties and improve woody yield.The research shows that heat stress resistant traits of trees are controlled by multi genes and multi-level regulatory networks,and the regulatory mechanism is very complex.The rise of ambient temperature can affect photosynthesis and tree growth,but the specific mechanism needs to be further explored and revealed.Therefore,it is of great significance to systematically study the mechanism of transcriptional regulation of forest trees under heat stress.Thus,this paper integrated time-course RNA-seq,s RNAseq,degradome sequencing analysis,weighted gene correlation network analysis,multigene association approaches and molecular biology experiments to explore systematically regulatory networks in perennial woody plants,poplar.Combined with the association mapping strategy,this paper analyzes the allelic variation effect of heat stress responsive genes and micro RNAs to explore the SNP markers which associated with photosynthesis related and growth traits,and further verifies the biological functions of the key regulatory modules by molecular biology approaches,and finally reveals the genetic regulatory network of miRNA m RNA in response to heat stress on photosynthesis and growth.The main research results and conclusions are as follows:(1)The physiological and biochemical indexes,photosynthetic characteristics and endogenous hormones of Populus trichocarpa under heat stress treatment for 0,4,8,12,24,36 and 48 h were measured.The results showed that the indexes of photosynthesis reached the extreme value and changed significantly at 36 h of heat stress;With the change of heat stress treatment time,the peroxidase(SOD,POD,CAT)activity increased rapidly;The contents of abscisic acid,indole-3-acetic acid and growth hormone changed significantly under heat stress.The results showed that heat stress could significantly affect the physiology,photosynthesis,and growth characteristics of poplar.(2)By using time-course RNA-seq,11,417 heat stress responsive genes were detected in P.trichocarpa.Pathway enrichment analysis revealed that the differentially expressed genes were mainly involved in photosynthesis,photorespiration,enzyme activity regulation,secondary metabolism,and stimulate responses.Based on public databases,168 photosynthetic genes which differently expressed under heat stress were obtained,including92 light reaction genes,41 Calvin cycle genes and 35 photorespiration genes.Further,totally77 photosynthesis-related genes were identified by weighted network coexpression analysis and Map Man pathway analysis.The enrichment analysis of cis-elements located in the promoter regions resulted four regulatory elements: SORLIP1 AT,SORLIP2AT,MYB1 AT and WRKY.Combined with the whole genome distribution pattern of transposable elements(TEs),the gene expression level increased with the distance to the nearest TE,and this relationship was stronger when the TE closest to a gene was 24 nt si RNA targeted.When the distance from the nearest transposon was 1 kb,the gene expression level reached the maximum.(3)By using s RNA-seq,this study identified 179 miRNAs and 36 novel miRNAs in response to heat stress across different time points of P.trichocarpa.By adopting target gene prediction,179 known miRNAs interacted with 423 target genes,and 36 novel miRNAs interacted with 92 target genes.Functional annotation and pathway enrichment analysis of target genes showed that these genes were enriched in stress response and photosynthesis and growth pathway.In addition,385 lnc RNAs could be predicted as potential target genes of 203 miRNAs/novel miRNAs,of which 44 lnc RNAs were predicted as precursors of 41 miRNAs/novel miRNAs.The results show that these lnc RNAs can indirectly regulate the downstream target genes of miRNA by affecting the expression and transcription of miRNA precursors and the expression abundance of mature miRNA.(4)A three-layer gene regulatory network of photosynthesis under heat stress was constructed by BWERF method in poplar.There are 77 photosynthesis related genes,40factors(35 transcription factors;5 miRNAs)and 20 factors(5 transcription factors;15miRNAs)in the bottom,middle and top layers of the network,respectively,which forming585 pairs of regulatory pairs.Based on the genome resequencing data of 435 Populus tomentosa associated populations,genotype-phenotype association was used to associate11,834 common SNPs in 109 candidate genes and miRNAs(62 genes,13 TF,34 miRNAs)with 29 photosynthetic and growth phenotypic traits.A total of 114 significantly associated SNPs were identified(P ≤ 0.0001),explaining the phenotypic variation rate of 0.94-21.22%,with an average of 10.63%.Epistasis analyses identified 696 significant SNP-SNP association pairs upon 29 phenotypic traits(P ≤ 0.0001),involving 243 SNPs within 32 photosynthesis genes,5 TFs,and 10 miRNAs,supporting the notion that epistasis is the crucial composition of genetic effects for traits.All these findings provided evidence for the interaction between miRNA and target genes.(5)Association analysis showed that the SNPs located on miR396 a and its target gene GRF15 were significantly associated with 10 photosynthesis-related and growth traits.Epistasis analysis revealed that miR396a-GRF15 were the hub interaction in genetic regulation network,and potentially act as a negative regulatory module.Transgenic ox Pag GRF15 plants expressing a GRF15 m RNA that lacks miR396 a target sites exhibited enhanced heat tolerance and photosynthetic efficiency compared to wild-type plants.Together,these observations demonstrate that GRF15 plays a crucial role in responding to heat stress and provide a new,multifaceted approach for identifying regulatory nodes in plants.This study provided an important theoretical basis for poplar resistance breeding.This study analyzed the photosynthetic genetic regulation network of poplar under heat stress and the interaction of miRNA-m RNA in the hierarchical network,which had important scientific and theoretical significance for exploring the synergistic effect of stress resistance and tree growth.This study provided scientific theoretical guidance for miRNA cooperating with TFs to regulate tree growth under heat stress,and would provide theoretical support for tree molecular resistance breeding. |
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