Identification Of AGO,HAT/HDAC Gene Families And Their Interacting MiRNAs In Jatropha Curcas And Their Roles In Response And Adaptation To Low Temperature

Jatropha curcas L.is a kind of tropical and subtropical energy plants with comprehensive utilization value,and low temperature is one of the important environmental factors limiting its growth.According to the previous research in our laboratory,the cold resistance of J.curcas seedlings under 1°C was significantly enhanced after 2 d of chill hardening at 12°C.Argonaute(AGO)is the core component of RISC(RNA Induced Silencing Complex)and a key component of the functional pathway of miRNAs.Histone acetyltransferases(HATs)and histone deacetylases(HDACs)are a group of enzymes that regulate the level of histone acetylation in eukaryotic organisms by altering the structural state of chromatin for epigenetic labeling of gene expression.But the role of these three gene families and their interacting miRNAs in response and adaptation of J.curcas to low temperature stress has not been reported in related studies.Based on the sequencing data of transcriptome,miRNAome and degradome of J.curcas seedlings under chill hardening at 12°C for 48 h and low temperature stress at1°C for 5 d from our laboratory,this study analyzed bioinformatics of the AGO,HAT and HDAC gene families of J.curcas at the whole genome level,identified and detected the expression of their interacting miRNAs,and conducted yeast expression verification and transgenic tobacco overexpression verification of some key genes with high response to low temperature,and made preliminary functional analysis of these genes,and the main results are as follows.1.Identification of the Argonaute gene family and their interacting miRNAs in J.curcas and their possible roles at low temperature adaptationA total of 9 gene family members were identified in the whole genome of J.curcas,localized to eight linkage groups.This family can be divided into three subfamilies,all contains Pi Wi,Argo Mid,PAZ,Argo N conserved structural domains.Pi Wi structural domains contain Asp-Glu-Asp-Asp/His(DEDD/H),H798,and QF-V,which are highly conserved catalytic active sites.Expression analysis showed that JcAGO1,JcAGO4,JcAGO5,JcAGO10,and JcAGO16 had high abundance under low temperature,salt,and drought stresses.60 miRNAs targeting to 9 family members were identified.Most of the AGO family members showed up-regulated expression under low temperature treatment.Based on the digital expression profile results,most of the miRNAs showed a down-regulated expression trend under the low-temperature treatment.q RT-PCR validation of selected highly expressed miRNAs revealed that miR11036-x,miR11109-x,miR3440-x,miR7731-y showed a significant down-regulated expression trend relative to the controls.There is a negative regulation between the AGO gene family and its acting miRNAs under low temperature treatment.JcAGO1 and JcAGO4,which were highly responsive and highly expressed under low temperature treatment,were selected for tobacco overexpression validation,and positive transgenic lines were obtained respectively.2.Identification and low-temperature expression analysis of HAT/HDAC gene family and their interacting miRNAs in J.curcasA total of 10 HAT and 14 HDAC gene family members were identified in the whole genome of J.curcas,localized to 8 and 7 linkage groups,respectively.The former contains four subfamilies,GNAT,MYST,CBP and TAFII250,and the latter contains three subfamilies,RPD3/HDA1,HD and SIR2,which were analyzed by bioinformatics.The expression profile analysis showed that JcHAC1,JcHDA19,JcHDT1,JcHDT3 and JcSRT2 were significantly up-regulated,while JcHAC1 and JcHAG1 were significantly down-regulated during the chilling treatment.50 miRNAs targeting the J.curcas HAT gene family and 53 miRNAs targeting the J.curcas HDAC gene family were identified.q RT-PCR was performed to validate the genes with high response to low temperature treatment and some of the miRNAs they targeted,and JcHAG1 and JcHAC1,JcHAM1 and JcHAC2 were found to be co-expressed.JcHAG1 and JcHAC1,JcHAC1 and JcHAC1,JcHAC1 and JcHAM2 were positively correlated with down-regulated expression,JcHDT1 and JcHDA6 were positively correlated with up-regulated expression,JcHAG1 and JcHAG2,JcHAG1 and JcHAG3 were negatively correlated with expression,and miR5059-x and miR5072-x were negatively correlated with their target genes during low temperature stress.There was a highly significant negative relationship between miR5059-x and miR5072-x and their target genes during low temperature stress.In addition,Western blot detection of histone H3 modifications during chill hardening revealed an increase in acetylation levels at CH24 h.The results of yeast heterologous expression experiments showed that JcHDA6,JcHDA9,JcHDA19,and JcSRT2 recombinant yeast could tolerate 5% salt stress and 5%,10%,15%,and 20% osmotic stress,while JcHAG3 and JcHAM1 recombinant yeast showed the opposite trend.Meanwhile,overexpression of JcHAG1 and JcHDA19 in tobacco was also performed and positive lines had obtained.In summary,all the above findings can provide a theoretical basis for further studies on the biological functions of the AGO,HAT and HDAC gene families and the interactions of targeting miRNAs in J.curcas and reveal the role of these interactions in response and adaptation to low temperature,and provide some new ideas for the future enhancement of cold resistance by means of genetic engineering and molecular breeding in J.curcas.