Studies On The Mechanism Of LncRNA PL-in Candidate Target Gene TaPLATZ2 And Its Regulatory Protein TaWRKY55 In Wheat Saline-alkaline Resistance

Saline-alkaline soil is a major environmental problem affecting crop production.Characterizing saline-alkaline tolerance genes and breeding stress-tolerant crop cultivars is the most effective means to exploit and utilize saline-alkaline land.Shanrong No.4(SR4)is an alkaline-tolerant wheat line derived from asymmetric somatic hybridization between the common wheat cultivar Jinan 177(JN177)and tall wheatgrass.Previous studies have shown that SR4 has strong alkaline resistance and is an ideal material to study the mechanism of alkaline resistance in wheat.LncRNA is a class of noncoding RNA with a length more than 200 bp,which has a variety of biological functions.However,the roles of lncRNAs in alkaline stress responses remain unclear especially in wheat.Therefore,the role and mechanism of lncRNA in alkaline stress responses of wheat need further study.In this thesis,we characterized alkaline stress responsive lncRNAs from SR4 and JN177,analyzed the function of a few alkaline stress-responsive lncRNAs and predicted their target genes.We systematically studied the function and molecular network of TaPLTAZ2,a candidate target gene of lncRNA PL-in,in regulating salinealkaline stress tolerance of wheat.It provided theoretical basis and important gene sources for wheat saline-alkaline stress tolerance breeding.The results of this work were as follows:1.Functional analysis of lncRNAs in response to alkaline stress in wheatSequencing was employed to identify the lncRNAs associated with alkaline stress tolerance.Approximately 19,000 novel lncRNA sequences were detected in SR4 and JN177.Upon exposure to alkaline stress,SR4 differentially expressed 5,691 lncRNAs,whilst JN177 differentially expressed 5,932.We selected five of them(L0760,L6247,L0208,L2098,and L3065)and generated seedlings of transiently knocked down lines using the virus-induced gene-silencing(VIGS)method.Knockdown of L0760 and L2098 caused the plants to exhibit sensitivity to alkaline stress,whereas knockdown of L6247,L0208,and L3065 enhanced wheat resistance to alkaline stress.We constructed lncRNA-miRNA-target-mRNA networks and alkali-response-related lncRNA-targetmRNA association networks to analyse the functions of lncRNAs.Collectively,our results demonstrate that lncRNAs may perform different roles under alkaline stress conditions.2.Functional analysis of lncRNA PL-in and its candidate target gene TaPLTAZ2 in regulating saline-alkaline stress responses of wheatWe knocked down lncRNA PL-in using the VIGS method and found that knockdown of PL-in enhanced wheat tolerance to saline-alkaline stress.PL-in transcribed from the third intron of TaPLATZ2 and they had consistent transcription direction.Moreover,under saline-alkaline stress,the expression of TaPLATZ2 was significantly down-regulated in transient knockdown lines of PL-in,indicating that TaPLATZ2 might be the target gene of lncRNA PL-in.We found that saline-alkaline stress could induce the expression of TaPLATZ2,overexpression of TaPLATZ2 enhanced sensitivity of wheat to saline-alkaline stress,while knockdown of TaPLATZ2 enhanced the resistance of wheat to saline-alkaline stress,it indicited that TaPLTAZ2 negatively regulate saline-alkaline stress tolerance of wheat.To elucidate the molecular mechanism of TaPLATZ2 in regulating wheat saline-alkaline stress response,we analyzed the downstream gene of TaPLATZ2.EMSA,ChIP-qPCR and luciferase reporter gene experiments proved that TaPLATZ2 could inhibit the expression of SALTOVERLY SENSITIVE(SOS)signal pathway gene TaSOS3.The luciferase reporter gene experiment showed that TaPLATZ2 could significantly inhibit the expression of H+-ATPase coding gene TaHA-6B.We hypothesized PL-in regulated the expression of TaPLATZ2 under saline-alkaline stress,and TaPLATZ2 affected efflux of Na+ and H+by regulating the expression of TaSOS3 and TaHA-6B,thus particated in response to saline-alkaline stress in wheat.3.Functional analysis of TaPLATZ2 regulatory protein TaWRKY55 in response to abiotic stressesWRKY transcription factor plays an important role in plant growth and development as well as response to biotic and abiotic stresses.We found that TaWRKY55 could bind the promoter of TaPLATZ2 by screening the yeast one-hybrid library.EMSA,ChIP-qPCR and luciferase reporter gene experiments proved that TaWRKY55 could promote the expression of TaPLATZ2.Overexpression of TaWRKY55 enhanced sensitivity of wheat to saline,alkaline and ABA,while knockdown of TaWRKY55 enhanced the resistance of wheat to saline,alkaline and ABA,it indicited that TaWRKY55 negatively regulates salinity,alkalinity and ABA stress tolerance of wheat.We analyzed the expression of important genes in ABA signaling pathway in TaWRKY55 transgenic lines,and found that TaABI4 was upregulated in TaWRKY55 overexpression lines but downregulated in TaWRKY55 RNAi lines.Yeast one-hybrid,EMSA,ChIP-qPCR and luciferase reporter gene experiments proved that TaWRKY55 could bind and promote the expression of TaABI4.We found overexpression of TaABI4 enhanced the sensitivity of wheat to alkaline stress,and RNAi lines showed resistant to alkaline stress.We found that TaABI4 could bind the promoter and inhibited the expression of ROS scavening gene TaSOD2 to participate in the response to alkaline stress in wheat.