Biological Function Research Of The Regulating Factor HL111 Involved In Expression Regulation Of Heat Shock Response In Arabidopsis

In our preliminary study,a WD40 family of proteins HL111,which may play an important role in plant thermal stress tolerance,was identified to explore the mechanism of plant high temperature stress response.In this paper,the function and mechanism of HL111 gene to response stress were studied by phenotypic analysis and transcriptional sequencing in Arabidopsis thaliana.The following experimental results were obtained:(1)Luciferase activity and transcription of Luciferase gene in mutant hl111 were significantly up-regulated after treatment at 38? for 1h.The HL111 gene was transferred into the mutant hl111 through Agrobacterium tumefaciens-mediated transformation to obtain the transgenic complementary plants.The Luciferase activity and transcription level of the complementary plants could be restored to the wild type,which indicated that HL111 gene caused Luciferase brightened in mutant hl111 under high temperature.(2)After 16 h treatment at 38?,the survival rate of mutant hl111 was significantly decreased,suggesting high temperature sensitivity.The two functional deletion T-DNA insert mutant thl111-1 and thl111-2 of the mutant hl111 were treated at 38? for 18 h,the survival rate of the two groups was significantly lower than wild-type,showing the same high temperature sensitivity as the mutant.The survival rate of the transgenic complementary plants at 38? for 16h,the survival rate can be restored to the wild type,indicating that HL111 gene is necessary for plant heat tolerance.(3)During the growth and development,the growth rate of the mutant hl111 was slowed down,the number of leaves decreased,the plants became smaller and shorter.Transgenic complementary plants growth and development can be restored close to the wild type,indicating that HL111 gene mutation affect the growth and development of plants.(4)The expression of HL111 gene in wild type P-LUC was analyzed by qRT-PCR and it was found that high temperature inhibited the expression of HL111.The expression of HL111 gene was not effected at Oh,0.5h,1h,2h and 5h after high temperature treatment in mutant hllll and wild type.The expression level of HL111 gene was the lowest after treated at 38? for 1h.GUS staining analysis showed that HL111 gene expressed at all tissues in Arabidopsis thaliana,suggesting HL111 was a constitutive expressed gene.The results of subcellular localization showed that HL111 protein was expressed in the nucleus and cytoplasm in Arabidopsis thaliana.(5)The transcriptional levels of heat shock transcription factor Hsfs and heat shock protein Hsps in the mutant hl111 were analyzed by RNA sequencing and qRT-PCR analysis after treated at 38? for 1 h,which showed that the expression levels of most Hsfs and Hsps in the mutant hl111 were lower than those in wild-type.These suggested that HL111 gene regulated the high temperature tolerance by regulating the expression of these heat shock transcription factors and heat shock proteins.All data above showed that HL111 gene may act as a positive regulator of heat shock protein gene in the high temperature response pathway in Arabidopsis thaliana.The current research results provide a basis for elucidating the mechanism of plant high temperature tolerance.