Screening And Identification Of Arabidopsis Molecular Chaperone,HOT5 In Responses To High Temperature

With global warming becoming increasingly severe,the rise of average temperature has become an irreversible fact.High temperature,causes chronic damage to plants,not only affects seed germination,accelerates leaf senescence and grain maturity,but also has detrimental influence to pollen vitality,reduces fructification percentage.During the evolution of plant,a set of sophisticated signal transmission systems have been generated to adapt to high temperature.However,heat shock transcription factors play a key role in heat stress.A large number of studies have revealed the regulatory mechanism of heat shock transcription factors in response to heat stress.Nevertheless,it’s a complex process,and there must be some regulation by other genes.In order to explore other regulatory genes and improve the thermal regulation network,we conducted a series of experiments to obtain relevant genes and uncover their molecular mechanisms.Based on this,we implemented experiments and obtained the results as following:1 Total biomass RNA was extracted from Arabidopsis seedlings treated with heat shock at 38 ℃,and cDNA was synthesized with reverse transcription.The cDNA bands were princpally distributed between 100-3000 bp and transferred into yeast strain Y187 with vectors to construct the cDNA library for screening heat shock transcription factor interaction proteins.After calculation and statistics,the titer of this Arabidopsis seedlings cDNA library is estimated as 1.88×108 cfu/mL,and the size of insert fragments in 100-2000 bp,which indicated that this cDNA library can be used to the screening of heat shock transcription factor interaction proteins.2 Screening Interaction Proteins of HSFA1、HSFA2、HSFA3、HSFA7a、HSFB1、HSFB2、HSFB2a、HSFB3、BZIP28、NAC019 and DREB2A by using yeast two-hybrid library,we selected 25 potential candidate genes.3 We ordered some mutants of candidate genes,and obtained a mutant,hot5-1,which is sensitive to high temperature.The gene was named HOT5.4 To confirm the phenotype furtherly,other mutants of this gene were ordered and subjected to high temperature experiments.It was found that the hypocotyl and root length became shorter,which illustrate the crucial role of this gene in responses to highi temperature.5 It was found that the HOTS was promptly induced by high temperature and the expression level increased sharply through QRT-PCR experiments,which also proved the essential role of the gene in responses to high temperature.6 The interaction between HOT5 with bait protein was further verified by bimolecular fluorescence complementation experiment.According to previous research reports,plants respond to high temperature by renaturing denatured proteins through molecular chaperones.Therefore,we speculate that HOT5,as a chaperonin,regulates the response to high temperature by helping the correct folding of proteins.