Functional Analysis Of Long Nocoding RNA HAL6 For Heat Stress Tolerance In Arabidopsis

In recent years,extremly temperatures occur more frequently.Most kinds of plant species will face with the challenge of heat stress.It is important to study the effect of heat stress on plant growth and development,etc.Long noncoding RNA is a kind of noncoding RNA whose transcript length is usually longer than 200 nucleotides.Previous studies,the researchers found that lncRNAs play an important role in the animals and plants,but little is known about the function and regulatory mechanism of plant lncRNAs.In this study,we identified a function-unknown long noncoding RNA that positivity regulates the thermotolerance in arabidopsis thaliana.The results are as follows:(1)We sequenced six RNA samples from Arabidopsis seedlings with or without heat stress by using the method of chain specific transcriptome sequencing.After screening,we finally obtained 8095 lncRNAs,including the newly discovered 1057,accounts for 13.06%.The results indicate that a lot of lncRNAs in arabidopsis have not been identified so far.The chromosome distribution of new finding lncRNAs is no difference between positive and negative chain.Length is mainly concentrated in between 200 nt to 1500 nt,and the position of the lncRNA and adjacent mRNA relationship mainly is intergenetic.The GO analysis of adjacent mRNAs finds them in triglyceride,mitochondria transfer,etc.The KEGG analysis showed that some organic metabolic pathwayschange obviously after the heat shock.And the expression of 79 lncRNAs changed obviously after heat shock.Among them,the expression of HAL6 can be induced obviously after heat stress.(2)Sequence and RACE-PCR results showed that the HAL6 is consist of 448 nucleotides,and secondary structure prediction revealed that HAL6 forms four stem ring.HAL6 is located between a transposon(At2g13274)and a pseudogene(At2g13280).There are 7547 nucleotides between At2g13274 and At2g13280.Both 37 ? and 45 ? treatments can induce obviously the expression of HAL6 in Arabidopsis seedlings.HAL6 expressed in all detected tissues,and highest expressed in seeds.By using MS2 positioning system,we observed that HAL6 located in the cytoplasm.(3)The HAL6 overexpression lines showed more tolerance to heat stress than wild type.For the mature plants,survival rate of the overexpression plants was much higher than that of the wild type plants after heat treatment.In order to further determine the role of HAL6 in the heat stress,we constructed a knock-out lines of HAL6 and found that its germination rate significantly reduced under the condition of treatment.(4)In order to dissect the molecular mechanism,we tested the expression of several heat shock transcription factors and heat shock protein HSPs in the overexpression plants and wild type plants.The results showed that with or without heat stress,no differences were observed between this two genetype plants.However,in the mutants hsfa2 and hsp101,the expression of HAL6 was significantly reduced Compared with wild type no matter whether treated with heat shock.Together,these results showed that HAL6 may play roles in the downstream of HSFA2 and HSP101 in plants response to heat stress.(5)In order to excludewhether small peptides play a role in the process of heat resistance,we firstly analyzethe sequence of HAL6 by means of bioinformatics,and find that there is no internal ribosome to enter site(IRES).HAL6 may not encode a functional protein.When the start codon ATG of HAL6 were mutanted to ATT,it did not change plants thermaltolerance.These results suggest that HAL6 mediated heat resistance is not through a small peptide.