Cloning And Functional Analysis Of Heat Stress Memory Key Genes HabZIP60 And HaHsfA5 In Haloxylon Ammodendron Seedlings

Haloxylon ammodendron is a small xerophyte of Chenopodiaceae,which can adapt to heat stress and drought and other harsh environments and plays a significant role in maintaining the stability of ecological structure.Our previous study found Unigene sequences annotated as b ZIP60 and Hsf A5 from transcriptome analysis of H.ammodendron seedlings under heat stress,and named them as HabZIP60 and HaHsfA5.In this study,the cloning of HabZIP60 gene and its functional analysis in stress response,the functional analysis of HaHsfA5 gene in stress response,and the cloning and functional analysis of promoter were studied.The results were as follows:（1）The HabZIP60 gene was successfully cloned.The HabZIP60 m RNA was cut under the induction of dithiothreitol（DTT）,and there were two forms of uncut（U）and cut（S）.The subcellular localization results showed that HabZIP60 U protein was mainly located in the endoplasmic reticulum,and HabZIP60 S protein was mainly located in the nucleus.The one-month-old H.ammodendron seedlings were subjected to abiotic stress.Through real-time fluorescence quantitative analysis,it was found that the expression of HabZIP60 and its m RNA shear were not only induced by DTT,ABA,dehydration and Na Cl stress,but also had heat stress memory characteristics.It is speculated that HabZIP60 may play an important regulatory role in endoplasmic reticulum stress response induced by abiotic stress.Transgenic Arabidopsis showed that overexpression of HabZIP60 U increased salt tolerance and heat tolerance of Arabidopsis seeds,but no heat stress memory was found.The tolerance of Arabidopsis seeds to osmotic stress and ABA was not improved.Overexpression of HabZIP60 U increased the survival rate of Arabidopsis seedlings after secondary heat stress,suggesting that HabZIP60 U is involved in heat stress memory in seedling stage.Overexpression of HabZIP60 U reduced the sensitivity of Arabidopsis seedlings to osmotic stress,but did not reduce the sensitivity to salt.Under normal growth conditions,overexpression of HabZIP60 U had no significant effect on seed germination rate,rosette leaf number and plant height of Arabidopsis thaliana.（2）The plant expression vector of HaHsfA5 gene was constructed and transformed into A.thaliana.Overexpression of HaHsfA5 played a positive regulatory role in the early germination of Arabidopsis seeds,and enhanced the tolerance of Arabidopsis seeds to heat,salt and osmotic stress.Over-expression of HaHsfA5 increased the survival rate of transgenic lines at seedling stage after secondary heat stress,and also improved the heat resistance of transgenic Arabidopsis at adult stage,indicating that HaHsfA5 played a positive regulatory role in the adaptability to heat stress at seedling stage and adult stage.In addition,overexpression of HaHsfA5 reduced the sensitivity of Arabidopsis seedlings to salt stress and ABA,but it did not reduce the sensitivity of Arabidopsis seedlings to osmotic treatment.Under normal gro WTh conditions,overexpression of HaHsfA5 had no significant effect on root length,number of rosette leaves,plant height and water loss of Arabidopsis seedlings.（3）The promoter sequences upstream of 5’end of HabZIP60 and HaHsfA5 genes in H.ammodendron were successfully cloned by chromosome walking method,and the lengths were 1049 bp and 887 bp,respectively.Bioinformatics prediction analysis showed that in addition to the basic promoter TATA-box and CAAT-box and other specific core elements,many cis-acting elements related to stress were also found.The vector p CAMBIA1304-HabZIP60::GUS and p CAMBIA1304-HaHsfA5::GUS fused with GUS reporter gene were constructed,and p CAMBIA1304-HabZIP60::GUS was transformed into A.thaliana.The positive plants of T₁ generation were preliminarily screened.In conclusion,the b ZIP transcription factor gene HabZIP60 is involved in the endoplasmic reticulum stress induced by abiotic stress,and overexpression changes the response characteristics of transgenic Arabidopsis to high temperature,dehydration,Na Cl and other stresses.The overexpression of heat shock transcription factor gene HaHsfA5 improves the tolerance of transgenic Arabidopsis to high temperature and other abiotic stresses.The promoters of HabZIP60 and HaHsfA5 contain a variety of stress responsive cis-elements,which indicate that HabZIP60 and HaHsfA5 are important regulators in response to abiotic stress.The analysis of DNA methylation of HabZIP60 promoter by BSP showed that heat stress exercise reduced the methylation rate of HabZIP60 promoter under secondary heat stress,indicating that HabZIP60had the expression characteristics of heat stress memory genes.