Genome-wide Identification Of HSF Family In Longan (Dimocarpus Longan Lour.) And Expression Analysis In Response To Heat Stress

Longan?Dimocarpus longan Lour.?,an important economic fruit tree in China,is of edible and medicinal values.The fruit quality and yield are closely related to the quality of longan embryo development.Temperature is an important factor affecting the embryo development In facility cultivation.Hence,it is necessary for the longan industry development to study the influence of heat stress on the embryo development of longan.Heat shock transcription factor?HSF?is one of the most important regulatory factors involved in heat stress.In this study,embryogenic callus?EC?was used as material to observe the cell morphology and determine the proliferation rate and cell viability of longan EC under different heat treatment.At the same time,the cell of longan early somatic embryo at different stages was selected as the research object to measure the physiological,biochemical and metabolic indexes.On this basis,the whole genome identification,bioinformatics analysis of the longan DlHSF family and the codon usage preference analysis of all members of the family were carried out,and the expression patterns under growth and stress were studied.In this research,DlHsfA2,which is an important gene in response of the embryo development and the heat stress.Cloning,vector construction and transient expression studies were performed on one of these important member DlHsfA2.The main findings are as follows:?1?Cell morphology were observed,cell growth and cell viability were determined by heat stress?30?,35?and 40??treatment of longan EC.The results showed that as the temperature increases,cell color showed a trend of deepening,graining more and more serious,the texture is more tight,40?temperature extremes cells show that when the rough,some cells appear deformity,extreme high temperature will affect the cell growth and morphological changes.The amount of cell growth showed a trend of“first rise and then fall”,longan cell grows most under 35?,it shows the best growth rate at 35?,but almost without growing at 40?.In addition,under heat stress,the viability value showed a downward trendency with temperature increases,which is the lowest at 40?.At the same time,the physiological and biochemical index were determined at development stage of different embryogenic cells in longan.It was found that the content of MDA,Pro and SOD enzyme activity with temperature rise,peak at 40?.Compared with the25°C control,the MDA,Pro content and SOD activity of the cells in the globular embryo?GE?and embryo development stage?EC?were higher at 40°C.However,the cells at different developmental stages at 30°C and 35°C were higher than the control,but the increase was small,indicating that heat stress had different effects on the physiological,biochemical and metabolic processes of different developmental stages of somatic embryos.?2?20 DlHSF family members were identified from the longan genome.Bioinformatics analysis indicated that they contain a typical DBD conserved binding domain?DBD?,and they were all unstable protein without a signal peptide.The promoter contains heat stress,light and other response elements related to stress and endosperm,leaf morphology and other growth factors,indicating that DlHSF may be involved in a variety of stress and growth processes.Phylogenetic tree analysis indicated that HSF can be divided into three categories:DlHsfA,DlHsfB and DlHsfC.The results of real-time fluorescent quantitative PCR?qRT-PCR?showed that DlHSF may play a major role in EC and GE stages during the development of longan somatic embryos.Expression level analysis of different tissues and organs found that DlHSF may be related to the growth process of longan flower development.?3?Further the bias characteristics analysis of the DlHSF family codon usage revealed that the codon preference of the DlHSF family members when coding amino acids is relatively weak as a whole,and codon A/T?U?had stronger selection bias than G/C,and preferred to end with A/T?U?.The optimal codons for this family are 12 such as CUU and CAA.The codon bias of the DlHSF family is mainly affected by the mutational stress.Compared with the frequency of codon usage of model organisms such as Arabidopsis thaliana,Nicotiana sylvestris and Solanum lycopersicum.Eukaryotic expression system is more suitable as a heterologous expression system of this family,while Arabidopsis thaliana and Nicotiana sylvestris could be the ideal receptors for the genetic transformation of the family.?4?Different stress treatments are applied to the longan EC.qRT-PCR result showed that DlHsfA1d and DlHsfA8 have a higher expression.While,there are higher expression of DlHsfA4d,DlHsfA6b and DlHsfA8 in salt stress.The result suggestion that these genes may play a crucial part in these stress process.Under heat stress,the expression of DlHSF reached the highest at 40°C,it is the most intense response to heat stress.On this basis,we continue to use DlHSF as the research object and study the expression level of DlHSF family at 40?in different time?0,1,2,4,6,12 and 24 h?.qRT-PCR showed that DlHSF family members had different degrees of extraordinany expression at 0,1,2,4 and 6 h,but most of them had the highest expression at 2 h.Therefore,we treated langan cell for 2 h under heat stress at 40°C after 2h SA,ABA,Ca²⁺and H₂O₂ treatment.The results showed that the up-regulation of DlHSF gene expression was most obvious under the treatment of ABA and H₂O₂ after heat stress,indicating that among the molecular signal transduction pathways under heat stress,DlHSF mainly plays a role in H₂O₂ and ABA signal transduction pathways under heat stress.?5?To explore the function of DlHsfA2,the DlHsfA2 sequence was obtained by cloning.Sequence alignment showed that the ORF length of DlHsfA2 was 1 125 bp,and the number of encoded amino acids was 374aa.The evolutionary relationship indicates that DlHsfA2 is closely related to citrus CsHsfA2 and has the longest relationship with rice.At the same time,p1302-DlHsfA2-GFPlocalizationexpressionvectorand p1301-DlHsfA2-GUS overexpression vector were constructed.The results showed that DlHsfA2 detected green fluorescence in the nucleus,indicating that DlHsfA2 plays a vital in the nucleus,which is a nuclear protein,consistent with the prediction of WoLF PSORT.Transient transformation of p1301-DlHsfA2-GUS in longan EC revealed that the GUS reporter gene and the hygromycin gene were detected in the positive control and overexpression materials in the transgenic EC compared with the non-transgenic gene.Moreover,the qRT-PCR results showed that the expression level of the GUS reporter gene in the overexpressed EC was significantly increased compared with the positive control.