Cloning And Expression Analysis Of Small Heat Shock Protein Genes Of Pansy

Viola tricolor L.is a world-renowned flower bed flower with rich colors,long flowering and cold tolerance.However,the pansy are jealous and hot,and it is difficult to adapt to the high temperature and climatic conditions in summer in China,which makes it difficult to raise seedlings in summer and shorten the viewing period.The discovery of heat-resistant genes is important for understanding the thermal response mechanism.Heat shock protein(HSP)is an important component of the heat shock protein,and small molecule heat shock protein(sHSP)is one of the most important proteins in the heat shock protein family.In this experiment,the transcriptome data of heat-resistant treatment of DFM-16 at 42 °C for 1 h and 2 h was used as the background.In order to study the expression of different materials in other time periods,according to the transcriptome data,A preliminary analysis of the trichrome small molecule heat shock protein gene family was carried out to further understand the structure and function of the trichrome small molecule heat shock protein gene.The research results are as follows:1.Based on the analysis results of heat stress transcriptional group of pansy,15 shsp genes were screened.Real-time quantitative PCR was used to analyze the expression of 15 sHSP genes in two materials at 42 ℃for 1 h,2 h,6 h,12 h.The results showed that 15 sHSP genes were induced by heat stress and were pansy thermal response gene.The transcript level of the heat-resistant material DFM-16 under heat shock was mainly concentrated at 1 h and 12 h compared with the control,and there was no significant drop trend at 12 h.The highest level of transcription of the thermosensitive material 08 H under heat shock compared with the control was mainly concentrated at 6 h.Most of the genes showed significant regression at 12 h.Therefore,DFM-16 has a shorter and more rapid accumulation of sHSP gene under heat shock,and the sustained expression level is longer than that in 08 H.Therefore,we can speculate that DFM-16 is more heat-resistant than 08 H,possibly by sHSP gene.The effect of heat shock protein transcription levels.Only VtHSP17-2 and VtHSP22-1 have the same expression pattern of the same gene in different varieties.The expression patterns of the other 13 genes in different pansy materials were different.This indicates that the expression of the pansy small molecule heat shock protein family gene is highly specific in different plant materials,which is consistent with previous studies on other species.Bioinformatics analysis showed that 11 can access the complete open reading frame(ORF),which belongs to the hydrophilic protein non-secreted protein and has multiple phosphorylation sites.Six(VtHSP17.6,VtHSP17-1,VtHSP17-2,VtHSP17.8,VtHSP17.9-1,VtHSP17.9-2 and VtHSP26.4-1)belong to the Cytoplasmic family,two(VtHSP24,VtHSP26.3)belong to the Chloroplast family,two(vthsp13,vthsp26.4-1)belong to the Mitochondria family.2.Three sHSP genes(VtHSP18,VtHSP13.2,VtHSP17.5)were cloned from the heat-resistant inbred line DFM-16 by RACE technique,and one sHSP gene(H-VtHSP17.6)was cloned from the thermosensitive material 08 H.The homologous gene similarity rate of VtHSP17.5and H-VtHSP17.6 was 94.56%.VtHSP18 and VtHSP13.2 and their homologous sequences were not cloned in 08 H,which may be caused by mutation of the promoter or transcription factor.The two genes were not expressed in 08 H or the expression level was very low,so we speculated that VtHSP18 and VtHSP13.2 play an important role in the thermoresponsive mechanism.All fourproteins are hydrophilic non-secreted protein proteins containing multiple phosphorylation sites.Phylogenetic analysis of the sHSP gene family revealed that Pansy VtHSP13.2 belongs to the cytoplasmic VII(CVII)subclass;VtHSP18 belongs to the cytoplasmic I subclass(CI);VtHSP17.5,H-VtHSP17.6 chloroplast family(CP).According to the results of functional phylogenetic tree,it is speculated that VtHSP18,VtHSP17.5 and H-VtHSP17.6 also have the function of responding to high temperature stress,which can improve the heat tolerance of plants.VtHSP13.2 has multiple inductions,in addition to improving the high temperature tolerance of plants,it can also Improve plant resistance to low temperature and saline.3.The overexpression expression vector of VtHSP18 was constructed by double enzyme digestion using pJIBIA163-1300 vector,which laid a foundation for further study of VtHSP18 syndrome of small heat shock protein of pansy...