| Pansy was a perennial herbaceous ornamental flower of the genus Amaranthus,but most of the Pansy cultivars was not resistant to high temperatures,which seriously restricts the application of pansy.Therefore,the application of the heat-resistant mechanism of pansy to the pansy was studied.And the breeding of new heat-resistant varieties was of great significance.Based on the Pansy heat shock transcriptome database,16 VtHSP70 genes with significant differences in expression under high temperature conditions were screened.In this study,the heat-resistant pansy material DFM-16 and the heat-sensitive material 08 H were studied.The research contents are as follows:(1)The DNA fragments of 16 VtHSP70 genes were verified by PCR,and 14 genes which were consistent with the transcriptome results were obtained.The expression levels of 14 VtHSP70 genes in DFM-16 and 08 H were analyzed by real-time PCR at room temperature and heat shock at 42 ? for 1 h,2 h,6 h and 12 h.The results showed that the expression levels of 8 genes(VtHSP70-2,VtHSP70-4,VtHSP70-5,VtHSP70-8,VtHSP70-11,VtHSP70-14,VtHSP70-15,VtHSP70-16)were increased with the increase of heat stress time.The heat-resistant pansy material DFM-16 has always been higher than the heat-sensitive material 08 H.Among them,the VtHSP70-16 gene was not induced by high temperature in 08 H material,but the expression level was significantly increased under heat stress in DFM-16 material;Four genes(VtHSP70-3,VtHSP70-6,VtHSP70-9,VtHSP70-13)were transiently higher than the DFM-16 material in the 08 H material.From the degree of gene-induced expression after heat stress,the expression level of VtHSP70-6 gene reached the highest in two materials during heat shock treatment for 2 h.However,the DFM-16 reached 6554.2 times of the control,and the 08 H material reached 4513.0 times of the control.The expression level of VtHSP70-15 gene in heat treatment of 08 H material was 0.36 times of that of thecontrol,and it was 17.75 times of control of DFM-16 material.It is shown that the expression level of VtHSP70 gene was positively correlated with the heat tolerance of pansy.(2)The full-length sequence of 14 VtHSP70 genes was cloned by RACE technology,and the full length of three genes was obtained,which were VtHSP70-4,VtHSP70-8 and VtHSP70-16.The full length of VtHSP70-8 gene in DFM-16 and 08 H,the sequence length was 2275 bp and 2346 bp,respectively;The full length of VtHSP70-4 gene in 08 H material,the sequence length was 1749 bp;And the full length of VtHSP70-16 gene in DFM-16 and 08 H materials,the sequence length is 2042 bp.Bioinformatics analysis showed that the proteins predicted by the three genes were all hydrophilic proteins;The similarity of the amino acid sequence predicted by the three genes was 74.24 %,indicating that the VtHSP70 gene was highly conserved.(3)The cDNA and gDNA of VtHSP70-16 gene were amplified by PCR.Sequence analysis showed that the length of VtHSP70-16 cDNA in both materials was the same as that of open reading frame,encoding 649 amino acids,and the amino acid sequence similarity was 98.61 %.The putative amino acid sequence contains a characteristic sequence EEVD of cytoplasmic HSP70,and phylogenetic tree analysis also confirmed that VtHSP70-16 was localized in the cytoplasm.In addition,the VtHSP70-16 gDNA structures in the two materials are similar,both containing an intron,but the intron sequence has a 9 bp base difference.(4)The promoter sequence of VtHSP70-16 gene was cloned by genomic walking method.Sequence analysis showed that the VtHSP70 promoter sequence in the two materials had a large difference,and the similarity was 34.20 %.Component prediction analysis showed that the reinforcing elements in the DFM-16 material were 21 more than the 08 H material,indicating that the VtHSP70 gene promoter plays an important role in the heat resistance of the pansy. |
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