Cloning And Genetic Transformation Of Pansy Heat Shock Transcription Factor Gene VtHSFB2b

Pansy is a famous herbaceous flower in the world,it is known as "the queen of flower beds".It endures cold temperature and lives cool climate well,the suitable growth temperature is 14~25 ?.The high temperature in summer has an adverse effect on the growth and development of pansy,leading to a decrease in ornamental value.Long-term high temperature stress may even cause plant death.The molecular regulation mechanism of abiotic stress tolerance depends on the activation and regulation of specific related genes.Plant heat stress transcription factor(HSF)plays a vital role in response to high temperature stress and conferring stress tolerance.In this experiment,the pansy inbred line DFM-16 with good heat resistance and the heat sensitive inbred line 08 H were used as research materials,Using RACE technology to isolate VtHSFB2 b gene from leaves,the sequence structure of the two materials and the characteristics of the expression level of high temperature response were compared and analyzed,and the function of the gene was studied by subcellular localization and genetic transformation of Arabidopsis.Below are key research findings:1.The cDNA sequence of the VtHSFB2 b gene in DFM-16 material is 1439 bp,and the cDNA sequence of the VtHSFB2 b gene in 08 H material is 1397 bp.The open reading frames of the VtHSFB2 b gene in both materials have the same length of 975 bp,but there are differences of 24 bases.The 324 amino acids encoded by the two predicted that there were correspondingly 6 amino acid differences,and the amino acid sequence similarity reached 98.15%.VtHSFB2 b contains a conserved DBD domain of HSF.Compared with all members of the model plant Arabidopsis HSF,it has the highest homology and closest relationship with Arabidopsis HSFB2 b.Biological information analysis also shows that it has the same characteristics as heat shock transcription factors.2.Using real-time PCR technology to analyze the expression of VtHSFB2 b,the expression pattern of VtHSFB2 b gene in the leaves of the two materials was similar,and both were induced by high temperature stress.The expression level of VtHSFB2 b began to rise at 0.5 h of 42 ? heat shock treatment,reached the peak at 1 h,and the expression level of DFM-16 material was much higher than that of 08 H material.Subsequently,the expression level of the two decreased significantly at 2 h.At 12 h,the expression of DFM-16 material decreased to the same level as the control,but the expression level of 08 H material was still higher than that of the control.3.The analysis of transient expression in onion epidermal cells showed that VtHSFB2 b was located in the nucleus,which was consistent with the results predicted by online software,further indicating that the gene encoding protein may play a role in transcriptional regulation.Using pJIBIA163-1300 as a vector and VtHSFB2 b gene cDNA as a target gene fragment,the VtHSFB2b-pJIBIA163-1300 plant expression vector was constructed and transferred into Arabidopsis,and transgenic Arabidopsis T1 generation plants were successfully screened.