Construction Of A Plant Experessing Vector Of Small Heat Shock Protein Gene From Solanum Tuberosum And Stress-induced Experssion Analysis

The life cycle of plant can't be in suitable environment conditions completely,abiotic and biotic stress will company the whole life cycle.The heat shock proteins(HSPs),a group of highly conserved proteins in evolution which induce by external stress of the envrionment,can improve the plant's ability to resist stress of environment.It can enhance the ability of plant resistance to adversity conditions and play a very important role in the whole process of evolution of the plants and the environment for a long time.Early transcriptome sequencing results show that expression level of a HSP gene PGSC0003DMG400009255 named sHSF-F of Favorita up-regulated significantly after inoculation of Phytophthora infestans 24 hours.We cloned the gene and constructed the plant expression vector and analysis the sequence of the gene.Real-time fluorescence-based quantitative RT-PCR were carried out of sHSP-F under stress conditions such as P.infestans drought,high temperature,low temperature.The main results as follow:1.Gene cloning and sequence analysis: primers of Favorita's sHSF-F were designed according to the sequences of PGSC0003DMG400009255 gene and the primer 5 'end add the sequence of BamHI and SalI enzyme sites.The sHSP-F gene was obtained by RT-PCR from the RNA of Favorita which had been infected by P.infestans for more than 24 hours.The largest open reading frame(ORF)of sHSP-F is 594 bp and encodes 197 amino acids.The amino acid sequence of the gene was compared to other small heat shock proteins and bioinformatics analysis was conducted.The results showed that sHSF-F contain ACD_Sc Hsp26_like,HSP20 and IbpA three conserved domain.The ACD_Sc Hsp26_like domain is important in heat shock protein which plays an important role in resistance to environment stress.2.Plant expression vector construction and transformation of agrobacterium: the sHSP-F was cloned into expression vector pCAMBIA1301 m.By sequencing and enzyme digestion validation,it shows that sHSP-F gene had been cloned into expression vector which named p1301-sHP-F.Next we transform the p1301-sHSP-F into the agrobacterium LBA4404 by electric shock and heat shock.This work provides the basis for function study of this gene.3.Real-time fluorescence-based quantitative RT-PCR analysis of sHSP-F gene: single element such as the P.infestans,high temperature,low temperature,drought stress were tested,we find the expression level up-regulated apparently 24 and 48 hourses after inoculation P.infestans.During the high temperature stress of 42 ?,the express level of sHSP-F increase significantly after 2 hours,and still up-regulated after 6 hourses,finally the express drop down after 12 hours.While the 4 ?temperature stress,the expression of sHSP-F gradually up-regulated but not obviously;Under the drought stress,compared to the control group,three periods shows no obvious differences of the expression.