| High temperature,pathogens and other adversity stresses are the major adverse factors that affect the growth and development of plants and the yield of plant crops.In order to improve the ability of plant resistance or tolerance to the stresses and increase the yield of crops,it has been one of the most important subjects of the modern agriculture study using gene engineering to cultivate the stress tolerance crops,which will be benefited by a better understanding of molecular mechanism underlying the plant tolerance and resistance to the stresses.In the present study,we isolated and functionally characterized a thermotolerance-related gene CaHPR1,and a defense-related gene,CaPHD1.The main results are as followings:1.The full-length cDNA of CaHPR1 and CaPHD1 were isolated from the pepper normalized cDNA library.By sequencing,it turned out that the CaHPR1 encodes a protein of 386 amino acids with a predicted molecule mass of 42.27 KDa,CaPHD1 encodes a protein of 220 amino acids with a predicted molecule mass of 25.31 KDa.Their corresponding promoters of CaHPR1 and CaPHD1 were also cloned and the cis-elements possibly responsible for stress responsiveness were analyzed,and various stress or pathogen responsive cis-elements were found to be present in the two promoters,indicating CaHPR1 and CaPHD1 might be involved in the adversity stress response of pepper.2.By fusing to green fluorescent protein(GFP)reporter gene and transient expression in Nicotiana benthamiana,CaHPR1 was targeted both cytomembrane and nuclei,while CaPHD1 was targeted exclusively in the nuclei.3.The CaHPR1 and CaPHD1 transcriptional expression were analyzed by quantitative real-time polymerase chain reaction(qPCR)in the pepper plant challenged with Ralstonia solanacearum inoculation or heat stress treatment or exogenous applied salicylic acid(SA),methyl jasmonic acid(MeJA),abscisic acid(ABA)and ethephon(ETH).The results showed that CaHPR1 was transcriptionally up-regulated to different degree by exogenous phytohormone applications of ABA and high temperature stress treatment(HT).On the other hand,CaPHD1 was transcriptionally up-regulated to different degree by exogenous phytohormones applications of SA,MeJA,ABA and ETH as well as by inoculation of Ralstonia solanacearum.All these results suggest that CaHPR1 might be involved in pepper’s response to heat stress and ABA signaling,while CaPDHl might be involved in response to inoculation of Ralstonia solanacearum via signaling synergistically mediated by SA/jasmonic acid(JA)or ethylene(ET).4.To further test the role of CaHPR1 in pepper thermotolerance,CaHPRl overexpressing transgenic tobacco plants were developed,and the results showed that the thermotolerance in CaHPR1 over-expressing transgenic tobacco had significantly enhanced,companied with significantly transcriptional upregulation of thermotolerance-related genes(smallNtHSP、NtHSF2、NtHSP18、NtHSP70、NtHSP90 and NtHSP101)and reactive oxygen species(ROS)detoxification associated genes(NtAPX1、NtAPX2 and NtCAT1)compared to that in the control plants.By contrast,the thermotolerance in CaHPR1 silencing pepper by virus-induced-gene-silencing(VIGS)was significantly decreased coupled with significant downregulation of thermotolerance-related genes(CaHSP24、ER-smallCaHSP and Chl-smallCaHSP).This result strongly suggests CaHPR1 play an important role in thermotolerance of pepper.5.Our data showed that CaPHD1 silencing significantly increased susceptivity of pepper plants to Ralstonia solanacearum Inoculation,accompanied with down-regulated defense-related genes(CaNPR1、CaDEF1、CaACC oxidase、CaABR1、CaPRl and CaHIR1).By contrast,the transient overexpression of in CaPHD1 pepper leaves triggered significant HR cell death and upregulation of the tested immunity-associated marker genes.These data suggest that CaPHD1 act as positive regulator in pepper immunity against Ralstonia solanacearum infection. |
PDF Full Text Request