Studies On The Expression Profile And Transcriptional Regulation Of A Thermal Inducible Gene RCA1 In Rhododendron Hainanense

The resources of Rhododendron species are abundant in China,but its utilization rate is relatively low.The cities’high temperature in summer greatly limits the application and geographical distribution as most of the cultivars are sensitive to heat.Photosynthesis is one of the most sensitive physiological responses of plants to high temperature.The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase（Rubisco）is the key factor determines photosynthesis under heat stress.Rubisco is regulated by Rubisco activase（RCA）.RCA activates Rubisco by removing inhibitory sugar phosphates before Rubisco can fix CO₂.The low activation efficiency of Rubisco under heat stress is the main reason for limiting the photosynthetic rate as RCA is sensitive to heat.Rhododendron hainanense,a subgenus of azaleas,was explored to have a good performance in thermostability compared with other rhododendrons,and was an ideal material for studying heat regulatory mechanisms of Rhododendron.Previous researches on overexpression of RhRCA1 gene in transgenic Arabidopsis thaliana have shown that the function of RhRCA1 gene was associated with heat tolerance.Under this premise,the thermal-inducible regulation mechanism of RhRCA1 was conducted in this study,which contributes to the researches on the regulatory mechanisms of heat stress response in Rhododendron hainanense,and is of great significance for breeding cultivars with improved thermotolerance of Rhododendron.Three-year-old cutting seedlings of Rhododendron hainanense were used as the experimental material to analyze the expression pattern of RhRCA1 gene by qRT-PCR.We isolated the promoter of RhRCA1 gene and respectively transformed it into tobacco and Arabidopsis to investigate its activity,tissue specificity and thermal inducibility.In addition,we found many abiotic stress response cis-elements including RGAANNTTC,a cis-element related to heat response.According to this,we preformed yeast single hybrid and dual luciferase reporter system to further study transcription factors upstream RhRCA1 gene.The main results are as follow:1.In order to analysis the expression profile of RhRCA1 gene,plants were subjected to different pretreatments in light and temperature.We extracted RNA from different tissues and obtained c DNA.The qRT-PCR showed that RhRCA1 gene mainly expressed in green tissues,and the expression of RhRCA1 gene at room temperature was extremely low regardless of light or dark conditions,which implied that the expression of the RhRCA1 gene was tissue-specific and was less affected by light signal.Remarkable high expression of RhRCA1 was observed after high temperature treatment,indicating that the expression of RhRCA1 was significantly up-regulated by high temperature.2.To explore the upstream regulators of RhRCA1,the 1624 bp sequence of RhRCA1promoter was isolated.After analyzing the sequence of the promoter,we found that RhRCA1promoter contained a series of basic elements and cis-elements related to light signals and tissue specificity.The promoter of the RhRCA1 gene is also has a function in regulating JA signaling as relative cis-elements exist.Moreover,a large number of elements related to stress response were found in the promoter,including ARE,ABRE,TC-rich repeats,and RGAANNTTC,CACCT-box,which related to heat stress response.3.For expressional and characteristic analysis of the promoter of RhRCA1,the promoter and GUS fusion expression vector,prRCA1::GUS,was constructed and transformed into tobacco.Staining of the leaves of transformed tobacco showed that high temperature up-regulated the expression of RhRCA1 promoter.Meanwhile,another plant expression vector,pr RCA1::LUC,was constructed using the RhRCA1 promoter to drive Luciferase,and was transformed into tobacco.The results showed that RhRCA1 promoter could strongly respond to heat stress.The vector prRCA1::GUS was also transformed into Arabidopsis.Analysis of T₃generation transgenic Arabidopsis tissues with histochemical staining showed that heat stress had a significant impact on induction of RhRCA1 promoter expression in green tissues,including cotyledon,mature leaf,young stem,sepal and seedpods.These results indicated that the promoter of RhRCA1 is a both heat-inducible and tissue-specific promoter.4.In order to investigate the upstream regulators of RhRCA1,the yeast single-hybrid system was used to screen transcription factors that specifically bind to the cis-elements on the RhRCA1 promoter.A heat response element,RGAANNTTC,whose sequence is highly similar to the HSE（Heat shock element,HSE）.RGAANNTTC was named as HSE-like,and was selected to construct the pAbAi-HSE＿like plasmid and transformed into the yeast cell.Preliminary results prove that heat shock transcription factor HSFA2 can bind to HSE-like element.In order to verify the binding characteristics of HSFA2 and HSE-like,we cloned HSFA2 sequence from c DNA of Rhododendron hainanense and constructed p GADT7-HSFA2vector.The results showed that HSFA2 can bind to the HSE-like element on the RhRCA1promoter.Further study to verify the transcriptional control of prRCA1::LUC regulated by HSFA2,we constructed expression vectors 35S::HSFA2,transformed with prRCA1::LUC into tobacco.The assay of dual luciferase reporter system gave the evidence that HSFA2up-regulated the expression of the reporter gene,indicating the possibility of heat regulating RhRCA1 mediated heat response through HSFA2.