Cloning And Preliminary Function Analysis Of CrHSFA2 In Ponkan

HSF gene plays a key role in plant's resistance in large number of studies.We have found that HSFA2 gene express massively during protoplast isolation and culture in previous gene chip study.It was indicated that HSFA2 has regulation role for regeneration of protoplast.But there is little study on HSA2 function analysis.Based on this,this paper used citrus callus,roots,stems and leaves,cloned a specific gene and analysised its bioinformatics..We construct the over expression vectors of CrHSFA2 and introduced it into Agrobacterium tumefaciens EHA105 and preserved,and completed the transformed callus.We analyzed the transgenic callus protoplast's activity and yield of the,in order to understand the role of CrHSFA2 in Citrus.1.We cloned citrus CrHSFA2 gene.Its sequence length is 2047bp,including a coding 916 amino acid and 1150 bp open reading frame.The protein molecular weight of this gene is 92891.95Da,and isoelectric point is 6.74;It has 99.89 percent of similarity with orange.2.We analyzed the expression pattern of CrHSFA2 by method of real-time PCR.The result show that the expression of CrHSFA2 was decreased significantly at 6hours in the 40 ? in Pokan's roots,stems,leaves and callus.The expression level of CrHSFA2 gene in stems and leaves first rised then declined during the time off,others are showed steady tendency in other issue.The expression of CrHSFA2 in protoplast is higher than callus,during the isolation and culture.We found that the newly isolated protoplast showd hightest experssion,then declined during the culture.3.Several factors affecting transformation of Pokan callus were initially optimized.The highest transformation efficiency was obtained when the explant were infected for 20 minutes at OD600 of 0.6 and co-cultivated in 28?.4.We successfully constructed the gene's over expression vector for pSN1301-CrHSFA2.Then CrHSFA2 introduced into Agrobacterium tumefaciens EHA105 and preserved,and completed the transformed callus.5.we analysed capacity of protoplasts between Pokan transgenic callus and wild type during comparing their yield after isolation and activity in culture during 8 days.we found that the yield of photoprast from transgenic callus is hiher significantly than wild type.And transgenic callus protoplasts and wild type protoplasts both show a high viability.Viability of transgenic callus protoplasts' decreased rapidly during the culturing.The transgenic callus protoplasts' viability decreased either,the higher viability keeps during cultured time.Moreover,transgenic callus protoplasts' viability is more higher than wild type protoplasts and significant difference between two kind of protoplasts.