The Study On Differential Proteomics And The Interactions With Its Dna Under Pb²⁺and Cd²⁺ Stress For Bruguiera Gymnorhiza（L.） Lam. Leaves Harvested From Hainan

Mangrove is an important plant of coastal wetlands in our country with its unique habitat,diverse species,obvious ecological functions and good social benefits.It has shown great potential for development and utilization in many aspects such as ecological protection and pharmacological activity.In recent years,numerous studies have focused on the extraction and isolation of chemical constituents of mangrove plants and the characterization of their biological activities,there have been few studies on the proteomics or DNA of mangrove plants.Bruguiera gymnorhiza is a kind of medicinal mangrove plant widely distributed in Hainan province.It has many pharmacological activities such as anti-inflammatory,anticancer,anti-tumor,antioxidant and antibacterial,and it is widely used in the research and development of new drugs.From the perspective that modernization of traditional Chinese medicine requires systematic study of phytopharmacological mechanisms,it is necessary to identify and analyze the plant proteins and determine the DNA sequence since there may be proteins with certain pharmacological activities in Bruguiera gymnorhiza.In addition,they are vulnerable to the stress of various heavy metals in order to exert their protective effect on the ecological environment due to the characteristics of the special growth environment of mangrove plants.However,which plant proteins are key in this process,and what are the mechanisms underlying the interaction between heavy metals and DNA?These two scientific questions still remain to be addressed.In this paper,the medicinal mangrove plant of Hainan Bruguiera gymnorhiza was taken as the research object in order to establish the fingerprint of plant protein and identify the main functional proteins.The differential proteomics of leaves of Bruguiera gymnorhiza under heavy metals Pb²⁺and Cd²⁺stress were studied.The mechanism of interaction between Pb²⁺,Cd²⁺and DNA of Bruguiera gymnorhiza were confirmed.The results of this study not only explained the material basis of medicinal plants from the perspective of biological macromolecules combined with the chemical components of small molecules,but also helped to realize the modernization of traditional Chinese medicine of Bruguiera gymnorhiza better.Moreover,we can better explain the mechanism of ecological protection of Bruguiera gymnorhiza from the molecular level by determining the mechanism of Pb²⁺,Cd²⁺and DNA of Bruguiera gymnorhiza.（1）A review was conducted on the properties,application value,and non-biological stress of mangroves,and in conjunction with the significance of this study,the principles,methods,and differential proteomics of proteomics were briefly introduced.The main application technologies of proteomics were also described,as well as the interaction between heavy metal ions and leaves of Bruguiera gymnorhiza DNA.（2）The phenol extraction method under optimized conditions was used in combination with two-dimensional electrophoresis and mass spectrometry to obtain the protein fingerprint of Hainan Bruguiera gymnorhiza leaves at seedling and maturity stages.High-expressed proteins were screened using Image Master^TM 2D Platinum 7.0 analysis software and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS）.The results suggested that there were 60 highly expressed proteins,including differential isomerase,ribulose bisphosphate carboxylase and DNA ligase,among which 42 were identified.There were 55 highly expressed proteins,including aspartate carbamoyl transferase,heat shock protein,ribulose diphosphate carboxylase,among which 34 were identified.（3）Two-dimensional electrophoresis and biomass spectrometry were used to isolate,extract,identify and analyze the differentially expressed proteins in the leaves of Bruguiera gymnorhiza at different periods under heavy metal stress of Pb²⁺and Cd²⁺.The results indicated that there were 4 and 6 differentially stressed proteins of Pb²⁺and Cd²⁺in mature Bruguiera gymnorhiza leaves,and 14 and 5 differentially stressed proteins of Pb²⁺and Cd²⁺in seedling Bruguiera gymnorhiza leaves,respectively.（4）By using bioinformatics technology,the physicochemical properties,multiple sequence alignment,and preliminary functional exploration of normal Bruguiera gymnorhiza leaf proteins and differential proteins under Pb²⁺and Cd²⁺stress at different stages were analyzed by using the protein online databases and the appropriate analysis platforms.The results showed that most of proteins in Bruguiera gymnorhiza leaves belonged to unstable protein types,which were involved in various functions such as growth,development,genetics and plant stress resistance in Bruguiera gymnorhiza leaves.The differential proteins of Bruguiera gymnorhiza leaves in different periods of Pb²⁺and Cd²⁺stress were all related to plant functions such as plant growth,genetics and efficient photosynthesis,and most of them belonged to stable protein types.The four differential proteins had good multiple sequence similarity with their homologs.（5）DNA extracted from the leaf of Bruguiera gymnorhiza was qualitatively and quantitatively analyzed,and the interaction mechanisms between heavy metal ions Pb²⁺and Cd²⁺with Bruguiera gymnorhiza leaf DNA were determined using UV-VIS spectroscopy and fluorescence spectroscopy,respectively.The results showed that Pb²⁺and Cd²⁺could cause the red-shift of the characteristic absorption peak at 210 nm and obvious color enhancement effect of the DNA of Bruguiera gymnorhiza leaves,and lead to the fluorescence intensity at the wavelength of 319 nm increased gradually.The thermodynamic calculations showed that hydrogen bond and van der Waals force are the main types of interaction between Pb²⁺and DNA,while hydrophobic interaction is the main type of interaction between Cd²⁺and DNA.It was preliminarily determined that they interacted with the DNA of Bruguiera gymnorhiza leaf in the pattern of groove binding.