Studies On Mutant Of Light-harvesting Complex I (LHI) From Thiorhodovibrio Sp.970(Trv)

Photosynthetic bacteria(PSB)are the first prokaryotes to appear in nature and exist in various environments on earth.Its unique photosynthesis system can effectively convert solar energy into chemical energy,and the research on the light harvesting mechanism of photosynthetic bacteria has attracted extensive attention.The Thiorhodovibrio sp.970(Trv)strain in this study was different from other photosynthetic bacteria.The maximum absorption peak of common photosynthetic bacteria LHI-RC was around 880-920 nm,while the maximum absorption peak of Thiorhodovibrio sp.970 strain LHI-RC was around970 nm.By structural analysis,it was found that Thiorhodovibrio sp.970 strain contained two pufBA encoded LHI genes:pufB₁A₁ and pufB₂A₂.The puha and pufbalm of photosynthetic bacteria Rhodospirillum(R.)rubrum H2(R.rubrum H2)were deleted by mutation,which did not have the ability to directly form the cytoplasmic membrane(ICM).However,with the help of the expression vector ppucterm with puha gene,the ICM could still be formed to express the foreign gene,so R.rubrum H2 was selected for heterologous expression.Therefore,in this study,mutations of the amino acids that may bind to Ca²⁺in pufB₁A₁ and pufB₂A₂ genes were carried out to verify whether they had an effect on the characteristic absorption peak of Thiorhodovibrio sp.970 strain.In this study,two genes encoding LHI were mutated and heterologously expressed in Thiorhodovibrio sp.970 strain.pufB₁A₁ and pufB₂A₂ genes were cloned in vitro.Six amino acids of pufB₁A₁ and pufB₂A₂ genes(histidine,aspartate and alanine of pufB₁A₁ gene,histidine,leucine and tryptophan of pufB₂A₂ gene)were mutated by site-directed mutagenesis to construct the recombinant expression vector of ppucterm.Then the mutant recombinant expression vector was introduced into R.rubrum H2 for expression.Finally,UV spectrum analysis and scanning showed that the mutant recombinant strain had different degrees of blue shift.By comparison,it was found that the maximum absorption peak of histidine mutation of pufB₁A₁ did not change,the maximum absorption peak of aspartic acid mutation was blue shift at 9 nm,and the maximum absorption peak of alanine mutation was blue shift at 4 nm.The maximum absorption peaks of pufB₂A₂mutations were blue shifted at 5 nm for leucine,6 nm for tryptophan,and 15 nm for histidine.In this study,the heterologous expression of amino acid mutation associated with Ca²⁺binding of Thiorhodovibrio sp.970 strain was carried out,which is helpful for the study of the effect of LHI light absorption,and provides a reference for the study of LHI structure and protein function.