Functional Study Of At5g52970 Gene And Map-based Cloning Of Yellow Heart Mutant In Arabidopsis Thaliana

Photosynthesis is the basis of most life on earth,and it takes place in organelles called chloroplasts.There are an increasing number of studies on the functions of chloroplast in particular in electron transfer,water splitting,assembly and repair of PSⅡ（Photosystem Ⅱ）,etc.Chloroplast immunophilin CYP38（Cyclophilin 38）is involved in the assembly and sustenance of PSⅡ,but its mechanism is unclear.To identify the interaction proteins of CYP38,yeast two-hybrid assay was performed to screen the thylakoid lumen Mini-library,we found that the thylakoid lumen protein At5g52970 can interact with CYP38 in yeast.This thesis attempts to figure out the function of At5g52970 gene to explore whether it can assist CYP38 to involve in the assembly of PSⅡ.Firstly,the C-terminal of CYP38 interaction between At5g52970 was confirmed by one-to-one yeast two-hybrid,and the transient expression At5g52970 in tobacco and chloroplast stratification experiments confirmed that At5g52970 was localized in the thylakoid lumen of chloroplast.At5g52970 gene was expressed in roots,stems,flowers,young leaves and old leaves,but relatively high expression in young leaves.Secondly,we obtained the T-DNA insertion mutant of this gene through PCR identification and screening,which was confirmed to be a null mutant by the RNA and protein level.Based on the information provide on the NCBI（National Center for Biotechnology Information）website,we predicted that At5g52970 contains a phosphatase domain,which may have phosphatase activity.TLP18.3（Thylakoid lumen protein 18.3）is known as an acid phosphatase which located in the thylakoid lumen,we speculated that At5g52970 may have similar functions.To clarify its function,we constructed at5g52970×tlp18.3 and at5g52970×cyp38-2 double mutants,respectively.The results showed that the phenotypes of at5g52970,tlp18.3 mutants and at5g52970×tlp18.3 double mutant have no significantly different from Col under normal light,while at5g52970×cyp38-2 has similar phenotypes with cyp38-2,both plants have phenotypes such as dwarf,slow development,which indicated that At5g52970 functions may be reflected in other aspects;at the same time,the phenotype of the mutant is not significantly different with Col under fluctuating light conditions,which indicated that at5g52970 mutant is not sensitive to light.In order to explore the function of the At5g52970 gene,we transformed the p35S::52970CDS-GFP vector into the at5g52970 mutant and Col,and found several yellow plants in the T₁ generation,one of which was numbered as C5.Western Blot analysis showed that the protein level of At5g52970 in yellow plants was higher than Col.PCR detection showed that the yellow plant might be caused by the p35S::52970CDS-GFP vector.For verification,no yellow plants appeared after reuse of pro52970::52970g DNA and p35S::52970CDS vectors without GFP were transformed and screened respectively,western blot results showed that the protein level of transgenic positive plants of p35S::52970CDS was significantly higher than Col and p35S::52970CDS-GFP overexpressing yellow plants C5,which indicated that the yellow phenotype of C5 should be caused by the p35S::52970CDS-GFP vector.In order to further analyze the function of At5g52970,we measured the physiological and biochemical indexes of At5g52970.The phosphorylation level of at5g52970 mutant was slightly stronger than Col in vivo.And the F’v/F’m of at5g52970 mutant was higher than Col,while NPQ（Non-photochemical quenching）was lower than Col,indicating that the photosynthetic efficiency of at5g52970 mutant was slightly higher than Col.The BN-PAGE（Blue Native-polyacrylamide gel electrophoresis）analysis showed that the at5g52970 mutant causes slightly more aggregation of the LHCⅡ complex but slight lower of PSI M and PSⅡ D compared with Col,indicating that the photosynthetic efficiency of at5g52970 mutant was affected.At the same time,we obtained a yellow heart mutant plant yh（Yellow Heart,yh）in the processing of screening of tlp18.3 mutant.The DNA level identified that it still had the insertion of tlp18.3 T-DNA,after the tlp18.3 T-DNA was removed,the yh mutant still had the yellow heart phenotype,it was speculated that the yellow heart phenotype was caused by a new gene mutation.Genetic analysis showed that it was a single-gene recessive inheritance.We performed gene mapping of yh mutant by map-based cloning technology,the results showed that the mutant was located between INS1＿55＿342 and INS1＿56＿34 molecular markers on the first chromosome,and the interval size was 676kb.It was confirmed that the mutant may be an allele mutant of At1g64790 through whole genome sequencing.The results of m RNA level identification showed that the mutant band was smaller and weaker than Col,and it was further confirmed by sequencing that the base of yh mutant lacked 459 bp.Although the yellow heart phenotype of yh mutant was similar to that of At1g64790 weak mutant ila,their leaf edge shapes are different,indicating that yh mutant had certain functions in the development of chloroplasts and leaf traits,which provides genetic materials for subsequent leaf traits research.