Chlorophyll Fluorescence Characteristics And PAO Gene Family Analysis Of TILLING Mutants In Wheat

As an important biochemical process of plants,photosynthesis provides material basis and energy source for nature,and plays an important role in the global carbon cycle and other material cycles.As an effective probe for studying plant photosynthesis,the technology of chlorophyll fluorescence(CF)is considered a measure of apparent photosynthesis,a powerful tool for evaluating the impact of plant stress,and an effective way to obtain plant information without damaging.It can be used as an indicative and predictive tool for the management of plant physiological stress and recovery.As the most widely cultivated crop in the world,wheat is China’s main food source and plays an important role in ensuring global food and nutrition security.In the genome era,with the development of modern molecular genetic technology,genomics research has become the core of contemporary wheat genetic research.Combined with bioinformatics,plant phenoomics will accelerate the research of functional genomics and provide data support for the optimization of wheat germplasm resources.In this study,chlorophyll fluorescence characteristics of wild type plant Jimai 22 were analyzed to clarify its photochemical characteristics and provide preliminary analysis for the photosynthetic analysis of mutant.In the whole exon sequencing database of the TILLING mutant population of Jimai 22,the mutants of chlorophyll-related genes and mutant plants with significant phenotypic differences from the wild type were searched,and their phenotypes were classified and analyzed.The chlorophyll stable fluorescence parameters of different mutant strains were determined,and fast light curves were drawn to construct an efficient and high-throughput screening system for photosynthetic mutants.At the same time,based on wheat genome and transcriptome information,using bioinformatics and molecular biology research methods,the Ta PAO gene family of wheat demagnesia chlorophyllin oxygenase was identified,analyzed and verified.The main results are as follows:1.Analysis of photosynthetic characteristics of Ji Mai 22Chlorophyll fluorescence parameter technology,rapid fluorescence kinetics technology and chlorophyll fluorescence imaging technology were used to determine the photosynthetic characteristics of Ji Mai 22 at different leaf ages.It was found that the correlation between the potential photosynthetic capacity and the activity of PSⅡ was significantly increased during leaf development,and the non-radiative energy dissipation pathway mainly based on lutein cycle began to be established and played a role.The defense system was gradually improved,and the photosynthetic apparatus was gradually mature and stable.The effects of aging on chlorophyll fluorescence characteristics were analyzed by using the method of dark induced aging.The results showed that with the increase of dark treatment days,the electron transfer rate and photochemical efficiency of PSⅡ reaction center would decrease.Although the degree of photoinhibition increased gradually in the first three days,the leaves still operated efficiently and played the role of photoprotection depending on the energy dissipation pathway.The differences of PSI and PSII energy distribution among flag leaves,young leaves,functional leaves and functional leaves were systematically analyzed.In general,no matter in photosystem Ⅱand photosystem Ⅰ,electron transfer rate,actual quantum yield and photosynthetic activity were in the order of flag leaf > functional leaf,mature leaf > young leaf,and electron transfer rate of PSII in different leaves.The actual quantum yield and photosynthetic activity are slightly lower than PSI,PSI is sensitive to changes in environmental conditions,and is less affected.The electron transfer rate of leaves through photosystem Ⅱ increases with the increase of light intensity.Young leaves are sensitive to light,and their tolerance to strong light is weak.2.Analysis of PAO gene family Bioinformatics and its expression pattern in wheatThe PAO gene family members in wheat genome were identified and analyzed by bioinformatics.A total of 19 Ta PAO gene family members were identified.These Ta PAOs proteins are hydrophilic proteins,mainly located in plastids and distributed in chloroplasts and other organelles.The phylogenetic analysis showed that the homology relationship between wheat and rice was closer in the evolutionary process.Gene structure analysis showed that the structure of closely related genes was basically consistent with the conserved domain,with 9 conserved Motif3 as the characteristic motif.The cis-acting elements indicated that PAO gene family played a major role in the light response of wheat,and played a secondary role in the regulation of wheat growth and development,stress resistance and hormone response.Transcriptional data from different tissues showed that Traes CS5D02G0466800 and Traes CS3B02G574300 genes were tissue-specific,and Traes CS4D02G309000 and Traes CS4B02G311100 were expressed constitutively.The results of q RT-PCR analysis showed that the expression levels of the three subfamily genes reached the maximum at 6 h after the exogenous application of jasmonic acid in 2-week-old wheat seedlings,and Traes CS4A02G411000 had the greatest change under the dark induced senescence treatment in 2-week-old wheat leaves.The results of this study can provide information basis for further study of biological functions of wheat PAO gene family.3.Screening of TILLING mutants and their Photosynthetic characteristicsBased on the Jimai 22 TILLING mutant library constructed in the laboratory,200 mutant plants with obvious phenotypic differences from the wild type were observed and recorded,and their phenotypes were classified and photosynthetic characteristics were analyzed.The results showed that the photosynthetic efficiency of the leaf yellow plant was lower than that of the other mutant types,and the difference between the leaf yellow plant and the wild type was the largest.It can be used as the key object for studying photosynthetic mutants.The electron transfer ability of different mutant strains was short > whole leaf yellowing > spiral > narrow leaf.Using the 20 J TILLING mutant library constructed earlier in the laboratory,500 mutants with chlorophyll variation phenotype were selected as target materials to determine the slow dynamic curve,and the frequency of stable fluorescence values of Fv/Fm,Y(II),q P and ETR in the mutant library were analyzed.The results showed that most mutant strains had weaker photosynthetic capacity than the wild type.Photosynthetic efficiency and electron transport rate were improved by EMS mutagenesis in only a few mutant strains.The values of different fluorescence parameters of different TILLING mutant strains were compared with those of the wild type,and the chlorophyll fluorescence parameter database was constructed,which laid the foundation for the subsequent efficient and high-throughput screening of mutants.