Cloning And Functional Validation Of The Photoprotective Gene GmRIQ1

The soybean（Glycine max（L.）Merr）is one of the important crop in China.During the growth,They are exposed in the complex and variable environments which affects the normal growth,and even leads the production to decline.So,using the transgenic technology to cultivate soybean varieties with high-yielding,stable-yielding,high-quality with strong stress resistance has became the key target of soybean breeding.The RIQ genes are localized in Arabidopsis thaliana,,they are sharing a conserved domain DUF1118,and the amino acid sequence of the RIQ genes all have two transmembrane domains.It is predicted to have a transport peptide structure localized in chloroplasts at the N-terminal region.RIQ genes are known to have the ability to improve the plant photoprotection,promote the transformation of plants to non-photochemical quenching pathway,and improve the stress resistance of plants by quenching the excess light energy of plants by heat energy.Using the AtRIQ1 gene sequence to find the GmRIQ1 gene in the soybean genome database,the CDS full-length sequence of the GmRIQ1 gene was 585 bp.Constructing the pCAMBIA1302-GmRIQ1 expression vector to find the subcellular localization of the GmRIQ1gene;Constructing the pCAMBIA3301-GmRIQ1 expression vector to transform soybean varieties Dongnong 50 and Arabidopsis thaliana wild-type,obtaining 3 Arabidopsis thaliana overexpression strains of GmRIQ1 gene and 3 trans-GmRIQ1 soybean strains.The Arabidopsis thaliana overexpression strains of GmRIQ1 gene,Arabidopsis thaliana deletion mutants of GmRIQ1 gene and wild-type Arabidopsis thaliana and trans-GmRIQ1 soybean strains were treated with different photointensities,and the relationships between photosynthetic pigments,chlorophyll fluorescence coefficients,photosynthetic coefficients and other indexes related to photosynthesis mechanism and photoprotective photoinhibition regulation mechanism were compared and analyzed.Through the analysis of soluble sugar,the stress resistance of soybean strains of trans-GmRIQ1 gene was preliminarily identified,and it was determined that soybean GmRIQ1gene was related to the regulatory mechanism of photoprotection.This paper lays a theoretical foundation for researching the regulation mechanism of crop light protection and cultivating soybean varieties with high-yielding,stable-yielding,high-quality with strong stress resistance The main findings are as follows:1.The GmRIQ1 gene was successfully obtained by homologous cloning.Through bioinformatics prediction of the GmRIQ1 gene,the CDS sequence is 585bp,encoding 194 amino acids.The bioinformatics gene is located on chromosome 6,belongs to membrane protein and has high similarity with Glycine soja.2.Analyzing the gene-specific expression in soybean,the highest expression in leaves.3.Constructing the pCAMBIA1302-GmRIQ1 expression vector,and PEG-Ca²⁺-mediated t-ransformation,the location of GFP fluorescence signal was determined the RIQ1 gene’s pr-otein was located in the chloroplast of cells,and the GmRIQ1 gene was related to photop-rotection.4.Constructing the pCAMBIA1302-GmRIQ1 expression vector to transform into the wild type Arabidopsis thaliana and soybean varieties Dongnong 50.There are 3 Arabidopsis thaliana overexpression strains of GmRIQ1 gene（GmRIQ1-a）and 3 trans-GmRIQ1 soybean strains were obtained.5.Different light intensity has different effects on photosynthetic pigments,chlorophyll fluorescence,photosynthetic characteristics,etc.With the increase of light intensity,photosynthetic pigment,NPQ,photosynthetic rate（Pn）and stomatal conductance（Gs）were positively correlated with light intensity,among which Arabidopsis thaliana strains:GmRIQ1-a（GmRIQ1 Arabidopsis thaliana expression）>WT（wild type）>GmRIQ1-g（GmRIQ1 Arabidopsis thaliana deletion mutant）;soybean strains:GmRIQ1-a（GmRIQ1 gene soybean strain）>CK（Dongnong 50）;The intercellular carbon dioxide concentration（Ci）was inversely correlated with the light intensity,among which Arabidopsis thaliana strains:GmRIQ1-g>WT>GmRIQ1-a;soybean strains:CK>GmRIQ1-a,preliminarily showed the GmRIQ1 gene can improve the non-photochemical quenching ability of plants,inhibit the photosynthesis of plants,and improve the photoprotection ability of plants.6.Discussing the different effects of different light quality wavelengths on the photosynthetic pigments and non-photochemical quenching of different Arabidopsis thaliana lines.Under the treatment of red light and far-red light wavelengths,the photosynthetic pigment-s of the three Arabidopsis thaliana lines have different effects.There is little difference be-tween the pigment level and the chlorophyll fluorescence coefficient level;but the chlorophyll content under blue light:GmRIQ1-g>WT>GmRIQ1-a;and the zeaxanthin content:GmRIQ1-g>WT>GmRIQ1-a;NPQ coefficient:GmRIQ1-a>WT>GmRIQ1-g,these indicators indicate that the expression of GmRIQ1gene is more active under the blue light.7.Measuring the soluble sugar content of GmRIQ1-a under the strong light irradiation,the results show that the level of soluble sugar content:GmRIQ1-a is higher than WT,it was preliminarily judged that the stress resistance of the trans-GmRIQ1 soybean strains was higher than that of CK Dongnong 50.