Functional Verification Of Soybean GmRIQ2 Gene

Soybean is an important economic crop,a source of high-quality protein and commercial materials that people need every day,and plays a pivotal role in agriculture.However,at present,the total output of soybeans in China isn't extremely satisfying the daily demand for soybeans and their soybean products.It is necessary to import soybeans from abroad to maintain domestic supply demand.Soybeans are sensitive plants,which are susceptible to abiotic stresses such as high light,high temperature,drought,and salinity,and then affects the quality and yield of soybeans.Therefore,accelerating the cultivation of high-quality soybeans that are resistant to high light and high temperature,is of great significance for increasing the total yield of soybeans.In the early stage,the GmRIQ2 gene of soybean gene sequence was found and cloned in the soybean genome database based on the Arabidopsis AtRIQ2 gene sequence.Sequence analysis of the GmRIQ2 gene revealed that this gene was involved in the photoprotective mechanism of photosynthesis and was related to the non-photochemical quenching pathway.By constructing an expression vector,GmRIQ2 gene overexpressing plants?GmRIQ2-a?,GmRIQ2 gene RNAi interfering plants?GmRIQ2-RNAi?and GmRIQ2 gene deletion mutant plants?GmRIQ2-g?were obtained.Based on the obtained T₃ transgenic plants,the subcellular localization of the genes was identified and PCR molecular identification was performed.Positive plants of the GmRIQ2 gene overexpressing plants,the GmRIQ2 gene RNAi interfering plants and the GmRIQ2 gene deletion mutant plants and wild-type plants were tested for different light intensity,the relationship of related indicators of photoprotective mechanisms such as photosynthetic pigments?chlorophyll and zeaxanthin?,non-photochemical quenching,photosynthetic rate,transpiration rate,stomatal conductance and intercellular carbon dioxide concentration were compared.The influence of GmRIQ2gene expression on plant yield was analyzed by agronomic traits;the structure of GmRIQ2gene-encoded protein and the cis-acting element of upstream promoter were analyzed by bioinformatics,and the promoter activity was determined by transient expression.The promoter was predicted to affect the expression of GmRIQ2 gene,and it was clear that the soybean GmRIQ2 gene was a photoprotective-related gene.In order to study the regulation mechanism of plants light protection,it was a theoretical foundation for cultivating high-yield and high-quality soybean varieties with high light tolerance.The main results were as follows:1.Construction of pCAMBIA1302-GmRIQ2 recombinant vector,transfected into Arabidopsis protoplasts by polyethylene glycol?PEG?-Ca2⁺-mediated transformation,and gene was located in the chloroplast of the cell by observing the location of GFP fluorescence signal.It was preliminarily speculated that soybean GmRIQ2 gene was related to light protection.2.By cloning and activity analysis of the upstream promoter of GmRIQ2 gene,it was found that the upstream promoter of the gene contains ACE,ATCT-motif,Box 4?ATTAAT?and CATT-motif and other inducible promoter elements related to light response.It is presumed that the GmRIQ2 gene promoter is an inducible promoter.The transient expression of Agrobacterium rhizogenes transformed soybeans demonstrated its activity and the activity of the CaMV35S constitutive promoter was higher.3.73 plants of GmRIQ2-a were obtained from T₂ transgenic plants,23 plants were obtained from GmRIQ2-RNAi,and 66 plants were obtained from GmRIQ2-g by PCR identification of bar primers and 35S-bar primers.T₃ generation positive transgenic plants were obtained.Among them,18 plants of GmRIQ2-a,16 plants of GmRIQ2-RNAi,and 34plants of GmRIQ2-g were obtained.4.Different light intensities have different effects on photosynthetic pigments,non-photochemical quenching and photosynthetic parameters of different plants.With the increase of light intensity,the chlorophyll and zeaxanthin of the four plants increased,while the GmRIQ2-RNAi and GmRIQ2-g plants were always higher than the CK plants,and GmRIQ2-a was always lower than the CK plants.This indicates that the GmRIQ2 gene has the ability to reduce photosynthetic pigments.There was no obvious regularity of initial fluorescence?F0?of the four lines,which proved that the expression of GmRIQ2 gene had no effect on F0.The maximum photochemical quantum yield of photosystem II of the four lines decreased with increasing light intensity,indicating that the GmRIQ2 gene increased the light dissipation capacity of the plants.The non-photochemical quenching coefficients NPQ and qN of GmRIQ2-a were higher than GmRIQ2-RNAi,GmRIQ2-g and control plants,GmRIQ2-RNAi and GmRIQ2-g were lower than control plants,indicating that GmRIQ2-a gene can enhance non-photochemical quenching of plants.The photosynthetic rate?Pn?,transpiration rate?Tr?and stomatal conductance?Gs?increased with the increase of light intensity in four lines and GmRIQ2-RNAi and GmRIQ2-g>CK>GmRIQ2-a.and intercellular carbon dioxide concentration?Ci?is the opposite which decrease with increasing light intensity,and GmRIQ2-a>CK>GmRIQ2-RNAi and GmRIQ2-g.5.Through the analysis of the relationship between agronomic traits and yield traits,GmRIQ2 gene overexpressing plants were lower than GmRIQ2-RNAi interference and GmRIQ2 gene deletion mutant plants in plant height,number of main stem segments,number of pods per plant,number of seeds per plant and 100-grain weight,and the number of main stem segments was lower than that of the control plants.The results preliminary showed that the soybean GmRIQ2 gene is a negative production regulatory factor.