Identification Of Soybean SPS Gene Family And Preliminary Functional Verification Of GmSPSA-2 In Roots

Root is an important organ in the process of plant growth and development,the accumulation of plant root biomass and its growth and development are closely related to sugar metabolism.SPS is a key rate-limiting enzyme regulating sucrose synthesis and starch decomposition,and plays an important role in the distribution of photosynthates between starch and sucrose.In plants,SPS catalyzes UDPG and F6 P to form S6 P,which is then catalyzed by SPP to form sucrose.Previous studies have shown that Previous studies have shown that SPS genes are divided into four subfamilies A,B,C and D,and members of the SPS gene family are Previous studies have shown that SPS genes are divided into four subfamilies A,B,C and D,and members of the SPS gene family are spatio-temporal specific in plants.But at present,the research on the SPS gene family in soybean is not deep enough,therefore in this study,6 members of soybean SPS gene family were identified by bioinformatics analysis,and their phylogenetic analysis and gene structure characteristics were analyzed.The Gm SPSA-2 gene,a member of soybean SPS gene family with the highest expression in soybean root,was screened by gene expression pattern prediction and spatio-temporal expression analysis.The gene was cloned to construct overexpression vector and transformed into Arabidopsis thaliana to obtain T1 generation transgenic positive Arabidopsis thaliana plants,and overexpressed Gm SPSA-2 hairy roots were obtained by infecting soybean,and determining its phenotype to verify the effect of Gm SPSA-2 gene on Arabidopsis thaliana and soybean roots.The specific results are as follows:1.Six members of SPS gene family were identified in soybean,and they were divided into A,B and C subfamilies by phylogenetic analysis.LOC100127429(Glyma.1G161600)and LOC100798787(Glyma.1G109700)are members of subfamily A,named Gm SPSA-1 and Gm SPSA-2 respectively,which are located on chromosomes 13 and 17 respectively.LOC100806543(Glyma.1G029100),LOC100813168(Glyma.0G308600)and LOC100807527(Glyma.1G108100)are members of subfamily B,named Gm SPSB-1,Gm SPSB-2 and Gm SPSB-3 respectively,and they are located on chromosomes 14,8 and 18 respectively.LOC100786531(Glyma.0G323700),a member of C subfamily,named Gm SPSC,is located on chromosome 6.Collinear analysis showed that there was a collinear relationship between Gm SPSA-1 and Gm SPSA-2,a collinear relationship between Gm SPSB-1,Gm SPSB-2 and Gm SPSB-3,and no collinearity relationship between Gm SPSC.The number of exons of soybean SPS gene family members is 12-14,and the distribution of conserved motifs is similar.Except for Gm SPSA-2,there are a large number of cis-acting elements in the promoter region of all members of soybean SPS gene family.2.The spatio-temporal expression of soybean SPS gene family members was analyzed,and the results showed that the temporal and spatial expression of soybean SPS gene family members was specific.The gene with the highest relative expression in the root is Gm SPSA-2,whitch was relatively high in the reproductive growth period;The gene with the highest relative expression in stem and pod was Gm SPSC,which was relatively high in reproductive growth period.The gene with the highest relative expression in leaves,flowers and grains was Gm SPSB-1,which was relatively high in the reproductive growth period.3.PEarley Gate-Gm SPSA-2 plant overexpression vector was constructed and transformed into col wild type Arabidopsis thaliana plants.Quantitative analysis and phenotypic determination of T1 transgenic positive Arabidopsis thaliana and negative control Arabidopsis thaliana,the results show that: The expression of the target gene in the roots of overexpressing Gm SPSA-2 positive Arabidopsis thaliana was 6.54-8.38 times that of the negative control,its root length was 1.24-1.7times that of the negative control,its root weight was 1.5-1.67 times that of the negative control,and its starch consumption capacity was 3.1-4.1 times higher than that of the negative control.The overexpression vector was used to transform the hairy root of Dongnong 50.Quantitative analysis and phenotypic determination of Gm SPSA-2 positive soybean hairy root and negative control hairy root,the results showed that: The expression of target gene in soybean overexpressing Gm SPSA-2positive hairy root was 15.1-27.6 times higher than that in negative control hairy root,and its starch consumption capacity was 1.41-1.47 times higher than that in negative control hairy root.The above results suggest that Gm SPSA-2 gene can regulate starch consumption,thus affecting the growth and development of plant roots and the accumulation of biomass.