Studies On The GsPIP1-4 Gene From Wild Soybean (Glycine Soja L.) Enhanced Drought Tolerance In Soybean (Glycine Max L.)

Wild soybean(Glycine soja L.)is the ancestor of cultivated soybean(Glycine max L.),native to East Asia and widely distributed from eastern Russia to southern China,which makes wild soybeans rich in genetic diversity;In the process of domestication and utilization of wild soybean,many beneficial traits of wild soybean have been lost,especially genes related to biotic and abiotic stress resistance.As an important food and oil crop,cultivated soybeans are often subjected to multiple stresses during the growth process to reduce yield.Therefore,research on genes with stress resistance in wild soybeans is of great significance to improve soybean stress tolerance.GsPIP1-4 is a gene of aquaporin family,which is closely related to water transport.In this study,the wild soybean GsPIP1-4 gene was cloned and transferred into the cultivated soybean transgenic receptor Tianlong No.1 by Agrobacterium mediated method to study the effects of GsPIP1-4 gene on physiological and biochemical characteristics of soybean under hydroponic drought conditions.The main findings are as follows:1.Cloning and bioinformatics analysis of GsPIP1-4 gene.The primers were designed by adding Xba1 sites at both ends of the CDS sequence of the GsPIP1-4 gene.After sequencing,BLAST alignment was performed to verify that the complete CDS sequence of the gene was successfully cloned in wild soybeans.The GsPIP1-4 gene was combined with Arabidopsis,Oryza sativa,and Camellia sinensis.Bioinformatics analysis of homology multiple sequence alignment and phylogenetic tree(Figure 2.8)analysis of PIP1-4 genes showed that the sequence of soybean GsPIP1-4 gene is the same as that of cultivated soybean,Oryza sativa and Arabidopsis GsPIP1-4 gene.The source consistency is high,reflecting the high conservation of the protein sequence of the GsPIP1-4 gene(Figure 2.6).By analyzing the conserved domain of GsPIP1-4,it was found that the GsPIP1-4 protein belongs to the MIP family and contains two unique genes of the family.NPA(asparagine-proline-alanine)units and amphiphilic molecular channels(Figure 2.7);through the fusion expression test of GsPIP1-4 and green fluorescent protein GFP(Figure 2.10),the GsPIP1-4 gene was found localized on the cytoplasmic membrane;analysis of its expression pattern by fluorescence quantitative PCR revealed that GsPIP1-4 was higher expressed in wild soybean pods,flowers,and roots,but lower in leaves and stems;analyzed the response of GsPIP1-4 to drought stress,we found that during ABA treatment,its expression in leaves first decreased and then increased;when it was treated with PEG,its expression in leaves first increased and then decreased,indicating that drought stress can induce the expression of GsPIP1-4 gene.The above studies of GsPIP1-4 genes indicate that it belongs to the aquaporin family and may plays an important role in drought stress signaling.2.Analysis of drought resistance physiological function of GsPIP1-4 gene.In order to determine the physiological function of the GsPIP1-4 gene,we cloned this gene from wild soybean and constructed it on the vector p LM-B001 containing the Bar marker gene.Seven strains of T0 GsPIP1-4 transgenic soybean material were obtained through the soybean transformation system using glufosinate as a screening agent.L5,L12 and L15 strains with higher expression levels were used for drought treatment experiments,and wild type(NT)plants were used as controls.Under normal culture conditions,there was no difference in the growth of each transgenic line and non-transgenic lines,but after 20% PEG8000 simulated drought treatment,the reduction in dry weight and root length of the three GsPIP1-4 transgenic lines were less than Non-transgenic plants,and the relative water loss rate of the leaves of L5,L12 and L15 strains is lower than that of non-transgenic plants,in order to reduce water loss in the plant.Analyze the root morphological indicators of each line under normal and drought conditions,non-transgenic plants have more reductions in root length,root surface area,root volume,and number of branches than transgenic lines L5,L12,and L15.Under normal hydroponic conditions,the net photosynthetic rate(AN),transpiration rate(Tr),and stomatal conductance(Gs)of the transgenic lines were slightly higher than those of the control group,After drought,the photosynthetic indicators decreased but the three transgenic lines were still significantly higher than the non-transgenic lines;Under drought stress,the stomata of GsPIP1-4-oe soybean leaves maintained a higher open state than NT,consistent with the changes in stomatal conductance Gs and transpiration rate Tr previously determined;The content of proline and the activities of proline synthesis and degrading enzymes in the leaves of transgenic plants,and the activities of antioxidant enzymes such as SOD,POD,APX,CAT are also higher than NT;In the field environment,GsPIP1-4-oe soybean can increase the yield by increasing the number of branches and pods.In summary,GsPIP1-4 has important functions in regulating water balance and so on.The cultivation of anti-retroviral soybeans not only has scientific theoretical significance,but also has great application value.