Cloning And Salt Tolerance Analysis Of Polyamine Oxidase Gene GmPAO1 In Soybean

Soybean is an important oil crop and food crop in China.In recent years,the occurrence frequency of abiotic stress such as drought and salinization increases year by year,which seriously affects the quality and yield of soybean.Salinization is a serious problem in salinization of saline-alkali land in China.Studies have shown that the Polyamine oxidase（PAO）gene family plays an important role in improving salt tolerance in cotton,cucumber and other plants,but the functional verification of The PAO gene in soybean has been rarely reported.In the early stage,Gm PAO1 gene was screened by transcriptome sequencing,and the gene was cloned and its gene function was analyzed by RT-PCR technology.The main results are as follows:1.The target gene Gm PAO1 was cloned from Jinong 18 mutant M18 with an open reading frame of 582bp and encoding 193 amino acids.Fluorescence quantitative PCR results showed that Gm PAO1 gene was expressed in soybean roots,stems,leaves and germinating seeds.Under PEG-6000 and Na Cl solution stress,Gm PAO1 gene expression level increased first and then decreased,suggesting that Gm PAO1 gene could respond to drought and salt stress and was up-regulated.2.The plant overexpression vector p CAMBIA3301-Gm PAO1 and gene editing vector PCBSG015-Gm PAO1 were constructed.The expression vector was transformed into soybean receptor Jinong74 by agrobacterium-mediated method and pollen tube channel method,and 3 T₀overexpression positive plants and 3 CRISPR/Cas9 positive plants were obtained.After subgeneration,10 T₁ overexpression positive plants,8 CRISPR/Cas9 positive plants,19 T₂overexpression positive plants,and 14 CRISPR/Cas9 positive plants were obtained.3.Sequencing analysis of T₂generation gene editing plants showed that four lines were edited,with one base missing in target 1 of KO1,one base replacing in target 2 of KO2,one base replacing in target 1 and target 2 of KO3,and one base inserting in target 2 of KO4.4.Southern blot analysis showed that Gm PAO1 gene was mainly integrated into the T₂transgenic plant genome in a single copy mode,but the integration sites were different.The results of q RT-PCR showed the relative expression level of Gm PAO1 gene in T₂generation overexpressed plants was significantly higher than that in control plants,and the relative expression level of Gm PAO1 gene in T₂generation gene-edited plants was significantly lower than that in control plants.5.The T₂-generation positive soybean seeds were treated with Na Cl stress,and the results showed that Gm PAO1 overexpression increased the germination rate,germination potential,germination index and vigor index of seeds under salt stress,and also promoted the growth of soybean side roots.The positive plants at T₂ generation seedling stage were treated with different concentrations of Na Cl stress.The results showed that the overexpressed plants had developed roots,total root length,root volume and other related indexes were significantly higher than the control plants.In addition,the relative water content,chlorophyll content,SOD activity and POD activity of the overexpressed plants were significantly higher than the control plants.The content of MDA and H₂O₂ was significantly lower than that of the control lines,and the overexpression lines improved the salt tolerance of soybean.