Optimization Of Agrobacterium-Mediated Cotyledon Node Transformation In Soybean And Cloning And Functional Analysis Of A Soybean Aluminum-toxicity Tolerant Candidate Gene GmSTOP1

As an important source of vegetable oils and a high quality protein food,Soybean has always been one of the crops that gets attention from all over the world.In recent years,with the improvement of people’s living standard,the demand for domestic soybean is increasing,while the soybean production by traditional technology could not meet the needs of the domestic market.Genetic engineered soybean varieties including insect resistance,disease resistance,herbicide resistance,and high quality have been shown advantages over traditional varieties including easier farming and management.However,there still exits many outstanding problems in soybean transformation,such as low transformation efficiency,poor stability and poor repeatability,which is much behind the other crops such as rice in terms of transformation efficiency and scale.A stable regeneration system combining with high efficiency transformation methods are necessary to apply genetic engineering in soybean genetic breeding.In this study,several factors during Agrobacterium-mediated cotyledon node transformation,such as Agrobacterium concentrations,germination methods,Agrobacterium suspension culture,co-cultivation time,hormone concentrations during shoot elongation stage,had been optimized to improve the efficiency of infection and shoot elongation,and an improved soybean transformation method with improved transformation efficiency was established.Main results were as follows:（1）The concentration of Agrobacterium tumefaciens is an important factor affecting transformation efficiency.The rate of total GUS transient expression in cotyledon node was 97.0%,90.7%and 85.6%,respectively,when using Agrobacterium with OD₆₅₀ as 0.6,0.8 and 1.0 to infect cotyledonary nodes of Jack-purple soybean variety.The infection ability of Agrobacterium was significantly higher when OD₆₅₀ was 0.6 than 0.8 and 1.0（shortest significant ranges,SSR test P<0.05）.The results of Tianlong 1 variety were consistent with that of Jack-purple.Therefore,using the agrobacterium concentration of OD₆₅₀ as 0.6 could achieve higher infection efficiency.（2）The infection efficiency varied when cotyledon explants were prepared by four different germination methods,including 1-d imbibition,1-d germination,3-d germination and 5-d germination using two soybean varieties of Jack-purple and Tianlong 1.The results showed that the highest rate of total GUS transient expression in cotyledon node were 76.3%and 96.7%in 1-d imbibition using Jack-purple and Tianlong 1,respectively,which were significantly higher than the other three germination methods（SSR test P<0.05）.（3）Low concentrations of antioxidants can prevent cell necrosis and improve the infection efficiency of Agrobacterium.The rate of strong GUS transient expression in cotyledon node after adding DTT in resuspension medium CCM increased 16%and 35%for Jack-purple and Tianlong 1,respectively,compared with the control CCM,and the rate of total GUS transient expression increased by 13%and 3%as well,which indicates that adding DTT in resuspension medium CCM could improve the infection rate.（4）Under the same culturing conditions,there was no significant difference in the infection efficiency at four different co-cultivation time including 3,4,5 and 6 days,therefore 3 days could be used for co-cultivation.（5）There is a synergism of GA3 and IAA on the shoots growth and elongation.The shoot elongation rate of 12.2%was achieved when using the combination of 1.0 mg·L^-1 GA3 and 0.1 mg·L^-1 IAA in the shoot elongation medium.This rate was significantly higher than the other three combinations of different GA3 and IAA concentrations（SSR test P<0.05）,which is an 8.5%increase over the original laboratory protocol using a combination of 0.5 mg·L^-1 GA3 and 0.1 mg·L^-1 IAA.Large areas of red or yellow acidic soils,with a total of 200 million hm2,are widely distributed in South China,which covering 22.7 percent of the total land area and 28 percent of the total cultivated area in China.Aluminum-toxicity is one of the major factors that limits the growth and production of crops and the most important factor that influences the soybean yield in southern China in acidic soils.Therefore,the most effective method to solve the problem of aluminum toxicity in acidic soils are to breed soybean varieties with high aluminum-tolerance in addition to neutralizing the acidity of soil.AtSTOP1 transcription factors can regulate the expression of genes related to aluminum-toxicity tolerance mechanisms,which plays an important role in aluminum-toxicity tolerance in Arabidopsis.We cloned a STOP1-like gene located on chromosome 16 from the aluminum-toxicity tolerant soybean cultivar(Kefeng^-1)using RT-PCR,and designated as GmSTOP1.The length of GmSTOP1 coding DNA sequence was 1566 bp,which encoded 521 amino acids.Diverse cis-acting elements involved in hormone,heat and stress responses were discovered in the 1500 bp upstream region of GmSTOP1,such as ABRE,HSE,TC-rich repeats,and other elements.Protein structure prediction showed that it did not have any signal-peptide or transmembrane region,but contained four conservative Cys-2-His-2 zinc-finger domains.Phylogenetic analysis demonstrated that GmSTOP1 was similar to the putative STOP 1-like protein from Phaseolus vulgaris.Results of subcellular localization showed that GmSTOPl protein located in the cell nucleus.The transcripts of GmSTOP1 were detected in all organs tested including root,shoot apical meristem,stem,leaf,flower,pod and seed,with the highest level in seed.GmSTOPl was up-regulated in soybean roots by 25 μmol L^-1 AICl₃ treatment,and reached the highest relative expression level at 24 hours,which was about 9.2 times of the level in control(0 μmol L^-1AlCl₃).In addition,real-time PCR analysis showed that the expression of GmSTOP1 in soybean leaf and root was also up-regulated by ABA,NaCl and PEG,respectively.Under 15 μmol·L^-1AlCl₃（pH 4.3）treatment,the root lengths of two GmSTOPl overexpressing transgenic Arabidopsis lines were significantly（SSR test P<0.05）greater than the wild type,as well as the relative root growth on AICl₃ vs.pH 5.8 and pH 4.3 vs.pH 5.8.The taproot growth of the wild type and GmSTOP1 overexpression lines were all inhibited after 10 d treatment with different concentrations of NaCl(50,100,150 mmol·L^-1)and mannitol(100,200,300 mmol·L^-1)respectively,but the taproot lengths of GmSTOP1 overexpression lines were significantly（SSR test P<0.05）greater than the wild type.Under the highest concentration of NaCl and mannitol treatment,the average root length of wild-type was 3.8 mm and 9.3 mm,respectively,but the root lengths of the transgenic lines reached more than 6.6 mm and 14 mm,respectively.Combining with the real-time PCR results,we speculated that GmSTOP1 overexpression could improve plant tolerance to aluminum-toxicity,high salinity and osmosis stress.