Transformation Of OsHsp90/BsCspB Gene Into Soybean By Agrobacterium-mediated Method

Soybean is not only a kind of important oil crops,but one significant cash crop,which contains plenty of protein,lipids,minerals,etc.,besides the use of food crop,its straw could also be the nutrient to raise poultry.In recent years,the domestic soybean income has been comparatively small,while the production and earnings of other food crops continue to increase,making the soybean planting area further shrink and the production decrease year by year,while due to the increased demand annually,the supply and demand balance for domestic soybeans have heavily relied on the international market,bringing serious industrial crisis to our nation.The growth phenotype and the yield of soybean crop are easily influenced by the growing environment,and abiotic threats may cause the output reduction or even a total crop failure.As the traditional breeding cycle is pretty long,in order to speed up the cultivation of high quality new varieties,many scientific research workers have applied the complex methods combining the transgenic technology and traditional breeding methods to cultivate new varieties,aiming to make them grow normally under the adversity environments of saline,drought,low and high temperatures.The expansion of cultivated areas with the mass beach salt has helped to increasing the level of the total soybean production,lower the dependence on the international market and safeguard the food security in China.Taking the three soybean varieties of‘Jidou 12',‘Zhechun No.4'and‘Tianlong No.l',we'Ve separately studied the impacts of NaCl(salt)treatment on the seed germination and soybean seedling growth.The seed germination test showed that the low concentration salt treatment could stimulate the germination of soybean seeds;the high concentration salt treatment would obviously inhibit the seed germination,and distinct differences among different varieties exist,with‘Jidou 12,having a good performance.When soybean produces two leaves and one core,treatment and the soybean seeding once with the 100 mmol·L-1NaCl,and repeat one time in two days,then measure the stress-related physiological indexes after one week's treatment.The result has shown that under the salt stress,the soybean's chlorophyll content,root activity and dry weight are all lowered to some extent;the malondialdehyde content,peroxidase activity,superoxide dismutase activity and free proline content all increase.The rise and fall ranges of each indicator of different varieties is all different.Combining the seed germination and seedling tolerance test,here we can make the preliminary judgment that‘Jidou 12'is more salt-resistant than the other two varieties,followed by‘Tianlong No.l'.Taking the soybean variety‘Tianlong No.1'as the recipient material,we use the agrobacterium-mediated genetic transformation of soybean cotyledonary to import the resistance-related genes of OsHsp90 and BsCspB into the soybean genome,aiming to obtain the new soybean germ plasm with the stably inherited salt tolerance and drought resistances.Meanwhile,the reverse PCR technology has been used to make T-DNA integration site analysis of the transgenic soybean plants obtained from the transformation.The main results are as follows:The genetic transformation of OsHsp90/BsCspB gene mediated by the agrobacterium tumefaction:through the agrobacterium tumefaciens-mediated cotyledon method and taking the cotyledon of the soybean variety‘Tianlong No.l'as the recipient material,we'Ve made the soybean genetic transformation via the constructed dual-gene plant expression vector PCambia3301-OsHsp90/BsCspB.After the herbicide screening on the transgenic line J93,the PCR Detection on the screening marker Bar gene,as well as the PCR Detection on the target genes of OsHsp90/BsCspB,here we can initially determine that OsHsp90/BsCspB have be successfully integrated into the soybean genome.T-DNA integration site analysis based on the reverse PCR technology.Make the amplification of the unknown sequences on the soybean chromosomes flanked by the T-DNA integration site of the above-obtained J93 transgenic lines.The sequence alignmentanalysis of any further PCR Detection of the T-DNA integration site has shown that T-DNA region of J93 strain locates in the forward direction at 47,093,689 bp of the 8th chromosome 8 within the soybean genome.Carry out salt toleranceidentification on the transgenic T1 line and make the salt toleranceidentification on the transgenicsoybean strain by using different concentrations of NaCl to simulate different levels of salt stress.The result has shown that there was no significant difference between the transgenic plant and parental plant phenotypes under the 0 mmol · L-1 NaCltreatment,while the chlorophyll content,total fresh weight and total dry weight of the transgenic plants under NaCl stress are all higher than that of the parental plant.The fluorescence quantitative PCR result has shown that under the salt stress,the expression of the target gene displays a certain rising trend as time goes on.The above experimental results have shown that transgenic plants have strong salt tolerance,proving that the two genes of Hsp90 and CspB could improve the salt tolerance of transgenic soybeans.