Gene Cloning And Functional Analysis Of Superoxide Dismutase During Lettuce Seed Germination

Lettuce(Lactuca sativa L.),an herbaceous plant in the Compositae family and an important vegetable crop,is usually cultivated by seed sowing.It has been shown that the germination of the lettuce seeds requires radicle elongation growth and endosperm weakening.During the processes of radicle elongation growth and endosperm weakening,reactive oxygen species(ROS)play an indispensable role.Superoxide dismutase(SOD)is a kind of enzyme that is closely associated with ROS metabolism.SOD broadly exits in animals,plants and microbes.Currently,the relationship between SOD genes and seed germination is still unknown.In this Thesis,we used the seeds of lettuce as material.We measured the activity of the SOD in radicle and endosperm cap when the seeds are imbibed in water and ABA.We cloned two SOD genes,LsSOD1 and LsSOD2 from the lettuce seeds.We detected the expression levels of LsSOD1 and LsSOD2 in the radicle and endosperm cap of the seeds which were imbibed in water and ABA.In addition,the overexpression vectors were constructed,and genetic transformation was conducted using the agrobacterium-mediated method,thus obtaining the transgenic plants.The main research results are as follows:(1)The activities of SOD in the radicle and endosperm cap of seeds when they respectively imbibed in water and ABA were tested using the nitrotetrazolium blue chloride light reduction method.The results indicated that for the seeds imbibed in water,prior to the time point(15h)when seeds were beginning to complete germination,the SOD activities in the endosperm cap and radicle were both increased.At 15-17 h of imbibition,the SOD activities were slight increases in endosperm cap and radicle.At 17-21 h of imbibition,the SOD activity was still a slight increase in endosperm cap,while the SOD activity in radicle was significantly reduced.For seeds that had imbibed in the 10 mg/L ABA solution,after 13-17 h of imbibition,the SOD activities in the endosperm cap andradicle were reduced.After 17-21 h of imbibition,the SOD activity in the endosperm cap was significantly increased,while the SOD activity in radicle was somewhat reduced.In general,the SOD activities in endosperm cap and radicle at each time points of imbibition in the 10 mg/L ABA solution were all higher than imbibition in water.(2)We used reverse transcription PCR and rapid amplification of cDNA ends techniques,two SOD genes were cloned from the lettuce seeds: LsSOD1 and LsSOD2.(3)We used real-time quantitative PCR to analyze the expression levels of LsSOD1 and LsSOD2 in the endosperm cap and radicle when the seeds were imbibed in water and ABA.The results indicated that the relative expression levels of the two genes in endosperm cap and radicle were different.In the endosperm cap and radicle which had imbibed for 13-15 h in water,the expression levels of LsSOD1 and LsSOD2 were both greatly increased,and during 15h-17 h imbibition,the expression levels of LsSOD1 and LsSOD2 in the endosperm cap and the LsSOD2 in the radicle were greatly reduced,the expression level of LsSOD1 in the radicle had a slight increase,in the endosperm cap and radicle with 17-21 h of imbibition,the expression levels of LsSOD1 and LsSOD2 were somewhat increased.In the endosperm cap and radicle which had imbibed for 13-21 h in 10mg/L ABA,there were small changes in the expression levels of LsSOD1 but at 15 h of imbibition,there was a peak value,and the expression levels of LsSOD2 continuing to drop.(4)We constructed two over-expression vectors with 35 S constitutive promoter:pBI1.4-LsSOD1 and pBI1.4-LsSOD2.Two over-expression vectors with inducible promoter: pER8-LsSOD1 and pER8-LsSOD2.And we obtained positive transgenic plants by using agrobacterium-mediated method.Currently,the transgenic plants are growing.From the changes of enzyme activities and transcription levels,SOD may play a certain roles during the processes of radicle elongation and endosperm weakening during the germination of lettuce seeds.