Functional Analysis Of Genes Related To The Metabolism Of Reactive Oxygen Species In The Germination Of Lettuce Seeds

Reactive oxygen species（ROS）is a non-enzymatic factor that plays a role in radicle elongation and endosperm weakening during seed germination.NADPH oxidase（respiratory burst oxidase homolog,Rbohs）and peroxidase（POD）is the key enzymes to produce ROS.Previous studies in our laboratory have found that ROS is related to radicle elongation and endosperm weakening during lettuce seed germination.However,the function of genes encoding Rboh and POD during lettuce seed germination remains to be elucidated.In this paper,LsRboh C1 and LsRboh C2 were cloned from the imbibed seeds of lettuce（Lactuca sativa）,and Li Li,a master of our laboratory,cloned Ls POD42,which were almost identical with the sequence of the later published lettuce genome.In order to study the function of three genes during lettuce seed germination,bioinformatics analysis of three genes and their promoter sequences was carried out,and the sequence characteristics and regulation mode of these genes and proteins were predicted and interpreted.Then we constructed the over expression,RNAi and promoter activity analysis expression vectors of the three genes respectively,transformed lettuce cotyledons by Agrobacterium mediated method,obtained the corresponding T₂ generation transgenic seeds,and studied the germination rate,ROS and gene expression level of the transgenic seeds.Finally,q RT-PCR and GUS staining were used to analyze the temporal and spatial expression patterns of the three genes,and the effects of plant hormones,light,temperature and mechanically damage on seed germination and gene expression were studied.The main results are as follows:（1）Screened two LsRboh C of LsRbohs family with high expression during seed germinationNone of the thirteen Rbohs in lettuce predicted the existence of signal peptide,among which seven LsRbohs have 4-5 transmembrane domains.These seven LsRbohs are probably the plasma membrane Rbohs that produce O₂^·-.Semi quantitative RT-PCR analysis showed that only LsRboh C1,LsRboh C2 and LsRboh H（XM＿023882826）were expressed in the seeds,indicating that they may play a role in the process during lettuce seed germination,but the expression level of LsRboh H（XM＿023882826）in the seeds was very low,while the expression level of LsRboh C1 was very high.Conservative domain analysis show that LsRboh C1 and LsRboh C2 contain EF hands,transmembrane domains,FAD/NAD binding and other major conservative domains,indicating that they also have the function of producing superoxide radicals at the plasma membrane.（2）LsRboh C1 may play a role during lettuce seed germinationThe cis-acting elements of the LsRboh C1 gene promoter sequence was predicted and found that the GCN4-motif element involved in endosperm expression,which may suggest that LsRboh C1 plays an important role in the endosperm of lettuce seeds.The temporal and spatial expression patterns of LsRboh C1 were analyzed by semiquantitative RT-PCR,q RT-PCR and GUS staining of promoter linked reporter gene.The results showed that LsRboh C1was expressed in roots,stems,leaves,flowers and seeds,but at a higher level in roots,stems,leaves and seeds,especially in the endosperm cap during seed germination,and the expression in the endosperm cap would follow the imbibition time is extended and shows an upward trend.Most importantly,the overexpression/RNAi of the gene significantly increased/decreased the production of superoxide radicals in radicle and endosperm cap,and significantly promoted/delayed the lettuce seed germination.The results showed that LsRboh C1 plays a role in the lettuce seed germination.（3）LsRboh C2 may affect radicle elongation and root growthThe temporal and spatial expression patterns of the LsRboh C2 were analyzed by semiquantitative RT-PCR,q RT-PCR and GUS staining.The results showed that LsRboh C2was also expressed in roots,stems,leaves,flowers and seeds,but the highest expression was in roots,followed by stems.Besides,it was found that the expression was specific at the radicle tip of germinating seeds,and the expression in radicle would increase with the prolongation of imbibition time.The down regulation of the LsRboh C2 gene significantly reduced the production of superoxide anion free radicals in radicle and endosperm cap,delayed the germination process of lettuce seeds,and the seedlings did not grow roots after germination.The results showed that LsRboh C2 plays a role in the lettuce seed germination,and probably affected the seed germination by affecting the elongation of radicle.（4）Ls POD42 may act on seed germination by affecting endosperm cap weakening and radicle elongationThe q RT-PCR was used to analyze the temporal and spatial expression patterns of Ls POD42.It was found that Ls POD42 was expressed in roots,stems,leaves,flowers and seeds,but the highest expression was found in roots.Ls POD42 promoter also have GCN4-motif,a cis regulatory element involved in endosperm expression,which may suggest that Ls POD42,like Ls POD42,plays an important role in the endosperm of lettuce seeds.The expression level of Ls POD42 was higher in the radicle at the early stage of imbibition,but decreased at the late stage of germination.The expression level in the endosperm cap increased with the extension of imbibition time.The overexpression of this gene significantly increased the production of peroxidase in the endosperm cap and radicle,significantly promoted the germination process of lettuce seeds.However,the down regulation did not significantly reduce the production of peroxidase in the endosperm cap,and had no significant effect on the germination of seeds.The results showed that Ls POD42 may affect germination of lettuce seeds by affecting the endosperm weakening of the endosperm cap.（5）Effects of hormones,light,temperatures and mechanically damage on seed germination and expression of LsRboh C1 and LsRboh C2After the lettuce seeds were treated with ABA/GA,light/darkness,different temperatures,and mechanically damage,it was found that 10μM GA could promote lettuce seed germination,while 10μM ABA seriously inhibited its germination;lettuce seeds germinated faster under light than when there was no light;between 16°C-26°C,The higher the temperature is,the better the seed germination will be,while at 30°C,the seeds germination will be seriously inhibited at the later stage of imbibition,which indicates that the proper increase of temperature is beneficial to the seed germination,but the phenomenon of thermal dormancy will occur if the imbibition time is too long under high temperature;The slight mechanically damage of lettuce seeds can promote its germination.GUS staining was carried out on the endosperm cap or radicle of LsRboh C1pro:GUS and LsRboh C2pro:GUS transgenic lettuce seeds under different conditions.The results showed that the expression of LsRboh C1 and LsRboh C2 could be promoted by 10μM GA,light,proper increase the temperatures and mechanically damage,but inhibited by 10μM ABA.The results showed that LsRboh C1,LsRboh C2 and Ls POD42 affect the lettuce seed germination by regulating the metabolism of ROS,but there are differences in the mechanism:LsRboh C1 mainly affects the weakening of endosperm cap during seed germination,LsRboh C2 mainly affects radicle elongation during seed germination,and Ls POD42 may affect both endosperm cap weakening and radicle elongation during seed germination.In addition,hormones,light,temperatures and mechanically damage not only affect seed germination,but also regulate the expression of LsRboh C1,LsRboh C2 and Ls POD42.