Seeking The Major Genes And The Mechanism Controlling Somatic Embryogenesis In Gossypium Hirsutum

Plant somatic embryogenesis can serve as model systems to study zygote embryo development invitro. Among all the cotton transgenic techonologies, somatic embryogenesis-dependent agrobacteriummediated transformation was widely used. So far, cotton somatic embryogenesis was still very difficμlt,the low differentiation frequency from callus to embryogenic callus was a main limiting factor. Onlyfew varieties have a high differentiation frequency, most of varieties are hard to differentiate or not. Theresult is that transgenic techonology could not be applied in most of cotton varieties. Therefore, thefuture work is to found the key genes controlling callus differentiation and investigate the molecularmechamism. In our study, we utilized agrobacterium-dependent transgenic technology to determine thetarget genes function in controlling embryogenic callus induction. In conclusion, we obtained thefollowing results:(1)Through the analysis of suppression subtractive hybridization data, we found the target genesGhSERK1and GhMAPK16, which had relatively higher expression level in embryogenic callus. Thenwe ulitized electronic cloning and genome data to obtain the CDS sequence.we construct theover-expression vector to transform CRI24, the result was that GhSERK1had a higher expression levelin embryogenic callus, which shorten the process of callus developing into embryogenic callus and hadno significant influence on the differentiation frequency.while GhMAPK16did not had a higherexpression level in embryogenic callus, its overexpression had no effects on callus differentiation.(2) AtWuschel could promote embryogenic callus formation in Arabidopsis, we constructedAtWuschel overexpression vector to transform CRI12which was hard to differentiate. As a result, thedifferentiation frequency of callus was increased from0.61to47.75percent. To investigate themolecular mechanism, we took some experiments. Finally we found that it was possible to activate theauxin signal and cytokinin pathway to induce the downstream gene expression. These downstreamgenes including GhLEC1, GhLEC2and GhFUS3were closely related with somatic embryogenesis. Toinvestigate the function of GhLEC1, GhLEC2and GhFUS3, we constructed the interference vector tosilence these three genes and then transformed the interference vector to CRI24, the result was that thedifferentiation ability of CRI24was seriously inhibited.(3)In order to obtain the key genes controlling somatic embryogenesis, we constructed ahomogenization full-length cDNA library from callus to embryogenic callus. It lays a foundation fortransforming the full-length cDNA library to cotton and obtaining the key target genes controllingsomatic embryogenesis.In conclusion, we found that AtWuschel could increase callus differentiation frequency. Genesinvolved in auxin signal pathway, cytokinin signal pathway and the dowenstream genes GhLEC1,GhLEC2and GhFUS3had differential expression level between CRI24and CRI12, CRI41, Lu28.CRI24had a high differentiation frequency, while CRI12, CRI41and Lu28were hard to differentiate.According to the mentioned above, AtWuschel may regμlate the genes involved in auxin and cytokinin signal pathway and the downstream genes to increase the differentiation frequency of CRI12. Theseresults showed that genes involved in auxin and cytokinin signal pathway and the downstream genesGhLEC1, GhLEC2and GhFUS3may play critical roles in controlling callus differentiation.