Molecular Cloning And Application Of LBDs From Cotton(Gossypium Arboreum L.)

Cotton,as a kind of the most important commercial and oil crops in the world,plays an important role in national economy.A mature,stable and efficient regeneration system of cotton somatic cell is the basis of transgenic breeding.Somatic embryogenesis is widely used for transgenic cotton regeneration.Cotton somatic cells are firstly dedifferentiated into callus,and then embryogenic callus,then subsequent globular、heart、torpedo、cotyledonary embryo.In the end an entire plant is regenerated.This process is regulated mainly by auxin and cytokinins.Previous researches suggest that the early callus induction of dedifferentiation process is closely correlated to the ability of embryo formation.Cells in the process of dedifferentiation can probably obatin the information of regeneration.Therefore dedifferentiation determines a cell’s fate,finally influcing embryogeniccallus regeneration.Cell dedifferentiation,a very complicated process,is associated with the expression of many genes.The transcription factor LBD(the lateral organ boundaries domain),which is found exclusively in plants,encodes a highly conserved LOB domain.It may play an important role in regulating the development of de novo root organogenesis.Recent researches shows that LBD gene families are activated in the late stage of lateral root and adventitious root initiation,and callus formation may share similar regulatory mechanisms with adventitious root formation.LBD are recently reported to be important in regulating callus formation.However,the possible function of LBD in cotton has not been reported.We isolated five genes of LBD family from cotton(Gossypium arboreum L.)by using homology cloning techniques and the main results are as follows:1.The cloning and bioinformatics analysis of Ga LBDsBased on the amino acid sequence alignment of At LBD16,At LBD17,At LBD18 and At LBD29 of with cotton AA genome database,five genes with the highest similarity were chosen to performe the bioinformatics analysis,including sequences analysis,multiple sequence alignment and phylogenetic tree construction.The result indicated that the five genes belonged to LBD family and were named Ga LBD-1,Ga LBD-2,Ga LBD-3,Ga LBD-4,and Ga LBD-5.2.Expression pattern analysis of Ga LBDs genesThe results of q RT-PCR showed that the expression level of Ga LBD-1 、Ga LBD-2、Ga LBD-3、Ga LBD-4、Ga LBD-5 had dramatic and rapid increase with the processing time extension on CIM medium induction.Most Ga LBDs had a rapid increase after 72 h CIM induction.Expression level of Ga LBDs in embryogenic callus(EC)had a sifnificant increase after 72 h CIM induction.The expression level of Ga LBD-3 was to increase to 300 times after 72 h CIM induction.This result is similar to expression patterns of At LBD genes family in arabidopsis thaliana.LBD16,LBD17,LBD18 and LBD29 of arabidopsis thaliana are dramatically and rapidly induced on CIM.Overexpression of LBD genes is sufficient to trigger callus formation without exogenous phytohormones,LBD-triggered callus formation resembles callus induction on CIM by ectopic activation of the lateral root development pathway,and suppression of LBD function inhibits callus formation.3.Genetic transformation and functional verification of Ga LBD1～5To determine the function of Ga LBD1～5 during the callus initiation,the expression vectors p BI121-Ga LBDs were constructed and transformed cotton via Agrobacterium mediated reforming process.The results of this research were that Ga LBDs improved callus growth significantly and the over-expression of each of these five genes in cotton was sufficient to trigger callus formation without supplied phytohormones.Histopathological study also showed that over-expressed Ga LBDs could promote callus formation in 7 days after agrobacterium transformation.These results indicated that the lateral organ boundaries domain(LBD)transcription factors were very important in regulating callus formation.4.Research on Ga LBD2 genes as safe marker genes of independent screening plant transgenic cellsGa LBDs genes are transcription factors,which playing key roles in callus formation stage of cotton tissue culture.We did a systematic research on the Ga LBD in cotton(Gossypium arboreum L.)and developed an application technology system of positive screening.We found that when Ga LBDs were over-expressed,the transformed cotton cells were dedifferentiated into callus without supplied phytohormones,while the differentiation and proliferation of nontransformed cells without Ga LBDs were very slow and showed distinct phenotype.Based on the phenotype,normal callus tissues would be selected to be developed further.Then positive transgenic plants would be achieved.