Research On The Function Of Apoplast Calmodulin In The Growth And Development Of Plant By Using Transgenic Arabidopsis

The results obtained in our laboratory in the past decade years showed, apoplast calmodulin in plant kingdom may regulate a lot of growth and development process of plant, such as accelerating the proliferation of Angelica dahurica suspension cells and the proplast cell regeneration,startup the pollen germination of many plants' pollen and accelerating the elongation of the pollen tube,stimulating the redox of corn root' s cell, inducing the expression of light independent rbsS gene, and participating in the regulation of the restraining function of Al³⁺ to pollen tube germination. But most of these results are obtained in vitro by means of physiological and pharmaceutical approach.To get in vivo evidences that apoplast calmodulin con 1d regulate plant growth and development process, a chimeric secretion form of calmodulin binding peptide, which contains a signal peptide, a calmodulin binding domain and a C-myc epitope was constructed. The chimeric gene was introduced into Arabidopsis . It was expected that the overexpression of this chimeric protein could be secreted into cell wall and bound to apoplast calmodulin, which could reduce the apoplast calmoduin concentration to make an apoplast camodulin "antisense" plant.By observing the potential phenotype change of apoplast calmodulin "antisense" plant,the in vivo function of apoplast calmodulin on plant growth and developmental process could be speculated. To guide the CaM-binding peptide into apoplast space, the signal peptide from Brassica Campetris pollen coat protein SLR1æ¡žPs was employed. It has total 26 amino acid, and is a very classic form of signal peptide. To neutralize the function of CaM, the CaM binding domain peptide from bovine calcineurin A subunit was chosen because the pre-experiment demonstrated that bovine calcineurin could bind to calmodulin strongely in an acidic and high calcium concentration condition which is similar to the cell wall environment.In order to increase the binding ability of the chimeric secreted calmodulin binding peptide, the plasmid containing two copies of calmodulin binding domain were constructed. Finally, the chimeric gene was inserted into binary Ti vector pLBJ21,and cloned into Agrobacterium tumefaciensGV3101 by electroporation. The chimeric gene was introduced into Arabidopsis through the method of vacuum infiltration which is widely used in Arobidopsis tansformation . Transgenic plants were screened by means of kanamycin resistance and further identified by genomicPCR and Western blotting, Through the phenotype observation of transgenic plants, the speculation that apoplast calmodulin may play a certain role in regulating the development and growth of plants was made. More research work are still going on.