Functional Analysis Of Zl-PINs Gene Of Zizania Latifolia

The swollen stem of Zizania latifolia could be induced by the infection of Ustilago esculenta,following with the inhibition of tip growth of stem,and the development of flower organs and the lignification and differentiation of stem cell,which would be related to the change of IAA content in stem regulated by IAA polar transport.As the important carrier of the polar transport of IAA,PIN1 played an important role in plant growth and development.In this research,the ZL-PINs family genes related to IAA polar transport were cloned in Z.latifolia,and their expression in different tissues of Z.latifolia were compared.The regulation effect by the infection of U.esculenta on Zl-PINs gene was clarified in the stem of Z.latifolia.The swollen pattern of Zl-PINs gene was clarified during the process of stem development.The ZlPIN1 c gene which maybe play an important role in the development of swollen stem in Z.latifolia was screened.Based on the Immunofluorescence hybridization analysis with At PIN1 antibody,the location and expression of Zl-PIN1 protein was observed in the stem and fibrous root cell membrane bottom of Z.latifolia.The over-expression genetic transformation vector of Zl-PIN1 s were constructed.The subcellular localization of transient expression in tobacco further clarified the localization of ZlPIN1 c in cell membrane and endoplasmic reticulum.The transgenic lines of Arabidopsis with over-expression of Zl-PIN1 a,Zl-PIN1 b and Zl-PIN1 c were constructed and screened.Combined with exogenous IAA treatment,the promoting effect of Zl-PIN1 c over-expression on the growth and development of fibrous roots and stems of Arabidopsis was clarified,and the biological functional phenotypic differences of three Zl-PIN1 s genes in Arabidopsis were compared and analyzed.The theoretical basis of IAA regulation during swolling stem of Z.latifolia and the subsequent analysis of IAA regulation during swolling stem of Z.latifolia were provided.The main results are as follows:1.Full length cloning of Zl-PINs genes in Z.latifoliaThe full-length sequences of Zl-PIN2,Zl-PIN3 a,Zl-PIN4 and Zl-PIN9 genes of Z.latifolia were cloned,which had the unique domain of auxin membrane transporterMem_trans Domain.The full length of Zl-PIN2 c DNA was 1554 bp,encoding 517 amino acids.It was closely related to Oryza brachyantha with the amino acid sequence similarity of 90.52%.The full length of Zl-PIN3 a c DNA WAS 1743 bp,encoding 580 amino acids.It was closely related to Oryza brachyantha with the amino acid sequence similarity of 82.42%.The Zl-PIN4 c DNA had a length of 1149 bp and encodes 382 amino acids.It was closely related to Triticum urartu,and the amino acid sequence similaritywas 70.50%.The Zl-PIN9 c DNA had a length of 1287 bp and encodes 428 amino acids with.It was closely related to Triticum dicoccoides with the amino acid sequence similarity of 72.39%.2.Expression pattern of Zl-PINs gene in Z.latifoliaThe expression analysis of Z.latifolia among different plant tissues showed that the ZL-PINs was mainly expressed in the stem and root,but low in the leaf and leaf sheath.Zl-PIN1 a,Zl-PIN1 b and Zl-PIN1 c were identified as the main genes expressed in the stem.The expression of Zl-PIN1 c gene was the highest in the stem,which was significantly higher than that of other Zl-PIN genes(P < 0.05).It would be speculated that the Zl-PIN1 c could be an important gene related to the growth and development of stem in Z.latifolia.The Zl-PIN1 s gene expression in the stem of Z.latifolia was significantly inhibited by the infection of U.esculenta,which could be related to the inhibition of the growth and development of the stem top in Z.latifolia during the subsequent development.The analysis of expression patterns between the two pregnant Z.latifolia phenotypes showed that Zl-PIN1 c was the main gene for stem expansion and development in Z.latifolia.The stem elongation during stem swollen and development of normal Jiaobai would be related to the restoration of expression of Zl-PIN1 c.3.Localization of Zl-PIN1 in different tissues and cells of Z.latifoliaThe tissue sections of stem and fibrous root were detected by immunofluorescence hybridization with At PIN1 antibody.Microscopic observation showed that PIN1 protein in stem of Z.latifolia was located at the base of cell membrane in the cells around vascular bundle.PIN1 protein in cells of fibrous root meristem was located at the bottom of cell membrane base,while the expression of PIN1 protein was also observed in endoplasmic reticulum.The subcellular localization analysis of transient expression in tobacco showed that the Zl-PIN1 c was mainly expressed in the cell membrane and endoplasmic reticulum of mesophyll cells,which further verified the results of immuno-fluorescence localization analysis of Zl-PIN1.The analysis of localization of Zl-PIN1 was basically consistent with the reported study of localization of At PIN1.4.Functional analysis of Zl-PIN1 s in over-expressing transgenic ArabidopsisThe p FGC-Egfp-Zl-PIN1 s over-expression genetic transformation vector were constructed.The Arabidopsis transgenic positive line over-expressing Zl-PIN1 s were constructed and screened by the transformation method of Agrobacterium infecting,and its expressions were verified and analyzed.Through the treatments of exogenous IAA and the observations of root growth and development of transgenic Arabidopsis seedlings,it was clear that three Zl-PIN1 s genes significantly promoted the root length and fibrous root number of Arabidopsis,among which Zl-PIN1 c had the best effect.Based on the observation of above-ground biological phenotype,the promoting effect of Zl-PIN1 s overexpression on the stem development of Arabidopsis was analyzed.The flower branch development of Zl-PIN1 c over-expression transgenic lines was better than that of control line.The biological function of Zl-PIN1 c was preliminarily clarified,which would be beneficial for its regulation in the stem development of Z.latifolia.