Effect Of Organic Fertilizer Applications On Starch Biosynthesis And Overexpression Analysis Of Amylose Synthesis Related Gene NtGBSSI In Tobacco(Nicotiana Tabacum L.)

Starch content and its component are important factors for tobacco quality and style.In order to explore the changes and accumulations of the starch components at the ripening stage and research the effects of the amylose on the tobacco quality,we studied the starch biosynthesis under different organic mixed fertilizer treatments;Based on these results,we isolated Nt GBSS1 from tobacco and characterized the functions in starch synthesis.The results were as follows:1.The application of the organic mixed fertilizer could markedly promote the accumulation of starch granule in tobacco leaves.The number of starch granules increased and the volume became larger with the increase of the application amount of the organic fertilizers.Applying organic fertilizers could increase the total starch content and the amylopectin content,while the amylose content reduced in mature leaves.Expression level of starch biosynthesis-related genes is in accord with the change of starch content.The expression of the ADP glucose pyrophoshporylase(AGPase)did not change significantly at the ripening stage,Tthe granule bound starch synthase gene(GBSS1)declined,while the expression of the soluble starch synthase gene(SS1)enhanced with the increase of application rate of the organic mixed fertilizer.2.Constitutively overexpressing on Nt GBSS1 in tobacco: The Nt GBSS1 gene was cloned and inserted into the plant expression vector,then the plant constitutive overexpression vector p CAMBIA1305.1-35S-Nt GBSS1-GUS was constructed.we obtained resistant scions by screening on the MS medium with 8mg/L hygromycin(Hyg),l.5mg/L 6-BA and 0.15mg/LNAA by Agrobacterium-mediated transformation method,and the scions striked root on the MS medium with the same screening pressure and 0.1mg/L NAA,two weeks later,it became seedlings.By RT-PCR,GUS histochemical assay,12 positive plants are gained.3.Comparative analysis of transgenic plants:We transplanted the positive plants to the field and observed the morphological changes;then determined the activity of the GBSS,the amylose content and the total starch content.The results showed that the constitutive overexpression of Nt GBSS1 did not change the tobacco phenotype,the total starch content was not affected while the amylose content increase significantly,so the proportion of the amylose accounts for the total starch improved markedly and the starch components changed.