The Study Of MeFtsZ1 Gene Regulate Amyloplast Division In Cassava

Cassava is an important food crop in tropical and subtropical regions,whose tuber root is rich in starch accumulation;and cassava starch has a wide range of uses in industry.The size of the starch granule is one of the important factors that affect the starch properties and industrial applications.Amyloplast as a plant storage and synthesis organ for starch grains,its volume is closely related to the size of the starch granule.Amyloplasts and chloroplasts are developed from proplastids,their division and proliferation are regulated under plastid division related proteins which their expression changes disturb plastid division,and result in larger plastids.FtsZ proteins(Filamenting temperature-sensitive mutant Z)can be formed FtsZ ring(Z ring)in plastid division site,which provide power to plastid division.Thus,FtsZs are the key proteins for plastid division.We have cloned three FtsZ genes(MeFtsZ1,MeFtsZ2-1 and MeFtsZ2-2)from cassava genome.Their expressions were associated with the starch accumulation during cassava tuber root development.In this study,in order to investigate the function of MeFtsZ genes in the process of cassava plastid division,first,the three MeFtsZ genes were over expressed in Arabidopsis;then MeFtsZl gene was over-and antisense-expressed in cassava roots through root specific promoter.The results might be a genetic foundation for cassava starch quality improvement.The main results are as follows:1.Function analysis of the three MeFtsZ genes has been done in transgenic Arabidopsis and cassava.The three plant over-expressing vectors of MeFtsZ 1-GFP,MeFtsZ2-1-GFP and MeFtsZ2-2-GFP were transformed into Arabidopsis by vacuum infiltration.The results showed that over-expression of MeFtsZ gene in Arabidopsis could decrease chloroplast numbers,but increase their volumes.MeFtsZ proteins were distributed in Arabidopsis chloroplasts as dot,block or fiber filament forms.However the morphology and chlorophyll content of the transgenic Arabidopsis did not change in comparison with the wild type.These suggest that the presences of MeFtsZ proteins disturbe the formation of Z ring in chloroplasts of the transgenic Arabidopsis,and result in abnormal plastid division.2.The expressions of the genes that are related plastid division were analyzed in MeFtsZ overexpressed transgenic Arabidopsis.The results showed that over expressions of MeFtsZ genes in Arabidopsis could change the expression of the genes that are related plastid division,in which MeFtsZ2-1 and MeFtsZ2-2 could significantly increase the expressions of AtFtsZ1,AtFtsZ2-1,AtFtsZ2-2,AtARC5,AtARC6 and AtSulA;and significantly decreased the expressions of AtARC3,AtMinEl,AtCRL and AtMCDl genes;but did not change their expressions of AtMinDl,AtPDV1,AtPDV2,AtCDP1 AtCLMP.However,overexpression MeFtsZl gene in Arabidopsis displayed small changes for most of the genes that are related plastid division,except AtFtsZl and AtCRL gene expressions were significantly decreased,and the rest of the gene expressions had no significant changes.3.In order to identify the effects of MeFtsZ1 on amyloplast development,MeFtsZl gene was specifically over-and anti-expressed in cassava roots.The root specifical vectors of pVKH-SpoA-MeFtsZ1 and pVKH-SpoA-antiMeFtsZl were constructed;and then transformed into cassava varieties of SC8.14 regeneration lines of pVKH-SpoA-MeFtsZ1 and 13 regeneration lines of pVKH-SpoA-antiMeFtsZl were selected;in which four lines of pVKH-SpoA-A4eFtsZ1(named TR-OE1,TR-OE2,TR-OE3,TR-OE4,respectively)and five lines of pVKH-SpoA-antiMeFtsZl(named TR-T1,TR-T2,TR-T3,TR-T4,TR-T5)have been identified with gene insertion in cassava genome by PCR.4.The transgenic cassava of TR-OE1,TR-OE2,TR-OE3,TR-OE4 and TR-T1,TR-T2,TR-T3,TR-T4,TR-T5 were transplanted and cultivated in field.The observation with scanning electron microscope found that the volume of the amyloplasts and starch grains,and the number of amyloplasts with a single starch grains in TR-OE2 and TR-T5 were increased.Southern-blotting analysis showed that the gene has been integrated into cassava genome with single copy.qRT-PCR analysis showed that the MeFtsZl carried by pVKH-SpoA-MeFtsZ1 was increased its expression,and the MeFtsZl carried by pVKH-SpoA-antiMeFtsZl was decreased its expression in transgenic cassava roots.However,the expressions of the genes that are related plastid division had not significant changes.5.The growth of the F2 generation transgenic lines of TR-OE2 and TR-T5 was investigated.The results showed that the height,leaf length,leaf width,petiole length,leaf number,stem diameter,root length,root diameter,root number,ratio of dry matter in TR-OE2 and TR-T5 lines displayed no significant difference.6.The starch development in tuber roots of the transgenic lines of TR-OE2 and TR-T5 was investigated.The results showed that in comparison with the wild type SC8,the amylose and amylopectin content in tuber roots of TR-T5 and TR-OE2 had no significant difference;the starch content in tuber roots of TR-T5 showed a slightly decrease,and that had no significant difference in the line of TR-OE2.Starch granularity analysis showed that the volume of the starch grains in transgenic strains of TR-T5 and TR-OE2 was significantly increased.The diamter of the starch grains in TR-T5 between 20?30 ?m,or>30?m was increased 2 folds and 66 folds that of SC8,respectively.And the diamter of starch grains>30 ?m in TR-OE2 was 24 folds that of SC8.Thus,the size of starch granules in TR-OE2 and TR-T5 between 20?30 ?m,or>30 ?m was significantly higher than that of SC8.7.The enzyme activity of AGPase,GBSS,SSS,SBE that are related to starch synthesis has been detected in the transgenic lines of TR-OE2,TR-T5 and wild type SC8.The results showed that the activity of these enzymes were not significantly different,which indicates that over expression,or antisense suppression MeFtZ1 gene in cassava tuber roots does not affect the activity of the key enzymes for starch synthesis;and the increased starch granule size is not caused by the activity of these starch synthesis enzymes.8.The starch viscosity curve among transgenic lines TR-OE2,TR-T5 and SC8 has obvious difference,which the peak viscosity and the breskdown of TR-OE2 are significantly reduced,the pasting temperrture and the peak time are increased;however,the hot paste viscosity,the final viscosity and the consistence have no changes.For TR-T5,the peak viscosity,the hot paste viscosity and the final viscosity of TR-T5 are significantly decreased,and the pasting temperrture is increased;but,the breakdown,the consistenc and the peak time have no changes.These results showed that changed starch granule size might lead to the changes of the starch viscosity properties in cassava.9.These were obvious difference about starch gelatinization heat characteristic curve among TR-OE2,TR-T5 and SC8 root.The starch gelatinization onset temperature,peak temperature and end temperature of TR-OE2 were significantly higher than that of SC8.The starch gelatinization onset temperature and end temperature of TR-T5 were significantly belower than that of SC8,nevertheless the peak temperature of TR-T5 did not change.The starch endothermic enthalpy among TR-OE2,TR-T5 and SC8 had no significant difference.The above results showed that the change of starch granule size could result in the change of the thermodynamic properties in the process of starch gelatinization in cassava.