Elucidating The Role Of IAA-leucine Resistant 1-like Hydrolase In The Abscission Of Tomatoes

Auxin play an important role in the process of regulatory organs’ detachment.Combined state auxin which is catalyzed by auxin amide hydrolase(IAA-Leucine Resistant 1-like Hydrolase,ILR)can release free auxin(IAA).Although studies on the regulation mechanism of auxin on Tomato detachment have a certain foundation,it is not clear that the influence of the auxin amide hydrolase on its regulation.This study analyzes the combination state auxin and CHX influence on the detachment process of tomato pedicel.ILR gene space and time expression and the characteristics of encoding proteins were studied,on this basis,the ILR gene was silenced in the tomato and its’ influencing on pedicel abscission.The main results are as follows:1.Disposing tomato flower pedicel explants though using different concentrations of IAA-Ala,IAA-lie and IAA-Asp,the IAA-Ala,IAA-Ile can significantly delay the pedical abscission,due to ILR can’t be hydrolyzed,IAA-Asp can’t regulate detachment.It confirmed that ILR participated in the process of the tomato flower pedicel abscission.Joining and removing IAA-Ile at a different time confirmed that processing 8 hours earlier before detachment can slow down the abscission occurred.DR5 was further used to test the auxin dynamic transformation after treatment with IAA-Ile.The results of GUS::DR5 and auxin content showed that exogenous IAA-lle could effectively increase the content of auxin in the area.However,the treatment of synthetic inhibitor of protein synthesis CHX has confirmed that the addition of a sufficient amount of auxin could not delay the detachment.2.Due to information of ILR tomato auxin amide hydrolase family is less,we adopt the strategy of conservative sequence blast and searched NCBI and SOL after finding conservative sequence through the known species arabidopsis thaliana,rice protein,Chinese cabbage,and then got seven tomato ILR gene(ILR 1,ILR 2,ILR 3,ILR 4,ILR 5,ILR 6,ILR 7 mean ILR 1-7).Seven ILR have M20 activity center and M20 binding sites was found though further clustering analysis.Through cluster analysis with known protein sequences,we found ILR has great differences.The family protein may have specific function.In order to find out whether these seven proteins have activity,we conducted in vitro induced protein synthesis and detect its activity,confirm all ILR protein have activity.3.SlILR 1,3,5,6 and 7 have a higher expression level in abscission area were found through analyzing ILR tissue specificity by using qRT-PCR,and suggest that the five genes may play a key role in the abscission area growth and detachment process.In order to further determine which ILR is falling out specific genes,we used the auxin and ethylene to accelerate and delay detachment process,and to investigate the difference of ILR space and time expression in this process,the results showed that SlILR 1,5,6 expressed detachment specific expression pattern.4.We adopt VIGS virus silence strategy,according to the requirements of VIGS transgenic technology,built transgene recombinant plasmid and transformed agrobacterium-mediated,agrobacterium microbial to infect tomato cotyledons.Testing positive of plants by Using qRT-PCR,the separate SlILR 1,5 or 6 expression were reduced respectively,and conducted SlILR 1 + 5,SlILR 1 6+ SlILR 5 + 6 and SlILR 1 + 5 + 6.1n addition,although SlILR 3,7 have no specific expression,and showed increasing trend for expression,we also conduct the VIGS silence plants.To identify the positive transgenic plants,the loss rate and its loss rate under the condition of the IAA-lle medium were measured.Determination results showed that ILR transgenic plants loss rate is higher than control experiment,and the loss rate of SlILR 1,5,6 and 1+5,1+,1+5+6 is significantly higher than control experiment.Results show that ILR genes participate in and delay the detachment process of tomato flower pedicel,including SlILR 1,5,6 are key genes for detachment regulation.In conclusion,we got seven tomato ILR gene,respectively are ILR 1,ILR 2,ILR 3,ILR4,ILR 5,ILR 6,ILR 7.Seven ILR have M20 activity center and M20 binding sites was found though further clustering analysis.Through cluster analysis with known protein sequences,we found ILR has great differences,and this seven proteins all have activity,the family protein may have specific function.It confirmed that ILR participated in the process of the tomato flower pedicel abscission,exist insufficient of combine auxin and auxin amide hydrolase in abscission,:including SlILR 1,5,6 are key genes for detachment regulation.