Genome-wide Identification Of Aquaporin Family And The Transient Expression Assay System Setup Of Mesophyll Protoplasts In Wheat

At present,the world has been challenged by the frequently increasing in drought conditions.As one of the four crops,wheat plays an important role in maintaining world food security.Aquaporins（AQPs）is widely present in the membrane system of living cells.When the water channel open,the water molecules both in and out of the membrane can be transported along the direction from high to low across the membrane.AQPs can also directly effect on the normal moisture physiological environment,which is important for maintaining the development of roots,stems,leaves,floral organs and seeds in wheat.Varieties research works have confirmed that AQPs can influence plant drought tolerance levels directly or indirectly.Due to the complexity of drought resistance mechanism and their own genome,start from the aquaporins with relatively simple background to study the function and mechanism of drought resistance in wheat will provide clear data for revealing the network which control wheat drought resistance.1.This study was relied on the biological information method and 105 wheat AQPs genes were successfully identified based on the wheat genome sketch sequences from Ensembl plant database.The TaAQPs genes was classified and named according to the comparative analysis of gene structure and phylogenetic analysis of AQPs from rice,maize and wheat.Among them,53 genes belong to the PIP subfamily,31 genes belong to the TIP subgroup,20 genes belong to the NIP subfamily,and only 2 genes belong to the SIP subfamily.After a series analyzes of sequence characteristics,evolutionary relationship,gene structure,cis-regulatory elements and gene expression of wheat AQPs gene,many data were accumulated for the further study of TaAQPs.2.In order to validate the existence of the identified genes,we classified the wheat aquaporin genes into PIP1,PIP2,TIP,NIP and SIP groups,each group of genes were subjected to multiple sequence alignment.According to the homology and conserved domain,a total of 19 pairs primers were designed to amplify the aquaporin genes from five different wheat lines.The results showed that the identified genes were existed and another gene TaNIP4-1a was identified at the same time.3.Key aquaporin genes which may involved in wheat physiological activities or response to stress were selected for the further functional study.Limited to the complexity of wheat genetic transformation,we transformed GFP gene into the protoplasts which were isolated from half-winter wheat KN199 and gradually optimized the conditions of protoplast separation and transformation.The optimized conditions were as follows:the leaves from 10d seedling were selected to isolate the protoplasts,the optimal composition of the Enzyme solution was 1.5%cellulase+0.75%pectinase+0.6M mannitol+20mM KCl+10mM MES+10mM CaCl₂+0.1%BSA.5h was selected as the enzyme isolation time,the centrifugal conditions were set up to 500rpm,3min;the souece of PEG4000should be carefully selected and the optimization concentration of the plasmid was 1μg/μl.4.pGEM-Ubi-GFP-nos and pBinaryUbiGFPnosEVec202Nos plasmids with the size of 6229bp and 14515bp,respectively,were successfully transformed into mesophyll protoplasts of wheat based on the optimized conditions,the green fluorescence signal were successfully detected and the transforming efficiency were about 70%.The results indicated that the efficient wheat mesophyll protoplast transformation system was published.The establishment of this system will provide a solid basis for further analysis of aquaporin genes which involved in the wheat biology.