Function Of TaCPK34 In Response To Drought And Grain Starch Synthesis In Common Wheat(Triticum Aestivum L.)

Master candidate: Xu MengjunCalcium-dependent protein kinase(CPK)family play important roles in regulating growth,development and biological synthesis in higher plants,however,biological functions of some CPK members have not been characterized.In our previous study,abundance of Ta CPK34 protein was quickly upregulated in the grain organ of wheat plants during the grain filling stage accompanied with diverse abiotic stresses,and the folds of its abundance were positively related to the rates of grain starch synthesis.These inferred that it might play an important role in regulating abiotic tolerance and starch synthesis in wheat grains during grain filling period.To confirm this speculation,in the present study,the function of Ta CPK34 was identified by using the barley stripe mosaic virus-virus induced gene-silencing(BSMV-VIGS)method and others.The main results were summarized as follows:(1)The isolated Ta CPK34 gene encoded a putative protein with 518 amino acids,and it contained the four conserved domains.The analysis of phylogenetic tree indicated that Ta CPK34 belonged to subgroup ? of CPK superfamily.By using Agrobacterium-mediated transient transformation,Ta CPK34 protein was found to be located at the plasma membrane in N.benthamina leaves.The transcripts of Ta CPK34 significantly increased during 20% PEG-induced water deficiency,ABA and JA by using real-time quantitative PCR method,suggesting that Ta CPK34 might play important role in response to drought stress and could be one member of the hormone signalling pathways.(2)BSMV-VIGS-induced Ta CPK34 silencing wheat plants were obtained by using BSMV-VIGS method and they exhibited more sensitive phenotype to natural drought stress,suggesting that it could play key role in response to drought in wheat.Isobaric tagging for relative and absolute quantification(i TRAQ)proteomic method identified 39 protein species exhibiting significantly altered abundance in leaves of BSMV-VIGS-induced Ta CPK34 silencing wheat plants at 14 d after drought condition.The identified protein species were functionally related to stress and defense,carbohydrate metabolism,nucleotide metabolic,photosynthesis,transportation,protein metabolism and signal transduction,suggesting Ta CPK34 could participate the drought tolerance by regulating the genes encoding these proteins.(3)The function of Ta CPK34 in regulating the starch synthesis was also identified using the BSMV-VIGS technique under filed conditions.The BSMV-derived Ta CPK34 silencing vectorwas used to inoculate the wheat spikes grown in the arch plastic shed during the heading stage under field conditions.BSMV-derived green fluorescent protein(GFP)-silenced wheat plants were used as the control.Our data indicated that expression levels of Ta CPK34 gene in BSMV-Ta CPK34-infected wheat plants were significantly inhibited by 63.8%-97.8% at 15 d,20 d,26 d and 30 d after virus inoculation.Starch content,and grain weight,length and width in BSMV-Ta CPK34-infected wheat plants were also decreased significantly.Scanning electron microscope observation showed that morphology of starch granules in grains of BSMV-Ta CPK34-infected wheat plants became looser and their numbers became fewer.These results suggested that Ta CPK34 could function in regulating the synthesis of grain starch.