Isolation And Function Of Transcription Factors Regulating Expression Of The TaAGPL1 Gene In Bread Wheat(Triticum Aestivum L.)

ADP-glucose pyrophosphorylase(AGPase)is a key enzyme of starch synthesis in bread wheat,and it is composed of two geminative large subunits(Ta AGPL1 and Ta AGPL2)and two geminative small subunits(Ta AGPS1 and Ta AGPS2).Our previous research showed that the overexpression of Ta AGPL1 gene can obvious increased AGPase activity and the rate of starch accumulation in wheat grains,suggesting that Ta AGPL1 plays a central role in starch synthesis.In this study,the upstream regulator factors and their function in starch synthesis of bread wheat were identified via yeast one-hybrid screening and BSMV-VIGS technique,respectively.The main results are as follows:(1)Two regulator proteins(Tab HIL39 transcription factor and Ta PDIL1-2 chaperone)were screened out by yeast one-hybrid technique.The cloned Tab HLH39 gene encoded a putative protein with 486 amino acids and it contained four classic domains.Subcellular localization and transcriptional activation assays separately implied that Tab HLH39 was localized in the nucleus and showed strong transcriptional activation activity.The open reading of Ta PDIL1-2 gene with located on the 4BS chromosome encoded a putative protein with 486 amino acids and its protein sequence contained four classical thioredoxin domains.(2)BSMV-VIGS gene silencing technitic was uesd to inoculate to wheat spikes at anthesis under field condition.These experiment results showed that expression levels of the Tab HLH39 gene in BSMV-VIGS-Tab HLH39 scilence plants were notably reduced at 19 d,24 d,29 d and 34 d after inoculation.Grain length,width,weight and starch content in mature grains of the BSMV-VIGS-Tab HLH39 scilence plants were significantly decreased.Using q PCR method,our data showed that the expression levels of the Ta AGPL1 gene in BSMV-Tab HLH39 scilence wheat grains were significantly decreased at 14 d,19 d,24 d,29 d and 34 d after inoculation.These suggest that Tab HLH39 may participate in the starch synthase of bread wheat by positively regulating expression of the Ta AGPL1 gene.(3)Ta PDIL1-2 was screened out,and its binding to the Ta AGPL1-1D promoter was further verified using another yeast one-hybrid screen.Transiently silenced wheat plants of the Ta PDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions.In grains of BSMV-VIGS-Ta PDIL1-2-silenced wheat plants,the Ta AGPL1 gene transcription levels,grain starch contents,and 1000-kernel weights also significantly increased.As important chaperones involved in oxidative protein folding,PDIL proteins have been reported to form hetero-dimers with some transcription factors,and thus,our results suggested that Ta PDIL1-2 could indirectly and negatively regulate the expression of the Ta AGPL1 gene and function in starch biosynthesis.