Molecular Cloning And Functional Identiifcation Of TaBTF3in Wheat (Triticum Aestivum L.)

In this paper, the full length cDNA sequences of wheat BTF3gene (named TaBTF3)was first cloned by using the RACE (Rapid-amplification of cDNA ends, RACE)method, and the VIGS (Virus-induced gene silencing, VIGS) method was used toidentify the functions of TaBTF3gene.The main results were as follows:1. The first TaBTF3gene was cloned from wheat by RACE-PCR method, thefull-length cDNA sequence of the TaBTF3gene was882bp with an open readingframe (ORF) of534bp, and the predicted TaBTF3protein has177amino acids with acalculated molecular mass of20KD. The genomic DNA sequence of TaBTF3was1742bp, containing five exons and four introns, with all splicing sites complying withthe GT-AC. The Real-time fluorescent quantitative PCR analysis showed that theexpression of TaBTF3was constitutive, and the higher level of the transcript wasfound in booting flag leaves, and the expression levels of TaBTF3increasedsignificantly under PEG, cold and NaCl stress conditions, or treated by ABA, MeJAand SA. The subcellular localization of TaBTF3protein was examined by usingpolyethylene glycol (PEG)-mediated Arabidopsis protoplasts transformation method,gene copy number of TaBTF3was tested by using southern blotting. Results showedthat TaBTF3is a multi-copy gene and might be predominantly localized in thenucleus and cytoplasm.2. The stable and efficient VIGS system with barley strip mosaic virus (BSMV)vector was established in our laboratory. We successfully silenced the wheat PDSgene (VIGS reporter gene-TaPDS) by using this BSMV derived vector，which resultsin significantly photobleaching phenotype. The strong GFP fluorescence was detectedin both leaves and roots of the BSMV-GFP wheat plants, thus, the spreading ofBSMV is systematic, not only limited to the inoculation sites. The established VIGSsystem will be beneficial to the gene function analysis of TaBTF3in wheat for thenext step.3. The TaBTF3-VIGS and BSMV-GFP transgenic wheat plants were successfullyobtained by using the VIGS method, the identification and physiological parameterstest of the BSMV-VIGS plants were carried out. Results showed that silencing ofTaBTF3leads to a significant decrease in the chlorophyll content, but increased inMDA and H2O2contents. The transcript levels of chloroplast-and mitochondrial-encoded genes were examined by using the real-time quantitative PCRmethod. To further investigate the structural changes of the wheat mesophyll cells ofTaBTF3-VIGS plants, paraffin slices were prepared and observed under lightmicroscopy. Compared with the BSMV-GFP control plants, the expression levels ofthe chloroplast-and mitochondrial-encoded genes were significantly inhibited, andthe significant differences in the mesophyll cell structure were also observed, the cellswere smaller in size, irregularly shaped, disorderly and loosely arranged, and hadenlarged intercellular spaces. These results indicated that TaBTF3may involve in thegrowth and development of chloroplast, mitochondria and mesophyll cell in wheat.4. The transcript accumulation of stress-related genes were examined by using thereal-time quantitative PCR method， and the abiotic stresses tolerance ofTaBTF3-VIGS plants and BSMV-GFP control plants were examined. Results showedthat the transcript accumulation of stress-related genes was significantly reducedunder normal growth conditions. TaBTF3-silenced wheat plants exhibited markedlyhigher relative electrical conductivity and water loss rate, lower content of the freeproline and relative water content under stress conditions compared to theBSMV-GFP plants. Silencing of TaBTF3results in impaired tolerance to freeze anddrought stresses, the results above implied that TaBTF3may involve in the process ofabiotic stresses tolerance.