Molecuar Characterization And Expression Analysis Of Transcription Repressor Factor TaASP1 Gene In Wheat

Post-embryonic development of plant relies on the activity of meristems, so the fate of the cell in meristem is important for plant development and type. Transcription factors enhance or nhibit the target gene through the interaction with other proteins. Arabidopsis thaliana TOPLESS (TPL) gene encodes a transcription repressor factor, belonging to TUP1 transcription repressor factor family. For now, the successfully cloned maize RAMOSA1 ENHANCER LOCUS2 (REL2) and rice ABERRANT SPIKELETAND PANICLE1 (ASP1) are highly homologous to arabidopsis TPL, and can regulate many aspects of rice development process and physiological reaction, at the same time, they also play important roles in the maintenance of axillary meristem In this study, we successfully isolate ASP1 gene from common wheat through electronic cloning and RT-PCR technologies. Moreover, we carry on the researches of expression patterns and functional analysis of ASP1 gene in order to discover its role in wheat spike development. The conclusions of this research are as follows:1. We first search the NCBI wheat EST databases using the OsASPl gene (AK111830) cDNA sequence as information probe. Then, the stitching 3591bp EST sequence of high homology which conyains intact ORFs is used as template to design 4 pairs primer to clone wheat ASP! gene. Because the 4 pairs primer are overlapping, so we can access the complete cDNA across splicing. We ampliilcate the ASP1 gene from the Triticum aestivum "Chinese Spring" and the amplified fragments length is 1284bp,1098bp,1072bp and1500bp. The isolated ASP1 gene open reading frame (ORF) from common wheat is 3402 bp, encoding 1133 amino acids. The nucleotide sequence homology with OaASP1 is 89%, at the same time it shares the same feature regions with TPL transcription repressor factor family. So we believe this sequence encode wheat ASP1 protein, named as TaASPl.2. Bsaed on the acquired cDNA sequence of TaASP1 gene, we design 8 pairs overlap primers in exon region to isolate the ASP1 gene sequence from Triticum boeoticum PI10474 (T. boeoticum, AA). The sequence length we isolated from T. boeoticum is 1162bp,1355bp,1163bp,1244bp,1142bp,920bp,1331bp and 1064bp respectively. Across the splice, we find the cloned ASP1 gene sequence is 8076 bp, which include 25 exons and 24 introns. The sequence length of exon and intron is 3402 bp and 4674bp respectively.3. Then we analyse the protein sequence alignment betweent TaASP1 isolated from Triticum boeoticum PI 10474 (T. boeoticum, AA) with TPL subfamily in other plants. The protein sequence similarity of TaASP1 with OsASP1, ZmREL2 and AtTPL are 92.5%,90.73% and 65.51%. Domain prediction analysis shows that the putative primary structures of wheat ASP1 contain all conserved domains of TPL protein subfamily, including LisH, CTLH and WD40 repeats. The amino acid sequence of wheat ASP1 of LisH is the same as those of ZmREL2 and AtTPL, which sharing 96.77% simliarity to OsASPl. The amino acid sequence of wheat ASP1 of CTLH with OsASPl, ZmREL2 and AtTPL is 98.28%,98.28% and 82.76%, respectively.4. Through the PCR amplification from 21 kinds nullitetrasomics derived from Triticum aestivum "Chinese Spring" and effective use of wheat genomic tat-abase URGI, we initially propose that the TaASP1 is located on 7A,7B and 7D chromosome. Through sequence alignment analysis we find polymorphism region which is conservative between 7A chromosome and 7B chromosome, but lacks of about 100bp sequence in 7D chromosome. In order to prove the above hypothesis, we design primer in polymorphism region which locates upstream of the start codon to PCR amplificate in 21 kinds nullitetrasomics derived from Triticum aestivum "Chinese Spring". Result, one specific band is only amplified fromN7DT7B. We prove that TaASPl is a multi-copy gene and located on 7A,7B and 7D chromosome.5. Then we extract RNA of different development periods and tissues from Triticum aestivum "Chinese Spring",10-A and E89, using Actin as reference gene and QF/QR as the primer of TaASP1, we analysis the expression pattern through quantitative PCR. The result shows that there is different expression level of TaASPl in spike, stem, leaf, seed and root during Triticum aestivum "Chinese Spring" grain filling period, but TaASP1 mainly is expressed in spike and stem. TaASPl is expresses stably at different spike length, when the length is 0.5-3.Ocm, the expression continues to increase, but when the length is 3.0-6.Ocm, the expression decline slightly. When compared between 3 different materials, the expression level of 10-A in spike, stems and leaf is higher than Triticum aestivum "Chinese Spring" and E89, and expression level in stem is higher than spike and leaf, which support the function during internode elongation and spike development.6. The 1248bp sequence which contains the whole signal peptide lies to the downstream of the start codon is used as the target signal peptide. Add Bg/Ⅱ and Spe I restriction enzyme cutting site through PCR amplification. Then the signal peptide of TaASP1 is ligated to eukaryotic expression vector pCAMBIA1302 and we named the new eukaryotic recombinant plasmid as 35S:TaASP1-GFP. Then recombinant vector is transformed into onion epidermis cell by particle bombardment method. When excited by 475nm blue light, the green fluorescent protein (GFP) of the original plasmid pCAMBIA1302 in the control group can be detected in cell membrane, cytoplasm and nucleus, but GFP of the new eukaryotic recombinant plasmid 35S: TaASP1-GFP only can be detected in nucleus, showing that TaASPl exists in nucleus.7. The 1557 bp sequence which located in the upper area of start codon is regarded as the promoter of TaASP1. Then we replace the 35S promoter of eukaryotic expression vector pBI121 to the promoter of TaASPl gene, and we named the new eukaryotic recombinant plasmid as TaASP1:GUS. Next, we infect tobacco by agrobacterium LB4404 mediated transformation method. The result of GUS assay shows that the root, stem, leaf and leaf stalks are dyed blue in transgenic tobacco, suggesting that the promoter of TaASPl is successfully transformed and expressed in multiple organizations.