| Color as an important quality trait for flour and wheat-based products, and cloning and expression analysis of genes associated with flour color is of great importance in wheat quality improvement. In the present study, PPO and PSY genes were cloned by screening Xiaoyan54genomic BAC library using PCR markers. Expressions of PPO and PSY genes during grain development were analyzed by semi-quantitative RT-PCR and Western Blotting. Alternative splicing of Ppo-Al gene were detected and characterized. In addition, Z-ISO genes were cloned by the method of in silico cloning in combination with PCR amplification. The main results obtained in this study are summarized below.1. Seven BAC clones containing PPO genes were obtained by screening the Xiaoyan54genomic BAC library using PCR markers. Four BAC clones207-2-3,207-3-3,207-12-1and910-9-4were located on chromosome2A; BAC clone780-4-7was located on chromosome2B; two BAC clones1376-2-3and1376-4-1were located on chromosome2D. Three shotgun sub-clone libraries, containing3-6kb DNA fragments generated by Sau3A I random digestion, were constructed from BACs207-12-1,780-4-7and1376-2-3. Using PCR and DNA sequencing, genes located on chromosomes2A,2B and2D were cloned, with5,447-bp,10,073-bp and4,295-bp genomic DNA sequences, respectively. The coding sequences of Ppo-A1, Ppo-B1and Ppo-D1obtained from Xiaoyan54BAC clones are consistent with those from in silico cloning, confirming the availability of in silico cloning of genes in common wheat. Besides the full ORF (open reading frame) sequences, the promoters and3’UTR regions were also obtained from BAC clones.2. Compared with Ppo-Alb cultivars, significantly higher staining intensities and PPO activities were observed in Ppo-Ala genotypes in the samples harvested after28dpa (Days post anthesis) and in the seeds stored for1year at4℃. Ppo-D1a genotypes showed slightly higher PPO activity than Ppo-Dl b genotypes at7dpa.3. Expression patterns of Ppo-Al and Ppo-D1were determined by semi-quantitative RT-PCR for eight wheat cultivars at7,14,21,28and35dpa. Ppo-A1expressed in grains but not in leaves. Putative transcription level of Ppo-A1a allele was much higher than that of Ppo-Alb allele during grain development. However, there were no obvious differences at the early developmental (7dpa) and nearly mature (35dpa) stages. The highest expression levels of Ppo-A1occurred between14and28dpa in grains, and then decreased rapidly at35dpa. Ppo-D1b has a similar expression pattern as Ppo-Ala, and its expression level is higher than Ppo-D1a after7dpa. Ppo-D1a allele showed a higher expression level at7dpa, but decreased rapidly to a stably low level during grain development. Based on cDNA sequencing data, eight spliced mRNA isoforms were detected from Ppo-A1b genotypes resulted from a191-bp InDel in intron I and a C/G SNP in exon Ⅱ. Only the constitutively spliced isoform could produce a putative full-length PPO protein based on the ORF sequence whereas the other isoforms contain PTCs (Premature termination codons). One67kDa Ppo-A1protein was expressed in E. coli. Western Blotting was applied for detection of PPO proteins from the seeds of eight wheat cultivars, at7,14,21,28and35dpa.4. Seven BAC clones containing PSY genes were obtained by screening the Xiaoyan54genomic BAC library using PCR marker PSY22. These BACs were287-11-4,436-12-6,988-6-8,808-2-6,900-12-7,1083-5-5and1036-10-7. BACs808-2-6and1083-5-5were located on chromosome7B; BACs900-12-7and1036-10-7were located on chromosome7D. The locations of the other three BACs287-11-4,436-12-6and988-6-8are unknown. Using PCR and DNA sequencing, Psy-B1and Psy-D1genes were cloned from shotgun sub-clone library based on1083-5-5and900-12-7, with9,173bp and5,073bp, respectively, including the sequences of promoters and3’UTR regions.5. Using semi-quantitative RT-PCR, Psy-A1genotypes showed a wave expression pattern during grain development. The highest mRNA transcription was detected during early developmental stages, then the transcription decreased to a much lower level at21and28dpa, and increased again to a higher level at35dpa. Using the primer generated from exon Ⅲ and3’UTR regions, no obvious differences in mRNA abundance were detected between the Psy-A1a and Psy-A1b genotypes.6. Full-length genomic DNA sequences of Ziso-D1on chromosomes5D were cloned from common wheat. Parts of genomic DNA sequences of Ziso-A1and Ziso-B1on chromosomes5A and5B, respectively, were also obtained from common wheat. Ziso-D1with a3,250-bp ORF has four exons and three introns. Three allelic variants Ziso-A1a, Ziso-A1b and Ziso-A1c were found from common wheat cultivars Chinese Spring, Zhongyou9507and CA9632, respectively. |
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