Studies On Cloning Of HMW-GS 12 Gene And Regulation Of Subunit Accumulation In Spring Wheat

Wheat is one of the most important nutrition and energy source. Wheat quality affected directly the improvement of life level satisfaction to food, nutrition balance and development of flour and food industry. The high molecular weight glutenin was the major storage protein in wheat endosperm. Its composition, number and accumulation had closely relation with wheat quality. The studies aimed at investigating the accumulation and the expression of mRNA of HMW glutenin in grain filling in two cultivars (DN7742 and XKH9) with the same 2 and 12 subunit and different in processing quality, as well as influence of the transcriptional level regulation to the accumulation of HMW-GS. Genes of HMW-GS 12 and their upstream regulative sequence was cloned by PCR method and used to study on regulation mechanism of the accumulation of HMW glutenin subunit in molecule level.In this paper, the accumulation of HMW glutenin subunits was studied by SDS-PAGE gel electrophoresis. The HMW-GS formed gradually in grain formation. In addition, along with the process of grain filling, the type of subunits increased gradually. Once the type of subunit formed, it was not disappeared in the duration of grain filling. The HMW-GS initiated around 10 day post-anthesis. All HMW-GS were found around 14 day post-anthesis in DN7742. In XKH 9, it was 2 and 4 days later when compared with DN7742. The accumulation of HMW-GS increased gradually along with the process of grain filling. The accumulation rate of HMW-GS increased rapidly 20 days post-anthesis and reached the peak 24 days post-anthesis in DN7742 and XKH9, but the accumulation rate and the final accumulation level of HMW-GS was higher evidently in DN7742, when compared with XKH9, which might be one of the reasons that DN7742 with subunit 2 and 12 but in high quality.The study was carried out on the mRNA expression level of HMW-GS 12 during grain filling by RT-PCR. The mRNA accumulation of HMW-GS 12 initiated 6 days after anthesis and reached the peak 14 days after anthesis in DN7742, and 10 and 14 days, respectively in XKH9. In the various stage of grain-filling duration, the mRNA expression amount of HMW-GS 12 was higher markedly in the grain of DN7742 than that of XKH9. It was considered that the higher accumulation amount of mRNA was the foundation of the higher accumulation amount of the HMW-GS 12 in DN7742. It indicated that the transcriptional level regulation might be the major regulation ways in HMM-GS accumulation.The full-length fragments encoding HMW-GS 12 were cloned from wheat genomic DNA in DN7742 and XKH9 by PCR method, and were registered in GenBank (Reg. No: AY486484, AY486485). Sequencing analysis indicated that two fragments were 1980bp. There were four mutant sites between two sequences and resulted in one amino acid change. This fragment of DN7742 showed 99.5% identity to the previously reported sequence, contained 10 bases change and resulted in two amino acid deletion and one amino acid substitution. The DNA sequence of HMW-GS 12 gene of XKH9 and the reported sequence were aligned. The result showed that two sequences had 99.6% identity with ten bases different but only two aminoacid deletion. It could not confirm that whether protein structure and characteristic of the subunit was affected by the deletion and substitution of these amino acids. Cloning of genes encoding HMW-GS 12 and their sequences analysis of nucleotide and deduced amino acid indicated that there was more apparently different in comparing the sequence of two cultivars with the previously reported sequence. The results would be useful for studying the two kind of proteins characteristic by the expression in eukaryotes and the protein purification in the future.The upstream regulation region of HMW-GS 12 gene in DN7742 and XKH9 were cloned by PCR method. These sequences analysis showed that the two sequences were 917bp and 919bp in length, respectively, containing one TATA-box, three possible CAAT-like boxes, two GCN4 motifs, one E-box and two possible E-like boxes. It was speculated that the presence of GCN4 motif and E-box were in responsible of gene endosperm-specific expression. In addition, there was a cereal-box with 24bp in upstream region of HMW-GS 12 located in 476bp to 499bp. This sequence was high homology with -300 element that can be found in the seed storage protein of cereal crop, and the promoter region contained a regulatory element HMW enhancer that is 38bp in length. This sequence was highly conserved in all HMW glutenin promoters, beginning at position between 677 and 714bp. It was possible that the two DNA sequence motifs of the promoter were relevant to the amount of expression of HMW glutenin gene. Two upstream regulatory sequences of HMW-GS 12 gene were cloned in the study, which would be useful for further investigating the promoter function by deletion analysis. These two upstream sequences were cloned for the first time, and were registered in GenBank (Reg. No:AY486486, AY486487)Two plant expression vectors pUbPBIHG and pDNPPBIHG with HMW-GS 12 gene, driven by the constitute promoter Ubi and a native HMW-GS 12 gene promoter in DN7742 respectively and terminated by Tnos, were constructed. It was useful for the relationship between the accumulation of HMW-GS and wheat quality and improves wheat quality by the genetic engineering in the future.