Cloning And Marker Development Of TaGL3.1 In Wheat

Wheat is a very important food crop globally, in recent years, the farming land area can be used is gradually reduced, while the population is growing, so the demand for food is also growing. under the condition of limited land resources, how to improve the yield of wheat has become the main research targets of agricultural development in the future. Thousand grain weigh plays a crucial role to the yield of wheat,which is mainly determined by the grain length, width, thickness and plumpness and so on. Xinong817 and Chinese spring were selected as the study materials, using rice OsGL3.1 gene and collinear relationship of wheat diploid ancestors and wheat genome,and combining with EST sequences of wheat to homologous clone.The result are followed:1.The complete TaGL3.1 gene was isolated from common wheat by PCR technology. The results obtained in three different sequences, Which came from the three chromosomes of wheat.TaGL3.1 different genome except contained base substitution, also contained nucleotide insertions and deletions, which led to differences of the different wheat genome. The genes in Chinese Spring 5As,5BS,5DS the full-length DNA sequences were 9063 bp, 9136 bp, 9111 bp, three homologous gene sequence consistency reached 96.79%; in Xinong 817 5As, 5BS, 5DS the full-length DNA sequences were 9065 bp, 9134 bp, 9111 bp, three homologous gene sequence consistency reached 95.40%.2.The result of cDNA sequencing showed that the three sequences were relatively conservative in the region of exon. The total length of cDNA was 2769 bp, encoding 922 amino acids. After the Chinese Spring and Xinong 817 sequences for comparison, in which A sequence only replaced a single base substitution in the two wheat materials, has not resulted in amino acid changes,there was a A-base deletion at the 3155 bp in the B sequence of Xinong 817, there was no difference in the D sequence in two wheat varieties. The homology of TaGL3.1 gene and HvGL3.1 gene(AK375108.1) was as high as 98%.3.TaGL3.1 protein primary structure analysis showed that the protein is stable proteins. The theoretical isoelectric point(PI) of the TaGL3.1 protein was 5.86, which consistented with the theoretical isoelectric point(5.70) of the rice. The secondary structure was mainly composed of the random coli,extended strand, alpha helix, and beta turn.Subcellular localization analysis showed that 50.5% of the possibilities were in the cytoplasm. Structure function domain analysis showed that TaGL3.1 protein belongs MPP superfamily, which encoded a serine / threonine protein phosphatase gene(PP2Ac) and conservative Kelch repeat structure domain.Transmembrane domain analysis showed that TaGL3.1 was likely to be a membrane outer protein.Signal peptide prediction results showed that TaGL3.1 protein did not contain signal peptide.The three-dimensional structure of TaGL3.1 and OsGL3.1 almost overlapped, which indicated TaGL3.1 gene can regulate the grain length of wheat.4.TaGL3.1 amino acid sequence comparison with other species GL3.1 gene indicated that there was high homology in different species of GL3.1 protein,TaGL3.1 had 98% homology in amino acid sequence compared with Hordeum vulgare.The amino acid sequence of TaGL3.1 and other plants GL3.1 gene had no obvious InDel,which indicated TaGL3.1 was highly conservative. Phylogenetic analysis showed that TaGL3.1 was conservative in evolution, especially in cereal crops.5.Primers were designed according to genome sequence alignment results of TaGL3.1 in Chinese Spring and Xinong 817 and difference locus.This experiment tested part of the materials by hybridization F5 group of China Spring and Xinong 817 using molecular markers TaGL3.1-9,which showed that this gene may be associated with grain length.6.40 pairs of sequence related amplified polymorphism(SRAP) primers were screened with the 52 derivatives from Huanghuai trial test or region test. The results showed: 40 pairs of SRAP primer combinations produced 941 bands in 52 materials, of which 266 bands were polymorphic with 28.27% of polymorphism, average 6.65 polymorphic bands per primer pair. The range of polymorphic information content(PIC) was from 0.106 3 to 0.738 5, with an average of 0.477 5. The 52 materials could be divided into five categories in the genetic similarity coefficient of 0.571,which were basically same with population genetic structure analyses. The results obtained through cluster analysis matched with the genetic family tree. The results of the genetic contribution of Yumai 2 to its derivatives varieties indicated that Yumai 2 had the contribution ratios of 44.06%, 42.14% and 41.98% to its second, third and fourth generation derivatives, respectively.