Gene Cloning And Expression Analysis Of A γ-gliadin TaWG04 Related With Wheat Quality

Common wheat and its relatives are rich in species of the gliadin genes, which is significantly correlated with quality traits. This is a effective way to study gliadins’ structures and their relationship with quality by cloning and analysising its expression and function.This topic with high quality and strong gluten wheat Zhengmai 366 and typical weak gluten wheat Zhengmai 004 as experimental materials, using RNA sequencing data analysis combined with(q) RT-PCR screening to find differentially expressed genes, cloned a gamma-gliadin gene, TaWG04, and analyzed its expression of regulatory mechanism.The main results were as follows:(1) ten differentially expressed proteins were screened by RNA sequencing.(q) RT-PCR analysis showed that there are seven gliadin genes have strong expression in zhengmai 004, however Ta WG04 and TaWG05 have minimum amount of expression in Zhengmai 366.(2) By Rapid Amplification of cDNA Ends(RACE) and nested PCR to obtain the full-length cDNA sequence of TaWG04, we get a 1144 bp fragment, which contains 101 bp sequences of 5 ’end, 68 bp sequences of 3’ end, and 975 bp sequences of a complete open reading frame(A6).It can encode 324 amino acids, which contains a number of 21 amino acids of signal peptide sequence.(3) Through bioinformatics to find homologous sequences of TaWG04, we design three primers according to the conservative area.By cloning sequencing, we receive 33 gene sequences and construct phylogenetic tree by deduced amino acid sequence structure characteristics. The similarity of A6 and CS-1 nucleotide sequence is 99% and the homology similarity coefficient of amino acid sequence is 100, so Ta WG04 is a gamma-gliadin gene with multi gene family.(4)By using(q) RT-PCR, we analysis the temporal and spatial expression characteristics of TaWG04 gene in different stages(7 d, 14 d, 21 d, 28 d and 35 days post-anthesis) and different parts(the thief 48 h, 7 days of seedlings, roots, stems, leaves, stamen, pistil, embryo, endosperm) in seed development, the results showed that this gene is only expressed in the endosperm and expression can be detected in the 14 day after pollination, gradually increase to the maximum amount in 28 day and then decreased.(5) Choosing different strong and weak gluten wheat varieties,(q)RT-PCR results showed that the TaWG04 in weak gluten wheat varieties such as AK58, Jiaomai 266 and Yumai 50 have strong expression, while in the Xinmai26, Xinong 979, Gaocheng 8901 and other strong gluten wheat were hardly expressed. The results showed that Ta WG04 may be a negative regulator in the wheat gluten quality.(6)Expression primers were designed from the A6 sequence without signal peptide, we use BamHI and HindIII enzyme to digest the purpose fragment and pET32 a vector, then connect and conversion to E. coli Rosetta(DE3) cells, the prokaryotic expression vector of pET-32a-gamma-A6 were successfully construct. Using IPTG induction and purifing by affinity chromatography after SDS-PAGE, we obtained the concentration of protein is 4 mg/ml and found it is located in inclusion bodies.(7) Preparation polyclonal antibody and Western Blotting detection, we found the fusion protein stripe size is about 55 kd which is consistent with the antigen protein expression. Beyond that we obtained the concentration of polyclonal antibody is 1.33mg/ml, can specifically recognize the recombinant protein with good immunogenicity.(8) Choosing different strong and weak gluten wheat varieties, Western Blotting results showed that the Ta WG04 in strong gluten wheat varieties such as Zhengmai366, Xinong 979 were hardly expressed; while in the weak gluten wheat varietiesof AK58, Zhou8425 B, Zhoumai16, Zhoumai9 and Zhoumai13 have strong expression.The results showed that TaWG04 may be a negative regulator in the wheat gluten quality, Which is identical with the Real-time PCR.