Selection And Identification Of Homozygous Transgenic Wheat Lines Overexpressing Avenin-like B Gene

Avenin-like b proteins are a novel family of wheat storage protein, each containing18or19cystenine residues hypothesized to form inter-chain disulphide bonds that couldaffect the functional properties of wheat flour. However, the role of these proteins indetermining the processing quality of wheat flour has been not confirmed yet.In this study, we detected transgenic wheat plants and their progenies at the DNA andprotein molecular level based on the obtained single avenin-like b gene transgenic plant.PCR method was used to confirm the positive transgenic plants; Southern blot analysiswas performed to deteromine the copy numbers of avenin-like b gene in the transgenicwheat lines; Besides, SDS-PAGE and Western blot analyses were used to detect whetherthe exogenous target gene expressed and inherited stably in transgenic wheat lines;Selection of homozygous progeny with stable expression of Avenin-like b proteins wascarried out in five generations. We obtained homozygous transgenic wheat lines stablyoverexpressing and inheriting transgenic avenin-like b gene. Furthermore, we analyzed theexistence form of Avenin-like b protein in the wheat seed. The main results are as follows:(1) The experimental condition of polyclonal antibodies against wheat Aenin-like bproteins was optimized, which provided with the base for detecting the expression levelsof Avenin-like b protein in the transgenic wheat lines by Western blot;(2) Selection of homozygous progeny with stable expression of Avenin-like bproteins was carried out in five generations (T0-T4). Three homozygous transgenic wheatlines (Zhengmai9023) and two homozygous transgenic wheat lines (Emai12)overexpressing Avenin-like b proteins were obtained;(3) Sodium dodecyl sulfate sedimentation (SDSS) test and gluten index analysisdemonstrated that overexpression of Avenin-like b proteins in all transgenic wheat linessignificantly increased SDSS volume and the amount and quality of gluten compared withthe control lines.These results showed that the Avenin-like b protein overexpressed in thetransgenic wheat had positive effects on the processing quality of wheat flour;(4) The results of SDS-PAGE and Western blot analysis showed that Avenin-like bproteins existed predominantly in both oligomer and polymer fractions rather than in monomeric fraction. Moreover, the amounts of Avenin-like b proteins in the polymerswere higher in the transgenic lines compared to the control lines.