Study On The Genetic Stability Of Exogenous Mutant Avenin-like B Gene In Transgenic Wheat Plants

The most prominent feature of Avenin-like protein is the presence of abundant cysteine residues（18 or 19）.It is speculated that some cysteine residues of Avenin-like protein may be involved in the formation of intermolecular disulfide bonds which may affect the processing quality of wheat flour.Previous studies of our research group have confirmed that Avenin-like b protein has a significant positive effect on wheat processing quality,but its mechanism has not been clearly revealed.Therefore,it is of some theoretical and practical significance to further explore the mechanism of Avenin-like b protein affecting wheat processing quality.Avenin-like b protein has three non-conserved cysteine residues at its N-and C-terminal.In this study,the mutant avenin-like b gene was obtained by using the artificial site-directed mutagenesis strategy,leading to the tyrosine residue at the C-terminal of wild-type Avenin-like b protein replaced by a cysteine residue.And the eukaryotic expression vector of avenin-like b*gene was constructed and successfully transformed into scutellums of wheat line Zhengmai 9023 by particle bombardment.Some transgenic plants were obtained by the way of tissue culture.PCR and Southern blot techniques were used to confirm the integrations of the transgene and SDS-PAGE and Western blot analysis were performed to detect the expression of the target gene in order to obtain homozygous transgenic lines of wheat.The main results are as follows:（1）The mutant avenin-like b*gene was obtained by using the artificial site-directed mutagenesis strategy,and we constructed vector pLRPT-avel^*.（2）The avenin-like b*gene was successfully transformed into scutellums of wheat line Zhengmai 9023 by particle bombardment and about ten transgenic plants of wheat were obtained after induction,differentiation and rooting culture.（3）PCR and Southern blot analysis showed that the avenin-like b*gene was successfully integrated into the wheat genome in multiple copies（2,5）,SDS-PAGE and Western blot analysis confirmed that the avenin-like b*gene was stably expressed in wheat endosperm.Four homozygous transgenic lines were obtained by the identification of five consecutive generations（T₀-T₄）.