Cloning Of Quinoa ?-Amylase Inhibitor Gene And Research On Germination Characteristics Of Transgenic Rice

When encountering wet and rainy weather,mature but unharvested grains in field crops begin to germinate and grow on the plants.This phenomenon is called ear germination.Ear sprouting is very common,occurring in crops such as wheat,barley,corn and rice;and it occurs in a wide range of areas,occurring in crop growing areas all over the world.Ear sprouting leads to lower yields and deterioration of varieties,which often causes huge economic losses.Ear germination is related to many factors,such as temperature,humidity,and changes in plant hormone levels.The combined action of these factors makes the alpha-amylase in crop seeds activated.Current studies have found that ear sprouting is related to the level of?-amylase.Domestic research on it is mainly focused on wheat,barley,buckwheat,beans and other crops,but there is no report on quinoa.In this study,the quinoa alpha-amylase inhibitor gene was successfully cloned from quinoa,and the function of the quinoa alpha-amylase inhibitor gene was studied through bioinformatics analysis and rice genetic transformation.The analysis shows that:(1)Using the method of homologous cloning,a new quinoa?-amylase inhibitor gene was amplified by RT-PCR.The gene sequence was 696 bp in length.The obtained quinoa?-amylase inhibitor gene was named For Cq AI.(2)Through bioinformatics analysis,it is concluded that there is a 696 bp reading frame in the sequence,which encodes a protein composed of 229 amino acid residues.The predicted molecular weight of the protein is 24.33 k Da and the protein isoelectric point is 8.27.The homology analysis showed that the gene has high homology with quinoa(alpha-amylase/trypsin inhibitor)and sugar beet(Protein P21),and the gene has a transmembrane domain.By predicting the function of the protein,it is found that the protein belongs to the PR-5 protein family and has certain antifungal activity.(3)In this study,the quinoa alpha-amylase inhibitor gene Cq AI was successfully introduced into rice through the Agrobacterium-mediated method.By extracting the leaf DNA of the transformed tobacco plant,using the target gene primers for PCR detection,and GUS rapid detection,5 T₀ generation positive plants,and T1 generation seeds were harvested.(4)The determination of the inhibition rate of?-amylase inhibitor and the?-amylase activity of rice seeds at different treatment periods found that the?-amylase activity of the transgenic and wild-type seeds reached the highest when soaked for 24 hours.With the germination process and the passage of time,the?-amylase activity gradually decreased,while the inhibition rate of the?-amylase inhibitor of the transgenic seeds was significantly higher than that of the wild type within 48 h.It is preliminarily speculated that the transgenic rice seeds may inhibit the endogenous?-amylase activity of the seeds at the initial stage of germination.