Application Of A My32b Pro Mot Or In Regulation Of Resistanting Rice Pre-harvest Sprouting Induction System

In the later stage of cereal crops maturity,when the weather is continuously cloudy and rainy,their seeds often germinate on the ear.The phenomenon often called pre-harvest sprouting.China has a vast territory and a wide area of rice planting and production,due to the pre-harvest sprouting phenomenon that the seeds will lose the edible value,seed value and storage value,and are usually used as feed.It will bring huge economic losses.In this study,Firstly,we investigated the expression of barley aleurone layer specific promoter Amy32b in rice,and modified the TATA-box of Amy32b with the Tet O manipulation sequence.Then we called the modifieded Amy32b promoter as Amy32m promoter.At the same time,we analyzed the expression of Amy32m promoter in rice.Secondly,based on the Tet-on regulatory system,we constructed one rice pre-harvest sprouting resistance induction regulatory vectors by using Os Amy1A as the target gene.The Amy32b promoter and the Amy32m promoter were applied to the rice pre-harvest sprouting resistance regulation vector p3F.Finally,we verified the function of rice pre-harvest sprouting resistance induction regulatory vector p3F,and the results show that the vector p3F could slow down the seed germinatin rate to a certain extent The main results are as follows:（1）We choose 688bp sequence before ATG of Amy32b gene as the promoter region and then the promoter region was ligated with red fluorescent protein gene RFP into Minghui 86.The results showed that the Amy32b promoter was specifically expressed in the aleurone layer of rice seeds by observing the cross-cutting and longitudinal cutting of seeds.（2）Tet O sequence was anchored near Amy32b promoter TATA-box to transform Amy32b promoter into Amy32m promoter,then it was ligated with red fluorescent protein gene RFP into Minghui 86,and we observed its expression in rice to test whether its function was changed due to modification.The results showed that the function of Amy32m promoter was consistent with that of Amy32b promoter,and there was not any change.（3）In this study,we choose the Os Amy1A as the target gene and Tet-on regulatory system as the basis,the regulatory expression vector and response expression vector were constructed on the same vector p LJ02 to construct the rice pre-harvest sprouting resistance induction regulatory vector.If there is the inducer Dox,the r Tet R protein was drived by Amy32b promoter can specifically bind to the Tet O manipulation sequence in Amy32m,the expression of Amy32m promoter will be inhibited and then will lead to suppress the expression of antisense RNA expression of Os Amy1A gene driven by Amy32m,so that seeds can germinate normally.If there is not the inducer Dox,the normal expression of antisense RNA expression of Os Amy1A gene inhibited the expression of Os Amy1A,thus inhibiting the normal germination of seeds.We verified the germination of the rice pre-harvest sprouting resistance induction regulatory vector p3F transgenic seeds under Dox and dd H₂O.The results showed that the germination rate of seeds treated with dd H₂O was slower than that of seeds treated with Dox.In rice production and hybrid rice seed production,slowing down seed germination rate could also reduce yield loss and economic loss caused by pre-harvest sprouting to a certain extent,which could provide a reference for future research on regulating rice pre-harvest sprouting.