The Expression Patterns Of Protein Inhibitor Genes OsLTPL164 And OsLTPL151 Induced By Chilo Suppressalis Feeding And Interference Vector Construction

Chilo suppressalis,as one of the most harmful frequent pests in rice,has caused great economic loss in rice production.At present,the large quantities of chemical insecticides used in the prevention and treatment of Chilo suppressalis have caused the problems of pesticide residues,environmental pollution and food security.However,it is a safer and more efficient method to use genetic engineering to cultivate anti-insect rice.Therefore,the rice lines 1688、1665 and 1654 cultivated routinely in the northeast area were selected as experiment materials.The expression patterns of OsLTPL164 and OsLTPL151 genes under Chilo suppressalis feeding and mechanical damage were analyzed by RT-qRCR technology,and silencing vector and overexpression vector of protease inhibitor genes OsLTPL164 and OsLTPL151 were constructed through RNAi and overexpression technology.Lastly,α-amylase activity of different tissues in three rice strains were determined under Chilo suppressalis feeding and mechanical damage.This provides a theoretical basis for estimating the role of OsLTPL164 and OsLTPL151 in the rice resistant Chilo suppressalis and for using the two genes to construct transgenic rice in the future.The results are as follows:1.The secondary structure and expression spectrum analysis of the rice protease inhibitor gene OsLTPL164 and OsLTPL151.By protein analysis,the result could be knowned that Os LTPL164 protein was composed of 159 amino acids and 5 alpha helices,including 10 Cys,and the OsLTPL151 protein was composed of 96 amino acids and 4 alpha helices,including 8 Cys.The tissue expression patterns of the OsLTPL164 gene is consistent in three rice lines.The results showed that the transcript abundance of OsLTPL164 in leaf and stem of three rice lines were significantly higher than in root.The tissue expression patterns of the OsLTPL151 gene is not consistent in three rice lines The expression of OsLTPL151 in the stem and leaf was significantly higher than in root in the 1654 line,but in the 1688 and 1665 rice lines,the expression level of OsLTPL151 in root was significantly higher than in stem and leaf.In addition,the two genes in the same tissue among three rice lines have different expression patterns.Up-regulation of OsLTPL164 and OsLTPL151 genes is higher in 1665 than that in 1688 and 1654 in three lines by C.suppressalis infestation.Both OsLTPL164 and OsLTPL151 showed a rise and then decrease and then rise expression tendency at different time point after artificial infestation and mechanical injury treatments.2.The alpha-amylase activity analysis in rice under the C.suppressalis feeding and mechanical damage.In different tissues of three rice strains,the activity of alpha-amylase was higher in the stem and leaf than that in root;the activity of alpha-amylase in the same tissue between the three rices showed that 1654 line was higher than 1665 and 1688 line.The activity of alpha-amylase showed a decrease and then rise and then decrease tendency at different time point after artificial infestation and mechanical injury treatments.In addition,the enzyme activity level was significantly reduced after artificial infestation and mechanical injury treatments 3 h,and the lowest value at 12 h;the activity of alpha-amylase of 1654 decreased significantly compared with 1688 and 1665 after artificial infestation and mechanical injury.3.The construction of silencing and overexpression vectors of OsLTPL164 and OsLTPL151 genesBy RT-PCR cloning and sequencing,we obtained the forward and reverse interference fragment of OsLTPL164 gene for 243 bp,and the forward and reverse interference fragment of OsLTPL151 gene for 203 bp.Finally,the silencing and overexpression vectors of OsLTPL164 and OsLTPL151 genes were successfully constructed.The yellow and compacted callus granules were obtained successfully,which laid a foundation for further studies on the planting and function of transgenic rice.