A Study On Copy Number Detection Of Transgenic Rice Using QPCR And Copy Number Relationship With Agronomic Traits

qPCR is an important method for quantitative detection of nucleic acids,the correct choice of standards and build a qualified standard curve will ensure the accuracy of quantitative qPCR.This study was designed to investigate the association of Bar gene copy number and transgenic rice field performance difference in transgenic rice.The study was designed to study the consistency between the standard curves constructed by plasmid DNA standards genomic DNA standrds,and the impact on qPCR quantification accuracy of different standard curves constructed different standards.This study selected Transgenic rice genome DNA,not linearized pBS plasmid DNA and linearized plasmid DN Aas standard,which containing reference gene Sps and target gene Bar,the standards dilution to 106,105,104,103,102 five concentrations,three standard material and two gene construct six standard curves were compared.Amplification efficiency of Two standard curve constructed by linearized pBS plasmid DNA constructs was 98.28%and 100.32%,respectively,nearly 100%;the Ct value of target gene Bar and reference gene Sps of three standards in five concentrations were 2.74,2.76 and 2.77,respectively,basically the same.This study shows that both plasmid DNA standards and genomic DNA standards can be used for qPCR,when constructing a standard curve.But the standard curve equation slope and vertical intercept obtained were significantly different,so the two can not be combined,while the standards were selected,linearized plasmid DNA standards compared to non-linearized plasmid DNA and genomic DNA standards is more suitable to construct a standard curve.In this study,we using transgenic rice MH63(cry1C*/Bar)with different Bar gene copy number as material,using qPCR identified Bar gene copy number of 16 parent plants and 16 F1 generation populations including 152 individual plants.We measured 9 traits after 16 F1 generation populations harvested,including plant height,tiller number,spike number,spike length,total grains per plant,grain weight,setting rate,grain weight per plant and filled grains per plant.In this study,we using transgenic rice MH63(cry]C*/Bar)with different Bar gene copy number as material,using qPCR identified Bar gene copy number of 16 parent plants and 16 F1 generation populations including 152 individual plants.We measured 9 traits after 16 F1 generation poptulations harvested,including plant height,tiller number,spike number,spike length,total grains per plant,grain weight,setting rate,grain weight per plant and filled grains per plant.The study found that 16 parent plants Bar gene copy number between 0.51 to 7.54,F1 generation Bar gene copy number between 0-4,the 16 F1 generation populations Bar gene copy number changed in different degrees;plant height,tiller number,spike number,spike length,total grains per plant,grain weight,setting rate,grain weight per plant and filled grains per plant of F1 generation decreased-8.16%,-6.57%,-9.90%,-19.85%,-14.22%,-11.27%,-6.90%,-28.41%and-30.95%,compared with non-trnsgenic rice MH63.The study found that transgenic rice MH63(cry]C*/Bar)'s field performance was significantly lower non-under normal growing conditions,and plant height,ear length,grain weight,seed setting rate and total plant grain weight were significant negative correlation Bar gene copy number in transgenic rice.