A Competitive PCR-based Method To Detect A Single Copy Of T-DNA Insertion In Transformants

Gene diversity is the basis of the evolution of life and the differentiation of species.In plants,agrobacterium-mediated genetic transformation is widely used to produce mutants and the number of mutant genes is significantly related to the number of T-DNA inserted on the host chromosome.However,it is difficult to identify whether a transformed plant contains a single copy of T-DNA due to the lack of simple and reliable methods.This study established a detection method based on competitive PCR to detect single-copy T-DNA insertion using the model plant Arabidopsis thaliana.The conclusion is drawn as follow:(1)The insertion of single copy of T-DNA in transgenic plants cannot be accurately identified by Real-time PCR.The ratio of single-copy T-DNA(containing a GFP gene)inserted into the heterozygous transgenic plant seb19-L into the Pro HTR10 and GFP fragments was evaluated by real-time PCR using single-copy endogenous genome fragment Pro HTR10.The average value of the ration(3.43:1)in the study was much higher than that be expected(2:1),so it if difficult to determine that the T-DNA in the transgenic plant is a single copy insertion.(2)The insertion of single copy of T-DNA in DNA samples can be detected effectively by competitive PCR.It was found that the average gray scale ratio of the upper band and the lower band for DMG&DG plasmid with the target and the competitive template of 1:1 and that for 2�DMG&DG plasmid with the target and the competitive template of 2:1assembled by competitive PCR was 0.88:1 and 1.35:1 respectively,suggesting that competitive PCR can recognize single-copy inserted DNA samples.(3)The insertion of single copy of T-DNA in transgenic plants can be found using competitive PCR.The transgenic lines with a target and competitive template 1:1(B5/-&M2/-and B8/-&M3-)and a target and competitive template 2:1(B5/B5&M2-and B8/B8&M3/-)was cultivated through hybridization.Then its genomic was performed by competitive PCR.The results showed that:(1)the average gray-scale ratios of the two PCR products tested by competitive PCR using B5/-&M2/-and B8/-&M3-genomic DNA as templates were 0.86:1 and 0.85:1,respectively.(2)the average gray-scale ratios of the two PCR products tested by competitive PCR using B5/B5&M2 and B8/B8&M3/-genomic DNA as templates were 1.39: 1 and 1.28: 1,respectively.These results indicate that competitive PCR can be used to detect the insertion of single-copy T-DNA in transgenic plants.(4)The general competitor BHK-was established.A universal competitor suitable for most transgenic plants is particularly important for the widespread use of competitive PCR to detect the insertion of single-copy T-DNA in transgenic plants.We constructed a universal competitor BHK-containing the above three resistance gene fragments,considering that almost all transgenic plants contain kanamycin,hygromycin or herbicide resistance genes.It was found that the gray-scale ratios of the two PCR products detected by competitive PCR using plasmids BHK-& BHK+(the ratio of target to competitive template is 1:1)and BHK-&2�BHK+(the ratio of target to competitive template is 2:1)as templates were0.8~0.9:1 and 1.2~1.3:1,respectively,indicating that BHK-can be used as a general competitor.(5)A transgenic plant BHK-1 containing a single copy of the universal competitor BHKwas composed.We constructed a T-DNA plasmid containing the BHK-&Pro RPS5A:H2B-td Tomato fusion fragment,and detected it by fluorescence microscopy(Pro RPS5A:H2B-td Tomato fusion gene makes the nucleus of the transgenic plant show red fluorescence)to obtain a number of stable transformed plants BHK-for a universal competitive transgenic plant.A transgenic plant containing only a single copy of with the insertion of BHK-&Pro RPS5A: H2B-td Tomato was dissociated using competitive PCR to amplify the genome of the hybrid plant of BHK-and seb19-L,and it was named BHK--1.(6)The transgenic plants BHK�1:1,BHK�2:1,BHK�3:1 for blank control were constructed.We generated a control transgenic plant BHK�1:1,BHK�2:1,BHK�3:1 to eliminate the difference in the ratio between the two PCR products under different conditions.We can accurately determine the number of T-DNA insertions in the test plants by the difference between the control transgenic plants and the test plants.