Optimization Of Soybean TAIL-PCR And Detection Of Target Fragment

In the research field of molecular biology, operations like probe preparation for molecular hybridization and gene cloning usually require the separation with the unknown sequence of the known adjoining DNA. TAIL-PCR is the major technology for detecting the gene insertion site of the transgenic crops, mainly responsible for the amplification of nucleotide sequence next to the known sequence, that is the flanking sequence. As one of the major indicators of the molecular features, flanking sequence acquisition is the effective means of analyzing the T-DNA insertion sites in transgenic crops, as well as the important proof in applying the patent protection for the transgenic crops, which is of great significance to the determination of sites for foreign genes to insert into the crops. Flanking sequence can ascertain whether the foreign genes have integrated with the genome or not, also, it can judge whether the foreign genes can impact the crops genome. What is important, the flanking sequence can acknowledge the specific site of the foreign genes to the receptor genome. The analysis of flanking sequence will be in favor of the gene cloning, transgenic crops studying, new species breeding, etc. promoting the study of transgenic crops.In recent years, due to the advantages of TAIL-PCR in flanking sequence cloning, such as being convenient, high efficiency, good specificity, etc. It has been widely used in the full length cloning of genes and the study of flanking sequence cloning. It is true that TAIL-PCR possesses much advantages, it also has some disadvantages:(1) The sequence binding sites for integrating the primer and the targeted genes are limited.(2) TAIL-PCR respondence may include much primer combination, which may lead to nonspecific banding.(3) It’s difficult to control the size of the specific banding. Thus this thesis aimed to determine the insertion sites of the anti-disease gene HrpZpsg12 and the draught resistant gene BADH in the crops genes, and discussed their stability under different generations, by the means of building a mature technology system on the basis of the classic TAIL-PCR optimization.This study applied Molecular Biological Detection(PCR technology and Southern blot hybridization) to analyze the stability of transgenic soybean under different generations, and get the stable positive plants. In addition, this study analyzed the parameters like the annealing temperature, Taq enzyme dosage, template concentration, arbitrary degenerate, specificity circulation under low temperature, etc. that may affect the TAIL-PCR of transgenic soybeans by the means of single factor experiment. 4 levels of the 5 factors each were tested with the orthogonal test, and the best combination of various levels were selected to build the expected mature technology system of the soybeans TAIL-PCR. By this optimized technology system, this study analyzed the flanking sequence of the high generation single plant copy with TAIL-PCR method, and determined the specific insertion site for the T-DNA to the transgenic soybean genome.The result obtained from the study is as flows:(1) After the PCR detection to different generations of transgenic soybeans plant, the electrophoresis result showed that all generations of soybeans can amplify the target genes, with clear bands and high specificity. The Southern blot hybridization results showed that there are obvious hybridization signals in different generations, with the hybridization band not the same, which proved that the targeted fragment have integrated with the soybean genome. The stable inheritance and expression of the foreign genes in the soybean genome can be tested in the molecule level.(2) single factor results show that the annealing temperature, enzyme concentration, template concentration, arbitrary degenerate and the low temperature cycle at different levels are the result of the TAIL-PCR reaction. In the second reaction, when the annealing temperature is 62 ℃, enzyme concentration is 1.0 U / 30 mL, template concentration is 35 ng/μL, arbitrary degenerate is AD2 can amplify out clear bands; In the second reaction, when the annealing temperature is 63℃, enzyme concentration is 1.5 U / 30 mL, template concentration is 40 ng/μL, arbitrary degenerate is AD3 can get ideal result;The low temperature cycle reduce four more suitable for TAIL-PCR reaction.(3) Combining with the results of single factor.Using the orthogonal experiment, screening the best combination. Optimization of TAIL-PCR reaction system as follows: the second reaction system is annealing temperature 61 ℃,1.0U/30 mL Taq polymerase, 30 ng/μL template; Third reaction is annealing temperature 62 ℃, 0.5U/50 mL Taq polymerase, 40 ng/μL template.Using 5 lower specificity cycle, arbitrary degenerate primer is AD3, under these conditions amplification of the stripes are clear, stable and specificity are better it indicates that this system is suitable for TAIL-PCR reaction.(4) After analyzed the T-DNA flanking sequence to the three single positive plant copy of the HrpZpsg12 gene transferred from T4 generation, the No.2 sample got the expected result. The sequence showed that T-DNA was inserted in the Chromosome 5 of the soybean genome. T-DNA is in homologous sequence with the soybean’s basic group from 131084 to 131524. Similarly, after analyzed the T-DNA flanking sequence to the four samples of the BADH gene transferred from T6 generation, only No.3 sample got the satisfied band. According to the sequence results, the T-DNA inserted the 18896 bp of the soybean genome, and the built TAIL-PCR is proved to possess versatility and stability.