The Study On Detection Technology Of Transgenic Bt Rice Huahui No.1

With the rapid development of transgenic technology, more and more transgenic crops have been created and commercializated, it is necessary to establish the accurate, reliable systems of technology and methods for identification of the GMOs. The researches were focused on the transgenic Bt rice Huahui No.1, systems of detection studies were done on the integration and transcriptional level, and unintended effects were also preliminarily studied. The main results were as follows:The system of technology was optimized and developed to detect the transgenic components on the DNA level in transgenic Bt rice. Based on the specific structures of rice endogenous reference gene Sucrose Phosphate Synthase and the elements of CaMV35S, NOS, HPT, CrylAb/Ac of pGL2RC7and pFHBTl, which are used for Huahui No.1as the transgenic vector plasmid, a series of primers were designed for PCR, and then the target DNA sequences of Huahui No.1could be accurately detected by qualitative PCR. The results of sensitivity detection show that0.05ng/μL is the limit of detection of DNA concentration, which is equivalent to100copies haploid rice genomic DNA, which shows that the primers were quietly specific, accurate and sensitive.The temporal and spatial expression of exogenous marker gene and the target gene in Huahui No.1was detected on the transcriptional level. The results of RT-PCR show that the fragment of antibiotic marker gene has been detected in the genomic DNA, but it was not expressed on the transcriptional level, the target gene was expressed stablely. The results that quantitative temporal and spatial expression of the target gene was analyzed by qRT-PCR show that the insect gene CrylAb/Ac in Huahui No.1was expressed in all different tissues during the whole growth period, but the amount of expression was different. We compared the expression levels of the insect gene in different period and in different tissues by the relative quantitative method, using the account of expression in the stem of seedling stage as a reference, the account in the ear bract of heading stage and grain filling stage is more than10times higher than the account in the stem of seedling stage; The account in the stem of grain filling stage and jointing stag is more than5times higher than the account in the stem of seedling stage; The account in the stem of heading stage, leaf of grain filling stage, leaf of heading stage, top leaf of jointing stage, stem of tillering stage, middle leaf of jointing stage, fringe of grain filling stage is more than2times higher than the account in the stem of seedling stage; The account in the leaf of seedling stage, leaf of tillering stage and blow leaf of jointing stage is slightly higher than the account in the stem of seedling stage; The account in the fringe of heading stage is lower than the account in the stem of seedling stage.The genes of key enzyme in the metabolic pathway of nutritional components and Anti-nutrients were detected on the transcriptional level in the leaf and fringe of grain filling stage Huahui No.1, to identify the unintended effects. The results show that:the transformation of Bt gene make the expression account of the key enzyme gene significantly decrease on the transcriptional level in the leaf and fringe of grain filling stage of the rice Huahui No.1. We compared the expression levels of the gene of key enzyme in the leaf and fringe of grain filling stage of transgenic rice Huahui No.l and its non-transgenic counterparts Minghui63by the relative quantitative method, using the account of expression in the leaf of grain filling stage of Minghui63as a reference, the account of expression of SPS in the leaf of grain filling stage of Huahui No.1is down by19%, the account in the fringe of grain filling stage is down by24%; the account of expression of ADPGase in the leaf of grain filling stage of Huahui No.1is not changed, the account in the fringe of grain filling stage is down by86%; the account of expression of GME in the leaf of grain filling stage of Huahui No.1is down by57%, the account in the fringe of grain filling stage is down by21%; the account of expression of PK in the leaf of grain filling stage of Huahui No.l is down by14%, the account in the fringe of grain filling stage is down by38%; the account of expression of PDX in the leaf of grain filling stage of Huahui No.l is down by82%, the account in the fringe of grain filling stage is down by60%; the account of expression of MIPS in the leaf of grain filling stage of Huahui No.l is improved by128%, the account in the fringe of grain filling stage is down by67%, which shows that the changes of metabolic processes in plants on transcriptional level might be caused by the transformation of foreign genes, it is of significance to study on unintended effects for the assessment of safety of GMP, the unintended effects was preliminarily detected only on the transcriptional level in this study, but their accurate effect on the metabolic components is for further study.