| The studies of transgenic rice in China are leading level of the world. Some disease-resistance and insect-resistant transgenic rice lines have entered the stage of application for the production of planting bio-security certificate. Because of their GMO safety and other issues, no lines has been approved of commercial production applications.The molecular analysis can be an important aspect of genetically modified crops biological safety evaluation, which is required in genetically modified organisms safety regulations whether in China or in other countries internationally. In this research, chromosome location of T-DNA inserted in RSV- resistant transgenic rice is by Tail-PCR, Genome walking and Southern hybridization. And we could study whether there are differences in gene expression in transgenic rice KRSV by DEG sequencing and Swath sequencing. The safety assessment of genetically modified rice is to lay the foundation for the promotion of anti-RSV. The results and conclusions are as follows:(1) Genome DNA of transgenic rice KRSV-1 and non-transgenic rice was single-enzyme digested with Hind III and Eco RI. The result of southern hybridization was that genetically modified material showed a single band while non-GM material showed no band. The result indicated that exogenous gene in KRSV-1 is a single copy.(2) Unknown flanking sequence of T-DNA in KRSV-1 was amplified by Hi Tail-PCR and genome walking. The results showed that the left border is flanked by a chromosome 2sequence and the right border is flanked by a chromosome 8 sequence and the right border is flanked by a chromosome 8 sequence. T-DNA carrying the 1.6kb DNA fragment of chromosome 8 was translocated to chromosome 2. The inserted site was at nucleotide positions between 2,443,520 and 22,443,553 of chromosome 2. A 32-bp deletion at the chromosome 8 locus and a 3-bp filler sequence 5’GCA is present at the junction of chromosome 2 and 8.(3) The results of Genome walking showed that the translocated chromosome 8 loci was restored to the native status. Blast analysis showed that the chromosome 2 breakpointwas located between two genes. The chromosome 8 translocated sequences was located in a pseudogene(16,434,112 and 16,434,890) and a hypothetical ORF(16,435,494- 16,435,712).(4) Extracted m RNA of tillering stage of rice leaf was analyzed by DGE sequencing.There are 7434 m RNAs expressing significantly(P <0.05) in genetically modified anti-RSV rice comparing with its wild rice. These m RNAs in transgenic rice distribute on all 12 chromosomes and do not focus on the inserted point in chromosome 2. It suggested that exogenous gene into rice occurred unintended effects. Further screening of the m RNA data,new protein and allergen-induced toxic protein m RNA were not discovered.(5) Seed proteins of anti-RSV transgenic rice and wild rice were analyzed by Swath sequencing. 1887 quantitative proteins were marked and there were 357 proteins expressing significantly in genetically modified anti-RSV rice comparing with its wild rice. Comparing with wild rice, the expression of thermal protein is different very significantly and the expression of 20 s, 40 s, 60 s ribosomal protein, ATP synthesis associated transporter protein,several chaperones, gluten, histones, and several defense response proteins induced by pathogenic bacteria are quite different in transgenic rice. The expression of original allergens didn’t appear significantly different and new allergen protein was not retrieved. |
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