ABSTRACT
Stable transgenic rice line (named KRSV-1) with strong resistance against rice stripe virus was generated using the genesequence of disease-specific protein by RNA interference. Comprehensive safety assessment of transgenic plants has turnedinto a significant field of genetic modification food safety. In this study, a safety assessment of KRSV-1 was carried out in astepwise approach. The molecular analysis exhibited that KRSV-1 harbored one copy number of transgene, which wasintegrated into the intergenic non-coding region of chromosome 2 associated with inter-chromosomal translocations of 1.6-kb segments of chromosome 8. Then, transcriptomics and proteomics analyses were carried out to detect the unintendedeffects as a result of the integration of the transgene. Although 650 dramatically differentially expressed genes (DDEGs)and 357 differentially expressed proteins were detected between KRSV-1 and wild-type (WT) by transcriptomics andproteomics analyses, no harmful members in the form of toxic proteins and allergens were observed. Encouragingly, thenutritional compositions of seeds from KRSV-1 were comparable with WT seeds. The results of this entire study ofmolecular analysis, transcriptome and proteome profile of KRSV-1 revealed that no detrimental changes in the form of toxicproteins and allergens were detected in the transgenic rice line due to the integration of the transgene.
ABSTRACT
Objective Under the methodology of differential proteomics and bioinformatics,the impact of the exogenous gene on the expression of rice in the proteome is discussed,aiming to explore into the study of genetically modified rice in the proteomics. Methods The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63 (MH63),the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional poly?acrylamide gel(2D-PAGE)electrophoresis spectrum;then,the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results The comparing and matching of protein spots between transgenic Bt (cry1Ab/1Ac)rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-trans?genic rice as a reference,transgenic Bt rice held 18 relatively high and 10 relatively low expressions;mass spectrometry and bioinfor?matics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy me?tabolism,protein synthesis,redox stress response and other biological processes. Conclusion Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome;however,there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.
ABSTRACT
Objective: Under the methodology of differential proteomics and bioinformatics, the impact of the exogenous gene on the expression of rice in the proteome is discussed, aiming to explore into the study of genetically modified rice in the proteomics. Methods: The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63(MH63), the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional polyacrylamide gel(2D-PAGE) electrophoresis spectrum; then, the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results: The comparing and matching of protein spots between transgenic Bt(cry1Ab/1Ac) rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-transgenic rice as a reference, transgenic Bt rice held 18 relatively high and 10 relatively low expressions; mass spectrometry and bioinformatics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy metabolism, protein synthesis, redox stress response and other biological processes. Conclusion: Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome; however, there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.
ABSTRACT
Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.
Subject(s)
Animals , DNA Primers , Disease Resistance , Genetics , Oryza , Genetics , Plant Proteins , Genetics , Plants, Genetically Modified , Genetics , Polymerase Chain Reaction , Protein Serine-Threonine Kinases , Genetics , SeedsABSTRACT
A twin T-DNA system is a convenient strategy for creating selectable marker-free transgenic plants. The standard transformation plasmid, pCAMBIA 1300, was modified into a binary vector consisting of two separate T-DNAs, one of which contained the hygromycin phosphotransferase (hpt) marker gene. Using this binary vector, we constructed two vectors that expressed inverted-repeat (IR) structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene. Transgenic rice lines were obtained via Agrobacterium-mediated transformation. Seven independent clones harbouring both the hpt marker gene and the target genes (RSV CP or SP) were obtained in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T1 plants were 8.72% for pDTRSVCP and 12.33% for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were strongly resistant to RSV. A molecular analysis of the resistant transgenic plants confirmed the stable integration and expression of the target genes. The resistant transgenic plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, suggesting that RNAi induced the viral resistance.
ABSTRACT
The present study assessed the efficacy of a heat-inducible cre gene for conditional removal of the marker gene from a rice genome via Cre-lox recombination. A cre gene controlled by the soybean heat-shock promoter was introduced into the rice genome along with the recombination target (lox) construct. Cre-mediated recombination was expected to remove the marker gene and activate the promoter-less GUS gene. Six transgenic lines displayed well-regulated heat-inducible Cre activity in the callus. However, only one line that contained a single copy of the cre gene maintained this property in the regenerated plants and their progeny. Marker-free progeny were obtained from the plant that was heat-treated at the seedling stage, indicating the inheritance of the recombination ‘footprint’. The presence of the ‘footprint’ was verified by polymerase chain reaction and Southern analysis. Therefore, the cre gene controlled by the soybean heat-shock promoter is an effective tool for conditional removal of the marker gene in rice
ABSTRACT
Adiponectin is a polypeptide specifically secreted from human adipocytes, and its deficiency is closely linked to increased obesity and type II diabetes. There is an urgent demand for large-scale production of human adiponectin for pharmaceutical applications. Here, we report that we have successfully obtained a high-level of expression of modified genes encoding human adiponectin in transgenic rice. The 735 bp cDNA of the native human sequence was adopted to rice codon usage, fused to the translation initiation sequence in the N terminus and to the KDEL signal sequence in the C terminus. An amplification promoting sequence acting as an enhancer of transcription was also introduced to enhance gene expression. The presence of the transgene and mRNA transcripts was confirmed by PCR, Southern blot and RT-PCR. Western blot analysis revealed that a protein of approximately 30 kDa was produced in rice leaves. ELISA analysis was used to determine the amount of recombinant adiponectin in transformants with the modified gene in up to 0.32% of total soluble leaf protein. Our results establish the feasibility of high-level expression of recombinant human adiponectin in transgenic rice.