{"title":"利用实时定量 PCR 测量产生人干扰素-γ(hIFN-γ)的转基因烟草植物的转基因拷贝数","authors":"Reza Heidari Japelaghi, Raheem Haddad, Mostafa Valizadeh, Ebrahim Dorani Uliaie, Mokhtar Jalali Javaran","doi":"10.1007/s13562-024-00879-z","DOIUrl":null,"url":null,"abstract":"<p>In transgenic plants, the transgene copy numbers can highly affect the level of expression and genetic stability of the transgene. Hence, the first step in their characterization is the estimation of transgene copy numbers integrated in the plant genome. Quantitative real-time PCR (qRT-PCR) was used to determine the copy numbers of human interferon-γ (<i>hIFN</i>-γ) and hygromycin phosphortransferase II (<i>hpt</i>II) transgenes in the genome of the T<sub>0</sub> generation of 18 transgenic tobacco lines using the <i>axi</i>1 gene as an endogenous control. With optimized PCR conditions, we attained highly exact estimates of one, two, three, and/or four transgene copies in the T<sub>0</sub> transformants. Moreover, estimation of copy numbers of the <i>hIFN</i>-γ transgene and the <i>hpt</i>II selective marker gene indicated that rearrangements of the T-DNA has regularly happened in transgenic tobacco. Transgene copy number was also estimated using Southern blot analysis of gDNA derived from transformants. The transcript level and expression amount of recombinant hIFN-γ protein were evaluated in various events using RT-PCR and enzyme-linked immunosorbent assay (ELISA) techniques. A disagreement between the transcript level and the amount of recombinant protein with an inverse correlation between transgene copy number and expression level observed in some events, probably showing translational gene silencing and co-suppression or silencing, respectively. These results were also compared with segregation ratios of hygromycin-resistant phenotype in T<sub>1</sub> plants of each line and found to be, in general, consistent.</p>","PeriodicalId":16835,"journal":{"name":"Journal of Plant Biochemistry and Biotechnology","volume":"30 1","pages":""},"PeriodicalIF":1.6000,"publicationDate":"2024-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Measurement of transgene copy number in transgenic tobacco plants producing human interferon-γ (hIFN-γ) using quantitative real-time PCR\",\"authors\":\"Reza Heidari Japelaghi, Raheem Haddad, Mostafa Valizadeh, Ebrahim Dorani Uliaie, Mokhtar Jalali Javaran\",\"doi\":\"10.1007/s13562-024-00879-z\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>In transgenic plants, the transgene copy numbers can highly affect the level of expression and genetic stability of the transgene. Hence, the first step in their characterization is the estimation of transgene copy numbers integrated in the plant genome. Quantitative real-time PCR (qRT-PCR) was used to determine the copy numbers of human interferon-γ (<i>hIFN</i>-γ) and hygromycin phosphortransferase II (<i>hpt</i>II) transgenes in the genome of the T<sub>0</sub> generation of 18 transgenic tobacco lines using the <i>axi</i>1 gene as an endogenous control. With optimized PCR conditions, we attained highly exact estimates of one, two, three, and/or four transgene copies in the T<sub>0</sub> transformants. Moreover, estimation of copy numbers of the <i>hIFN</i>-γ transgene and the <i>hpt</i>II selective marker gene indicated that rearrangements of the T-DNA has regularly happened in transgenic tobacco. Transgene copy number was also estimated using Southern blot analysis of gDNA derived from transformants. The transcript level and expression amount of recombinant hIFN-γ protein were evaluated in various events using RT-PCR and enzyme-linked immunosorbent assay (ELISA) techniques. A disagreement between the transcript level and the amount of recombinant protein with an inverse correlation between transgene copy number and expression level observed in some events, probably showing translational gene silencing and co-suppression or silencing, respectively. These results were also compared with segregation ratios of hygromycin-resistant phenotype in T<sub>1</sub> plants of each line and found to be, in general, consistent.</p>\",\"PeriodicalId\":16835,\"journal\":{\"name\":\"Journal of Plant Biochemistry and Biotechnology\",\"volume\":\"30 1\",\"pages\":\"\"},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2024-03-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Plant Biochemistry and Biotechnology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s13562-024-00879-z\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Plant Biochemistry and Biotechnology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s13562-024-00879-z","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Measurement of transgene copy number in transgenic tobacco plants producing human interferon-γ (hIFN-γ) using quantitative real-time PCR
In transgenic plants, the transgene copy numbers can highly affect the level of expression and genetic stability of the transgene. Hence, the first step in their characterization is the estimation of transgene copy numbers integrated in the plant genome. Quantitative real-time PCR (qRT-PCR) was used to determine the copy numbers of human interferon-γ (hIFN-γ) and hygromycin phosphortransferase II (hptII) transgenes in the genome of the T0 generation of 18 transgenic tobacco lines using the axi1 gene as an endogenous control. With optimized PCR conditions, we attained highly exact estimates of one, two, three, and/or four transgene copies in the T0 transformants. Moreover, estimation of copy numbers of the hIFN-γ transgene and the hptII selective marker gene indicated that rearrangements of the T-DNA has regularly happened in transgenic tobacco. Transgene copy number was also estimated using Southern blot analysis of gDNA derived from transformants. The transcript level and expression amount of recombinant hIFN-γ protein were evaluated in various events using RT-PCR and enzyme-linked immunosorbent assay (ELISA) techniques. A disagreement between the transcript level and the amount of recombinant protein with an inverse correlation between transgene copy number and expression level observed in some events, probably showing translational gene silencing and co-suppression or silencing, respectively. These results were also compared with segregation ratios of hygromycin-resistant phenotype in T1 plants of each line and found to be, in general, consistent.
期刊介绍:
The Journal publishes review articles, research papers, short communications and commentaries in the areas of plant biochemistry, plant molecular biology, microbial and molecular genetics, DNA finger printing, micropropagation, and plant biotechnology including plant genetic engineering, new molecular tools and techniques, genomics & bioinformatics.