Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.

[1]  Cornelius S. Barry,et al.  Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato. , 1996, The Plant journal : for cell and molecular biology.

[2]  G. Hannon,et al.  The rest is silence. , 2001, RNA.

[3]  Nektarios Tavernarakis,et al.  Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing. , 2002, Nucleic acids research.

[4]  Y. Meyer,et al.  Preparation by two‐dimensional electrophoresis of proteins for antibody production: Antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts , 1988, Electrophoresis.

[5]  F. B. Abeles.  CHAPTER 9 – The Role of Ethylene in Agriculture , 1992 .

[6]  F. Grosveld,et al.  The tomato RNA-directed RNA polymerase has no effect on gene silencing by RNA interference in transgenic mice , 2002, Transgenic Research.

[7]  Y Tian,et al.  Cloning of 1-aminocyclopropane-1-carboxylate (ACC) synthetase cDNA and the inhibition of fruit ripening by its antisense RNA in transgenic tomato plants. , 1998, Chinese journal of biotechnology.

[8]  P. Sharp,et al.  RNA interference--2001. , 2001, Genes & development.

[9]  G. Tucker,et al.  The effect of chimeric transgene architecture on co-ordinated gene silencing , 1998, Planta.

[10]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[11]  J. Nap,et al.  Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco. , 2000, RNA.

[12]  N. Hoffman,et al.  Ethylene biosynthesis and its regulation in higher plants , 1984 .

[13]  H. Schöb,et al.  Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Olson,et al.  Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Sijen,et al.  Post‐transcriptional gene‐silencing: RNAs on the attack or on the defense? , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[16]  J. Kurreck.  Improvement through novel chemical modifications , 2003 .

[17]  Phillip D. Zamore,et al.  RNA interference: listening to the sound of silence , 2001, Nature Structural Biology.

[18]  S. Mango,et al.  Gene silencing in Caenorhabditis elegans by transitive RNA interference. , 2003, RNA.

[19]  Phillip D. Zamore,et al.  RNA Interference , 2000, Science.

[20]  P. Waterhouse,et al.  Construct design for efficient, effective and high-throughput gene silencing in plants. , 2001, The Plant journal : for cell and molecular biology.

[21]  Marc‐André D'Aoust,et al.  Antisense Inhibition of Tomato Fruit Sucrose Synthase Decreases Fruit Setting and the Sucrose Unloading Capacity of Young Fruit , 1999, Plant Cell.

[22]  B. Cullen,et al.  Sequence requirements for micro RNA processing and function in human cells. , 2003, RNA.

[23]  G. Tucker,et al.  Developmental Abnormalities and Reduced Fruit Softening in Tomato Plants Expressing an Antisense Rab11 GTPase Gene , 2001, The Plant Cell Online.

[24]  D. Grierson,et al.  Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. , 2002, Journal of experimental botany.

[25]  Harry Smith,et al.  Phytochromes and light signal perception by plants—an emerging synthesis , 2000, Nature.

[26]  Neil A. Smith,et al.  Gene expression: Total silencing by intron-spliced hairpin RNAs , 2000, Nature.

[27]  A. Fire,et al.  RNA-triggered gene silencing. , 1999, Trends in genetics : TIG.

[28]  H. Vaucheret,et al.  RNA Silencing in Plants--Defense and Counterdefense , 2001, Science.

[29]  Patrick J. Paddison,et al.  Stable suppression of gene expression by RNAi in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  D. Grierson,et al.  cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant , 1993, Plant Molecular Biology.

[31]  C. Hornyik,et al.  A viral protein suppresses RNA silencing and binds silencing‐generated, 21‐ to 25‐nucleotide double‐stranded RNAs , 2002, The EMBO journal.

[32]  V. Iglesias,et al.  High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Jens Kurreck,et al.  Antisense technologies. Improvement through novel chemical modifications. , 2003, European journal of biochemistry.

[34]  Michel Labouesse,et al.  RNA interference: genetic wand and genetic watchdog , 2000, Nature Cell Biology.

[35]  P. Genschik,et al.  Identification of a new cis-regulatory element in a Nicotiana tabacum polyubiquitin gene promoter , 1997, Molecular and General Genetics MGG.

[36]  D. Grierson,et al.  Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model. , 2000, Plant physiology.

[37]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[38]  E. Meyerowitz,et al.  Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  F. B. Abeles,et al.  Ethylene in Plant Biology , 2022 .