Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.     

Development of a virus induced gene silencing vector from a legumes infecting tobamovirus

Medicago truncatula, the model plant of legumes, is well characterized, but there is only a little knowledge about it as a viral host. Viral vectors can be used for expressing foreign genes or for virus-induced gene silencing (VIGS), what is a fast and powerful tool to determine gene functions in plants. Viral vectors effective on Nicotiana benthamiana have been constructed from a number of viruses, however, only few of them were effective in other plants. A Tobamovirus, Sunnhemp mosaic virus (SHMV) systemically infects Medicago truncatula without causing severe symptoms. To set up a viral vector for Medicago truncatula, we prepared an infectious cDNA clone of SHMV. We constructed two VIGS vectors differing in the promoter element to drive foreign gene expression. The vectors were effective both in the expression and in the silencing of a transgene Green Fluorescent Protein (GFP) and in silencing of an endogenous gene Phytoene desaturase (PDS) on N. benthamiana. Still only one of the vectors was able to successfully silence the endogenous Chlorata 42 gene in M. truncatula.     

病毒诱导烟草的基因沉默

病毒诱导基因沉默是利用RNA介导病毒防卫机制的一项技术。构建含有目的基因片段的人工改造病毒载体,通过农杆菌侵染导致植物内源目的基因沉默。为建立病毒诱导基因沉默体系,选用烟草脆裂病毒（TRV）和烟草为实验材料。构建了八氢番茄红素去饱和酶基因（PDS）的基因沉默病毒载体,病毒载体侵染结果显示目的基因PDS沉默导致烟草幼苗出现光漂白现象。采用RT-PCR的方法检测目的基因PDS的沉默效果,结果显示PDS基因mRNA被显著降解。该体系的建市有利于将来对植物基因进行高通量功能分析。     

Silencing of a meristematic gene using geminivirus-derived vectors

Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants.     

采用玉米Ubi-1启动子获得低拷贝转基因玉米植株

通过基因枪粒子轰击和草丁膦（PPT）选择获得可育的玉米转基因植株,并分析了外源基因在转化体中的拷贝数与启动子之间的关系。用玉米Ubi-1启动子驱动外源基因,玉米转化体中外源基因的拷贝数较低;可能的原因为Ubi-1启动子通过与其内部同源序列发生重组而被定点整合进玉米基因组,共转化的两种质粒DNA在整合至玉米染色体DNA之前已重构成为一个整体。结果显示使用某一植物自身基因的启动子可以降低外源基因在该物种转基因个体中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因玉米种子。     

A modified viral satellite DNA that suppresses gene expression in plants

DNAbeta is a type of single-stranded (ss) circular satellite DNA found in association with monopartite-genome begomoviruses, such as Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10). Y10 DNAbeta is required for symptom expression in plants but depends on TYLCCNV-Y10 genomic DNA (DNA-A) for replication and encapsidation. When we converted DNAbeta into a gene-silencing vector (modified DNAbeta (DNAmbeta)) by replacing its C1 open-reading frame (ORF) with a multiple cloning site (MCS), it was replicated but no longer induced symptoms in association with TYLCCNV-Y10 DNA-A, so allowing the effects of gene inserts to be recognized easily. Insertion into DNAmbeta of sequences from any of the three host genes (proliferating cell nuclear antigen (PCNA), phytoene desaturase (PDS), and sulfur (Su)), or from a transgene (green fluorescent protein (GFP)), resulted in silencing of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than a month and was associated with decreased levels of mRNA of the gene targeted. Although DNAmbeta probably does not enter meristematic tissue, the PCNA gene could be silenced there. DNAmbeta was an effective silencing vector in tested N. glutinosa, N. tabacum Samsun (NN or nn), and Lycopersicon esculentum plants, and was able to silence two genes simultaneously. This satellite DNA vector-based form of virus-induced gene silencing (VIGS) promises to be applicable to other begomovirus/DNAbeta systems, which are recently reported to occur in several dicotyledonous crop species, thereby providing a powerful approach to gene discovery and the analysis of gene function in these crops.     

Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.