Development of a Virus-Induced Gene Silencing System for Cotton and Its Application for Functional Genomics in Fiber.

摘要

Cotton is an economically important crop plant grown worldwide for the long, spinnable fibers that derive from the surface of the cottonseed. The large genome of cultivated cotton has complicated both traditional breeding and genome assembly. Biotechnological improvements have been hampered by cotton's recalcitrance to transformation. An improved understanding of the function of genes unique to the cotton fiber could guide transformation-based initiatives leading to improvements in fiber qualities.;Virus-induced gene silencing (VIGS) is a reverse-genetics technique that can be used to investigate gene function by producing a rapid, sequence-specific knockdown (silenced) phenotype for a target gene without the need for transformation. Here I describe the development of a set of cotton VIGS vectors for delivery by Agrobacterium or biolistics, optimization of environmental conditions for vegetative gene silencing, and characterization of both transgene and endogenous gene silencing in cotton fiber.;The objectives of this work were to: (1) develop a VIGS vector from the bipartite geminivirus Cotton leaf crumple virus (CLCrV) and (2) characterize its ability to silence genes in cotton fiber. Standard molecular cloning techniques were used to replace the coat protein gene of the CLCrV A component with a poly-linker that would allow insertion of up to 750-bp of targeting sequence. The chlorophyll biosynthetic gene Magnesium Chelatase (ChlI) was used as a visible marker for vegetative gene silencing. Following particle bombardment of cotton cotyledons, I observed the onset of ChlI silencing in new growth beginning between 12 and 21 days post infection. Silencing of ChlI was significantly better at 22°/18°C (day/night) than at 30°/26°C and the viral vector showed over a 10-fold increase in accumulation at the lower temperature regime.;The CLCrV-B component was tested as an insertion vector for carrying short gene silencing fragments. A 28-bp ChlI insert in the CLCrV-B component produced limited silencing that was correlated with GFP expression when it was cobombarded with the CLCrV-A component carrying a GFP. Silencing became systemic when a wildtype CLCrV-A component was used. A 21-bp ChlI fragment failed to induce silencing while a 55-bp fragment failed to produce an infection. Additionally, I assembled and tested two low cost particle delivery devices from common, off-the-shelf components. These guns will make molecular techniques, such as RNAi and transient expression, readily available to a greater audience.;To characterize the vector's ability to silence genes in cotton fiber, I silenced a 35S:mGFP5-ER reporter gene (a gift from K. Rathore). Vegetative GFP silencing in cotton resulted in a sectored phenotype, similar to what was observed for the endogenous ChlI gene. I developed a 96-well assay for comparing GFP fluorescence in fibers from different ovules. This assay suggested that silencing within a locule was uniform. I used quantitative RT-PCR to demonstrate a 4.3 fold reduction in GFP transcript in cotton fiber at 15 days post anthesis.;To test endogenous gene silencing in cotton fiber, I targeted the alpha-expansin 1 (GhEXPA1) gene. Expansins play a role in cell wall loosening during cell expansion. Silencing of GhExpA1 was not evident at 12 Days Post Anthesis (DPA) but there was a 5.6-fold reduction in transcript at 16 DPA compared to plants inoculated with CLCrV carrying a non-homologous insert as a control. This resulted in an 8.8% reduction in the length of mature cotton fibers. These results verify the usefulness of VIGS for investigating gene function in cotton fiber but suggest that the cotton fiber is only susceptible to silencing at certain times in development.