| MicroRNAs (miRNAs) are 21～24 nucleotide(nt) long, non-coding small RNAs that exist in almost all eukaryotes. They act by binding to the target mRNAs to negatively regulate gene expression. MiR159 is a kind of highly conserved miRNA which may exist in almost all land plants. Preliminary studies showed that miR159 may have certain correlation with anther and seed germation in some plants. Some studies have already showed that the quantity of miR159 expressed in tomato leaves is relatively higher than other miRNAs, but its factual function in tomato is still unknown. In recently years, researches on siRNA-mediated gene silencing became a hot point, and related studies implied that although siRNAs had some effect in gene-silencing, but owe to its lack of stability in expression and insufficient accuracy in identifying its target sequence, there is still much deficiency and uncertainty in its further application. miRNAs are endogenous, which differ from the siRNAs in their origin, and they have the advantage such as high expression quantity, more stable and unique to the target RNAs compared with siRNAs, so they are thought to be the new focal point in gene silencing related researches.Cucumber mosaic virus (CMV) is a typical tripartite positive single strand RNA (+ssRNA) virus. It has a broad range of hosts and mainly causes host symptoms such as mosaic, dwarf, green islands, chlorisis, yellowing, etc. So it can disturb the normal growth and development of plants. Tomato (Lycopersicon esculentum Mill.) is an indispensable vegetable and fruit in people's daily life, studies on improving its output and quality have been a hot point for a long time. CMV is one of the main causes that lead to the virus diseases in tomatoes, every year the reduction of output caused by CMV has brought serious influence on the nation's economic development and on the agriculture and industry production, so the researches on tomatoes'genetic transformation related with CMV resistance has both important significance on scientific research and economics.In this research we intergrated the above researches such as miRNA mediated gene-silencing and anti-virus related transgene studies in tomato, and transformed the tomato by the mediation of artificial miR159. In this way we preliminarily established the anti-CMV transformation system mediated by amiR159 in tomato. The main work in this paper can be separated into three parts. Firstly, by studies on the tissue culture, and antibiotic sensitive selection culture concentration for the fast regeneration of transgenic receptor tomato—Micro Tom, , we found out the proper explant and culture conditions for callus and shoot induction, and we also found out the optimal culture media for callus and root induction. By these results we set up foundations for Micro Tom's genetic transformation. The ingredient of the callus induction medium is: MS+0.25 mg/L IAA+2mg/L 6-BA, and for the root induction is: MS+0.5 mg/L IBA, while the antibiotic sensitive contration combinations for efficient selecting antibiotic-resistant seedlings is 50 mg/L kanamycin+200mg/L cephamycin. Secondly, we construct the plasmid containing the pre-artificial miR159 (pre-amiR159) named pBI121-(pre-amiR159), then we used the plasmid to transform agrobacterium EHA105 and tomato Micro Tom, and got some antibiotic plantlets, so that we build up the preliminary genetic transformation system mediated by amiR159 in tomato. The process for plasmid constructing is, choose a 21bp long sequence from the ORF in the RNA1 of CMV-Fny as the target of amiR159, and design the pre-amiR159 according to the sequence complementary to it, then synthesize the cDNA of the pre-amiR159 and connect it to the corresponding sites in the plasmid pBI121. The main process for the genetic transformation of tomatoes is, use the transformed agrobacterium to infect the explant , and select the antibiotic-resistant plantlets in antibiotic selection culture medium, etc. We got antibiotic-resistant plantlets in this process. In the course of antibiotic-resistant plantlets selections we investigated and optimized treatment conditions such as the pretreatment of the explant, the concentration of agrobacterium used in infection, the time used in agrobacterium infection, etc. Thirdly, by identifying the successfully transformed tomato from the antibiotic-resistant ones through molecular biology methods, then inoculate them with target virus, we got the comprehensive results for the transformed tomatoes'anti-CMV property. So we validated the effectiveness of the tomato transformation system in resisting CMV guided by amiR159. The molecular biology identifying process mainly refers to the PCR and Southern Bloting techniques in detecting the target sequence in the genome from antibiotic-resistant seedlings, the RT-PCR technique refers to the identifying of the target RNA in the antibiotic-resistant seedlings. In these processes we identified several transformed tomatoes. The virus inoculation experiment is to inoculate CMV-Fny to the transformed tomatoes, and test their anti-virus functions a few days later. By comparing with tomatoes not inoculated, we found that the transformed tomatoes showed resistance to the target virus CMV-Fny to some extent , and their main traits compared with the common tomatoes in that they had less symptoms or even not infected, the growth conditions of the whole plants are relatively better, etc. By integrating these three main results, we preliminarily selected and erected the genetic transformation system for the amiR159 mediated CMV resistance in tomato. |
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