Construction And Analysis Of Activation Tagging Population In Tobacco (Nicotina Tabacum L.)

Tobacco(Nicotina tabacum L.) is an important economic crop and a dicotyledonous model plant for research. Recently, the draft genomes of N. sylvestris and N. tomentosiformis have been assembled. In the tobacco post-sequencing era, analysis of genetic mutations is believed to be one of the most effective ways to investigate gene function. As a part of the Tobacco Genome Initiative (TGI), a population of approximately 100,000 independent activation tagging lines was established. Phenotypic screening was performed for the activation tagging lines as well as the distributions of T-DNA insertions and expression analysis of the genes adjacent to the activation tag.1. The donor material used for generating tobacco mutant pools was Ncotiana tabacum L. (Honghuadajinyuan), a key cultivar planted widely in China. An activation-tagging T-DNA harboring the 35 S enhancer was introduced into the tobacco genome using an optimized Agrobacterium-mediated transformation approach. In our transformation approach, it was effective that using the antibiotic Timentin to elimination of A. tumefaciens at concentrations ranging from 100-150 mg/L. The co-cultivation and sub-cultivation in the dark is believed to be critical to the success of the optimized transformation protocol. A high positive result of PCR testing in transformed plants was also observed under the selective presence of Basta. Southern blot analysis showed that about 88%of individual lines tested carried 1-2 T-DNA copies with an average of 1.6 per line. Thus, approximately 136,000 T-DNA inserts were made.2. Phenotypic screening had been performed for activation lines for 3 years, approximately 1,000 mutant lines with obvious phenotype changes havd been identified. These phenotypes included abnormalities in leaf and flower morphology, plant height, flowering time, branching and fertility. In the T1 generation planted in 2012,311 mutant lines with obvious phenotype changes have been identified in 4105 mutant lines. When 234 seeds from these lines were germinated,59 lines (1/4) showed the same phenotypes as the parent Tl transgenic plants of which 36 lines (1/6) showed dominant or semi-dominant phenotype.3. To facilitate the use of these tagged lines, we isolated genomic sequences flanking the inserted T-DNA via a fusion primer and nested integrated PCR (FPNI-PCR). Till now,1,417 sequences have been amplified from 1,257 T1 transgenic lines. These isolated sequences were analyzed by the BLASTn homology search program, using the Nicotiana tomentosiformis and Nicotiana sylvestris scaffold sequences.998 FSTs (flanking sequence tags) was generated and represent 963 distinct insertion sites. Distributions of T-DNA insertions showed that higher insertion frequency correlates well with the regions of higher gene density in genome. Distribution of FSTs in genic and intergenic regions was also performed and a higher integration frequency was observed in genie region and the region of 5 kb away from a gene. The T-DNA insertion bias the UTR region, and 5’UTR had a higher frequency of insertions than 3’UTR..4. To monitor the perturbation of gene expression in our population caused by the activation tag, fifteen T2 transgenic lines that showed the same phenotypes as the T1 transgenic parent lines were selected at random. The expression of those genes closest to the T-DNA integration sites was analyzed by RT-PCR using gene-specific primers. Fifteen candidate genes were established and 7 candidate genes was observed in greater expression fashion. The distance between the T-DNA enhancer sequence and ATGs position varies from 750 bp to 13.1 kb. One gene close to the LB of the T-DNA can also be activated as well as the last 6 genes close to the RB of the T-DNA. Gene activation was observed both downstream and upstream of the enhancers and both to the right and to the left of the T-DNA. No correlation was observed between the degree of activation and distance.