The Construcion And Analysis Of Roots Suppression Subtraction Hybridization CDNA Library Before And After Tobacco Topping

The nicotine is a tobacco-specific alkaloid, a secondary metabolite synthesized in the root.The biosynthesis pathway of nicotine has been studied quite clearly.That is the pyrrole ring is formed from the arginine or ornithine path-way, and the pyrimidine ring is formed from the aspartate path-way, and then both rings condensed into nicotine. After topping, the content of nicotine accumulated, but the reason why the content increased and the regulation mechanism of nicotine are not clear. The induced feature of alkaloid biosynthesis showed that the alkaloid synthesis process must be the result of signal transduction. Although many key genes involved in alkaloid biosynthesis have been cloned, the regulation genes which is confirmed and involved in alkaloid biosynthesis are few. Therefore, suppression subtractive hybridization was adapted to construct a cDNA library of root tip before and after topping and screen differentially expressed genes, which has great potential to reveal the molecular basis referred to the impact of topping on the nicotine contentIn this study, Plant material is nicotiana tabacum L. (ecotype K326) topped after 24h were taken as material, the topped plants as the experimental group (tester), untopped as control group (driver) and conversely. The SSH-cDNA library of root tip in tobacco before and after topping was constructed by high sensitivity and high output rate of suppression subtractive hybridization, and then forward and reverse subtractive library were got, each containing 1000 clones. Positive clones obtained through screening of the library were sequenced, then the result was analyzed on bioinformatics and it is expected that the molecular mechanism of nicotine biosynthesis was explored at the level of genomic.The major findings:1.SSH was adapted to construct a cDNA library of root tip successfully before and after topping. After cloning and transforming, the size of inserting cDNA was between 200 and 700bp confirmed through colony PCR.It was consistent with the expected requirements, and lain a foundation for high efficient screening of differentially expressed genes.2.450 clones of hybridization signal significantly different were screened from 1300 clones initially verified by colony PCR using reverse Northern hybridization. The 450 clones were sequenced, then the results were analyzed with ESTs Bioinformatics, including some genes and transcription factors involved in regulating nicotine synthesis were found. Three ESTs that code PMT, ODC, MADS-box respectively were chosen to analyze the expression pattern before and after tobacco topping with RT-PCR, and the result indicated that expression level of the three ESTs were higher after topping.3. After functional annotation on the non-redundant EST sequences, we found many genes involved in metabolic activity in plants, such as synthesis of the plant hormones, the plant secondary metabolism. The results showed that the library we built was satisfactory and achieved the desired goals, and established the foundation for further study on the molecular mechanism of nicotine biosynthesis.