Silencing MYB Gene Of Flue-cured Tobacco Using VIGS Technique

VIGS technique is a rapid and effective method of characterizing gene functions, which involves a virus delivery vector carrying a short sequence of targeted gene to infect a young plant, and in a few weeks natural defense mechanisms of the plant result in silencing of the targeted gene.The flower color of tobacco mainly determined by flavonoids. Recent research demonstrates the flavonoid biosynthetic pathway of Higher plants is regulated by MYB transcription factor.The research constructed the gene silencing vector of VIGS technique, by searching the MYB gene of transcription factor family in the database. And using the Agrobacterium-mediated transformation to introduced the vector to Yunyan87and transgenic tobacco Y3. By optimizing the infection system, detecting targeted gene expression level, we got the the tobacco which is silenced by VIGS.The results showd that:1. Successfully constructed6silencing vectors with targeted MYB gene, pCAM:pGR106-S74、pCAM:pGR106-S77、pCAM:pGR106-S79. pCAM:pGR106-S85, pCAM:pGR106-S87�'�pCAM:pGR106-S89,after inserting selective targeted MYB gene to viral vector, and confirmed it by the digestion-detection and the sequences results comparison.2. The result shows that the growth of tobacco and Agrobacterium infection system has a great effect on infection effect. Because Agrobacterium-mediated infected epigeal cotyledon, the symptom of virus appear on the tobacco seeding of Yunyan87. The speed of infection is slower than the lower true leaf, and the infection effect is worse than the lower true leaf. Because the three leaves tobacco is too small, the leave appeared bacterial infections after infected, then all of three leaves tobacco wilted. So, to the Agrobacterium infection system is best for choosing the lower true leaf to infected, and infect on the four leaves or five leaves.3. Most transgenic tobacco Y3which is successfully infected has the situation that the targeted gene expression level decreased. The MYB gene expression level of the tobacco which is infected by silencing vector pCAM:pGR106-S79apparently decreased. The decline in multiples reached4.5-10.The research successfully constructed the system of VIGS silencing vector, optimized the infection system, defined the best infection period of tobacco, and got the infected tobacco which targeted gene expression leves declined, and provide the precondition to extend the identification of gene function using VIGS.