Tobacco(Nictina Tabacum L) Resistance Breeding To PVY, Resistance Genes Clone And Analysis

Potato virus Y (PVY) is one of the most important tobacco diseases in China, As the expand ofgreenhouse vegetable cultivation and the potato planting area in recently years, the occurrenceand damage of PVY to tobacco production have appeared sharp upward trend. A greateconomic loss was taken as the outbreak epidemic of PVY in Heilongjiang, Liaoning,Shandong, Anhui and Yunnan tobacco planting areas successively. In view of the actualsituation of lack of PVY resistance tobacco variety in our country, the PVY resistance sourceselection, genetic analysis for PVY resistance, cloning of PVY resistance gene and PVYresistance breeding have been done in this research, which contents including followings:1.17and114tobacco germ plasma resources were identified for PVY-resistance usingthe artificial inoculation methods in greenhouse and field disease nursery respectively. Thetobacco varieties, C151and CV91, were selected out as high resistance to potato virus Y, andthe varieties, including CV87，87414and NC55were confirmed medium resistance to PVY,which were appeared to horizontal resistance controlled by multiple genes.2. The PVY resistance varieties including C151、CV91、CV87and NC55were geneticanalyzed using hybrid with other materials, the results indicate that C151`PVY-resistance wascontrolled by few genes, which belong to incomplete dominant. CV91`PVY-resistance wascontrolled by few recessive gene，which is impressible of dominant gene susceptible to PVY，it will be appeared resistance hydride with varieties without dominant gene susceptible to PVY.F2generation of CV87Hybrid combination with IslandGold and Longjiang851respectivelyappeared difference in the rate, none disease: slight disease: heavy disease, which performanceof continuous distribution. Which controlled by the majority of minor genes，should be additivegene. The plants rate of none disease: slight disease: heavy disease in the F2generation ofNC55×9625combination≈8-5-3，which performance of continuous distribution. NC55wasshowed controlled by the majority of minor genes.3. Using CV91as a parents, a high resistance to PVY variety, flue-cured tobacco varietiesLongjiang911and hybride LJ237were bred, and LJ237was confirmed as high resistance toPVY and longjiang911was confirmed as medium resistance PVY. In the same time, LJ981have been bred using CV87as a parents.4. The idea concentrating more minor horizontal resistance genes in a variety was putforward in this article. Making use of medium horizontal resistance genes, setting properhybrid groups, concentrating more resistance genes and getting stronger resistance variety thanits parents is possible in their hybrid latter generations. The use of horizontal resistance germplasma resources should be appreciable.5. C151, a variety high resistance to PVY, were used as the experimental materials, Thetobacco leaf SSH Library induced by PVY has been build usingSuppression Subtractive Hybridization, The differential expression fragments in the tobaccoleaf inoculated by PVY were sequenced more than280ESTs. The obtained280ESTs weresequence analyzed and function predicted through blasting in NCBI’s Blast Web site, amongof them,96fragments were predicted to be RuBP carboxylase related to photosynthesis,12fragments were believed to have relative with the chloroplast DNA in Nicotiana sylvestris andNicotiana tomentosiformis，and there are78fragments were predicted to be relate to PVY resistance,：15fragments for Lipid invertase gene,8fragments for mutant catalase gene，26fragments for protease inhibitor gene，26fragments for Ferredoxin gene and Iron sulfur proteingene,3fragments for Glycosyltransferase genes，9fragments for Glycine rich proteinprecursor protein,86fragments for other function genes。6. Real-time PCR analysis was applied to analyze the dynamic expression patterns of fivegenes at five times after inoculation of PVY1-72hours. The results showed that all of that fivesequences were induced expression fragments, the full-length gene relative to PVY resistancewas obtained by the strategy of Rapid amplification of cDNA ends to the five sequences.7. Using homologous sequence method, the translation initiation factor eIF4E which wasrelative to PVY resistance was cloned in tobacco variety CV91.8. Through blast the eIF4E (above) gene sequence with others, two mutant sits have beenfound, which are not happened in susceptibility varieties, which possibly influences virustranslating and then cause the variety to occur resistance.