| N.tomentosiformis Goodspeed is categorized to Nicotiana, Subgenus Tabacum, Section Tomentosae and its somatic cell chromosome number is 2n=12II. Nicotiana tabacum (SSTT) origins from the hybrid of the ancestor of N.sylvestris (SS) and ancestor of N.tomentosiformis (TT) or N.otophora (TT) after the chromosomes being doubled naturally. For the narrow genentic background of tobacco varieties and shortage of molecular markers with polymorphism, the development of tobacco breeding is limited. Based on the N.tomentosiformis Goodspeed genome DNA datum, SSR sequences were searched by the software SSRFINDER with the searching condition of length of motifs 2～5bp and repeats of motifs 10～60. The information of SSR sequences was analysed and SSR primers were designed by the software PRIMER 5.0 , based on the conserved sequences of both sides of SSR.Axillary bud will grow quickly after decapitation during tobacco leaf production. Growing of axillary bud will consume a large number of nutrients.If the axillary bud had not been removed in time , the yield of tobacco leaf would reduce and the quality of tobacco leaf would decline.Axillary bud is initiated by the the formation of axillary meristems,which develop postembryonicly.Many regulatory genens , whose encoding proteins belong to the plant specific protein family GRAS.Mutation or function missing of these genes would affect the formation of axillary meristems.The NtLS gene(EU935581) of tobacco was cloned by RACE technology in our lab.The sequences of NtLS highly similar with that of LS/LAS/MOC1 ,which regulate the formation of axillary meristems.In order to difine the fuction of NtLS ,the interference fragment was choosen from NtLS and the RNAi expression vector was constructed and transformed into Nicotiana tabacum W38 by Agrobacterium-mediated transformation using the leaves disc methods.The main results as followed:(1) By searching N. tomentosiformis Goodspeed genome DNA with SSRFINDER ,6441 SSRs were identified from 5227 scaffold. Total length about 300kb of SSRs occupied approximately 0.12‰of genome DNA. The average distance between SSRs was about 400kb. Dinucleotide and trinucleotide motifs were most abundant classes, 45.2% and 49.6% respectively. The average length of dinucleotide, trinucleotide, tetranucleotide and pentanucleotide motifs are 39bp,53bp,66bp and 75bp respectively.(2) The number of 10～20 repeats,21～30 repeats,31～40 repeats,41～50 repeats and 51～60 repeats are 4809(74.6%), 963(15.0%), 349(5.4%), 205(3.2%) and 113(1.8%) respectively.(3) CT is the most abundant type in the 11 types of dinucleotide motifs,GAA is the most abundant type in the 51 types of trinucleotide motifs,AAAG is the most abundant type in the 44 tetranucleotide motifs and CGGGA is the most abundant type in the 45 types of pentanucleotide motifs.The GC content of all SSR sequences is 33.5%.(4) Based on the conserved sequences of both sides of SSR, SSR primers were designed by the software PRIMER 5.0. There are 6020 SSR primers avaible, which meet the primer searching conditions.It provides a base for the development and application of SSR molecular markers in tobacco.(5) The interference fragment was choosen from NtLS and the RNAi expression vector pART27-FR was constructed successfully by using the pKANNIBAL vector and pART27 vector.(6) The the RNAi expression vector pART27-FR was transformed into Nicotiana tabacum W38 by Agrobacterium-mediated transformation using the leaves disc methods.Testing results show that we gained positive transformants.It provides genetic materials for the research of the formation of axillary bud and regulatory mechanism in tobacco. |
