In order to obtain the full-length sequence of NtOR and reveal its biological function,the full-length cDNA and genomic DNA sequences of NtOR were cloned from cv.K326 using homologous cloning method,and logged in GenBank(accession numbers JN379458 and JN375578).The results of bioinformatics analysis showed that the full-length of genomic DNA and cDNA were 5027 bp and 1188 bp,encoding 317 amino acid residues.NtOR possessed 2 transmembrane domains,1 signal peptide cleavage site,5 glycosylation sites and 11 phosphorylation sites,and the secondary structure was mainly composed of 12 α-helices and 20 β-sheets,subcellular located on plasma membrane.The phylogenetic and BLAST analyses indicated that NtOR was the orthologous gene of SlOR and ViOR.Microarray results in GENEVESTIGATOR database revealed that NtOR was expressed at the highest level in cotyledon,followed by mature leaf,young shoot and flower,rather weak in other tissues and organs.

To prevent and control tobacco bacterial wilt disease with antagonistic bacterium against Ralstonia solanacearum,the microbial diversity in soil of tobacco bacterial wilt disease nursery was preliminarily analyzed by means of conservative fragment amplification,construction of library and bio-information comparison.The results indicated that there existed abundant microbial communities including not only Burkholderia fungorum,Pseudomonas graminis,Aspergillus niger and Fusarium oxysporum,but also some uncultivable bacterium in the soil.The analysis of microbial diversity in the soil could provide some information about microbial community structure for the study on colonization and proliferation process in soil in the application of antagonistic bacterium against Ralstonia solanacearum.

Genetic diversity of 276 flue-cured tobacco germplasm of worldwide origin was evaluated based on 16 selective polymorphic primer pairs.Results showed that a total of 611 alleles were amplified by using 16 SRAP primer pairs,with an average of 38.19 alleles per primer pair.Flue-cured tobacco accessions from abroad showed higher diversity than those from China.Flue-cured tobacco from Guizhou province showed the lowest diversity.Results obtained by NJ clustering,PCA,and STRUCTURE analysis were identical,which indicated that flue-cured tobacco were a closely related group consistent with geographic origins and genetic relationship.Unique and deficient alleles in each population were detected and plenty of complementary alleles among geographic populations were found.Foreign accessions had the most potential to broaden the genetic basis of flue-cured tobacco accessions from China.

Adopting a homology cloning method,the full length cDNA sequence of tobacco phytoene synthase gene(named T-psy1) was cloned from flue-cured tobacco cv.K326,the T-psy1 gene,GenBank accession number HM345582,included 1521bp, with coding protein containing 441 amino acids,protein accession number ADK25054.The results of BLAST search and phylogenetic analysis showed that T-psy1 presented high genetic homologies with the phytoene synthase of ornamental tobacco,tomato and pepper,which were 95%,91% and 86%,respectively;and the coding protein consistences of T-psy1 gene with phytoene synthase gene of ornamental tobacco,tomato and Elaeagnus umbellate were 97%,90% and 74%,respectively.

ISSR marker was used to find genetic relationship between 12 local tobacco varieties(strains) and 5 major tobacco varieties of Guizhou in this study.The result showed that 23 ISSR primers tested could produce distinct fingerprints among 17 varieties(strains) of tobacco.An acceptable DNA polymorphic fingerprints was constructed with 9 primers which could be used to identify these tobacco varieties(strains),all varieties(strains) were distinguished from each other and showed genetic diversity.The result from the analysis were used by NTSYS 2.0.The result showed that genetic similarity of these cultivated varieties(strains) from 0.212 7 to 0.787 2 by SimQual and these 17 varieties(strains) of tobacco could be divided into three groups by UPGMA clustering analysis.

Demethylmenaquinone methyltransferase,the final step of menaquinone biosynthesis,whick convert dimethylmenaquinone(DMK) to menaquinone(MK),furthermore,it also regulates RNA metabolism,which inhibits the endonuclease activity and thereby leads to increase the half-life and abundance of RNAs.From the Normalized DNA library of the flue-cured tobacco cultivar Nanjiang-3,the full length cDNA sequence of tobacco demethylmenaquinone methyltransferase gene(named T-DM1) was cloned and its sequence was analysed,and its GenBank accession is HO663886.Analysis by the software ORF finder and ProtParam showed that the T-DM1 included 741 bp,with a 501 bp open reading frame(ORF)which encoding a protein of 166 amino acids,with a calculated molecular weight(Mw)of 17.77 kV and a isoelectric point(pI) value of 5.70.The results of Blast and phylogenetic analysis showed that T-DM1 gene had homology with the demethylmenaquinone methyltransferase gene of some plants,T-DM1 encoded protein had high identity with the demethylmenaquinone methyltransferase protein of Gossypium hirsutum,Zea mays and Arabidopsis thaliana,which were 96%,86%,85%,respectively.

In this paper,the total DNA was extracted from the soil microbes of tobacco diseased-fields by SDS-CTAB method.The size of the obtained DNA after purification was about 21 kb,and the value of OD260/OD280was higher than 1.70.The PCR amplification with bacterial and fungal conserved sequence primers showed that the amplified bands with the expected size could be detected by agarose gel electrophoresis.

Objective: In order to obtain information of functional genes,a normalized cDNA libray from Flue-cured tobacco variety of Nanjiang-3(Nicotiana tobacum cv.Nanjiang-3) was constructed.Method: RNA was extracted from leavies and flowers of Nanjiang-3 according to RNeasy Plant Mini Kit manipulation,the first cDNA was synthesized by reverse transcriptase.Double cDNA obtianed through long-distance PCR(LD-PCR) amplification method as template,the normalized cDNA library of Nanjiang-3 was constructed by the SMART technology which based on the double strand specific nuclease(Duplex-Specific Nuclease,DSN).Result: The normalized cDNA library was constructed,which contained 1.26×106 independent clones,its recombination ratio was 97.47%,average insertion size was above 1.2kb.Random selected 48 clones were tested by PCR,then 20 positive clones were sequenced and BLAST,the results showed that many genes and expression sequence tags(ESTs) were contained in the cDNA pool.Conclusion: Basis material information of research for genetic resources and gene function of Nanjiang-3 were provided by the cDNA library.

Nicotinamidase is one of key enzymes in the pathway of nicotina biosynthesis in tobacco.In plants,by the catalyzation of nicotinamidase,the nicotinamide was converted into nicotinate which is one of important substrates in the biosythesis of nicotina.From the full-length cDNA library of the flue-cured tobacco cultivars Nanjiang-3,the full length cDNA sequence of tobacco nicotinamidase gene(named T-nic1)was cloned and its sequence was analyzed.Analysis by the software ORF finder and ProtParam showed that the T-nic1 included 841 bp,which encoding a protein of 162 amino acids,and its GenBank accession is HQ448853.The results of Blast and phylogenetic analysis showed that T-nic1 gene had homology with the nicotinamidase gene of some high plants,T-nic1-encoded protein had high identity with the nicotinamidase protein of Arabidopsis thaliana and Populus trichocarpa,which is 71%,67%,respectively.

Long terminal repeat（LTR）-retrotransposons are genetic elements and important for the plant genomic organization.This review described the structure,genomic organization,expression,regulation,and discusses their contributions to plant genomic research.

From the normalized DNA library of the flue-cured tobacco cultivar Nanjiang-3,the full length cDNA sequence of tobacco which is highly homologous to the gene SNAREs was isolated,(named as T-NTGP2)cloned and its sequence was analysed,with GenBank accession number HO663897.Analysis by the software ORF finder and ProtParam showed that the T-NTGP2 with a 597 bp open reading frame(ORF)which encode a protein of 199 amino acids,with a calculated molecular weight(Mw)of 22.59 kD and a isoelectric point(pI)value of 6.96.The results of Blast and phylogenetic analysis showed that T-NTGP2 gene had homology with the SNARE protein Ykt6 gene of some plants,and T-NTGP2 encoded protein had high identity with the Ykt6 protein of Nicotiana tabacum,Populus trichocarpa and Ricinus communis,with identity of 99%,91%,91%,respectively.The result of the conserved domains analysis indicated that T-NTGP2 has a signature motif for vesicle-associated membrane proteins(VAMP),which implied that T-NTGP2 would be a YKT-like SNAREs longins.

The use of phage for bacterial control has already a long history.In recent years,as one of the prevention and treatment measures of bacterial diseases of crops,the use of phage therapy instead of traditional pesticides is calling more and more attentions,this review summarized the newest application of phage for bacterial diseases and its future prospect on corps.

A dwarf mutant with abundant sucker was observed in populations of second generation of Nicotiana tabacum(L) cv.GT11 which experienced space mutation in a recoverable satellite,the Chinese third unmanned spacecraft "Shen-Zhou III",and these heredities were stable.It was named as T-srd,Tobacco of Sucker-rich and Dwarf mutant.The suckers of T-srd emerged at the first leaf node in 5 weeks after sowing.In field,the emergence of sucker was orderly from bottom to top,and under the control of the apical bud,which had no sucker until the node laid under half of the stalk height at first flower stage.At 90 days after transplanting,the plant height,length of internodes,and stem girth of T-srd were lower than those of GT11.There was no sucker-rich and dwarf phenotype in F1 population,and the segregation ratio in F2 population of seedling between sucker-rich and sucker-free phenotype in Chi-square test followed the Mendel Genetic Law.Those results suggest that the sucker-rich and dwarf trait is a qualitative character which belongs to recessive mutation controlled by single gene.

The dry seeds of tobacco(Nicotiana tabacum) cultivar GT11 were carried to the space by recoverable airship"Shenzhou No.3" for 162 h in 2002.After space flight,a sucker-rich and dwarf mutant(T-srd) was obtained from the seedling population of the recoverable GT11.Major characteristics of T-srd phenotype included branchysm of stalk in the field and early emergence of suckers in seed bed.Amplified fragment length polymorphism(AFLP) analysis showed that the genetic materials DNA were different between T-srd and GT11.This tobacco mutant provided a useful material for the research on axillary bud regulation of tobacco.

The T-phylloplanin encoding protein was analysed by informatics tools provided on internet. The entirely T-phylloplanin gene has 861 bp containing a complete open reading frame that encodes 111 amino acids with a transmembrane signal peptide,several N-glycosylation and casein kinase II phosphorylation sites. The molecular weight of the protein encoded by T-phy lloplhin gene is 11.30 kD,and iso-electric point is 7.74. The T-phylloplanin encoding protein has highest similarity with the protein featuring defensive function secreted by short stalk glandular hair,thus indicating the worth for further study in tobacco resistance system.

In this research,242 683 expressed sequence tags(ESTs) were analyzed by software,indicating that 9 339 SSR were found in 211 728 non-redundant tobacco ESTs,and the average distance between SSR was approximately 14.21 kb,accounting for 4.41% of ESTs,including 216 kinds of repeat motifs were mined out.Trimeric repeats,accounting for 50.34%,was dominant in all SSR,followed by dimeric and mono-meric repeats,23.00% and 16.48%,while the frequency for other repeat type was below 5% each.The most abundant motif was A/T,accounting for 14.68% in all repeat types,followed by AT/TA,AG/TC,AAG/TTC,accounting for 10.49%,9.48%,6.85%,respectively.10 pairs of primers for EST-SSR randomly designed were subjected to PCR for DNAs from six tobacco varieties and one pair of primers showed polymorphisms.The result provided a base for the development and application of EST-SSR markers in tobacco.

In order to supply some information to the study about relations between the morphology and functions of glandular hairs on tobacco leaves,the micro-morphology of glandular hairs on the surface of leaves of the tobacco cultivar Nicotiana tabacum K346 were observed by scanning electron microscopy.The long stalk glandulars on the surface of tobacco leaves commonly have several cylindrical nodes on the parts of stalk,the short stalk glandulars have thicker stalk with one short cylindrical nodes;the head of glandulars,the main of the secretive function parts,which has complex structure with larger than its stalk,especially the head of the short stalk glandular,wrinkly around with obviously convex and concave;the morphlogy of glandular hairs on tobacco leaves have relations with its secretive functions,the information of glandular hairs micro-morphology may benefit to the study on the relation between the function and morphology.

P1T10(GenBank Access No.EB102896),an expression sequence tag(EST) which related to fungal-resistant was selected from a SSH(Suppression Subtractive Hybridization) pool.The complete cDNA of T-Phylloplanin gene(GenBank Access No.DQ451214) was gained by RACE(Rapid Amplification of cDNA End) with primers designed according to the sequence of P1T10.With the EditSeq program(version 5.01) of DNA STAR software T-Phylloplanin was analyzed,the results indicated that it contained a complete ORF(open reading frame) and a protein containing 110 amino acids was encoded whose weight was 11.3kD and pI(isoelectric point) was 7.74(GenBank Access No.ABE03627).The homology analysis by BLAST indicated that the similarity was 93% between T-phylloplanin and the complete cDNA gene(GenBank Access No.AY705384) of a resistant protein from tobacco leaf surface.Semi-quantitative RT-PCR analysis indicated that the T-phylloplanin expression was higher in mutant T-cldf leaf than in tobacco K346.

Mutants of tobacco are very important materials in tobacco functional genomics.In this paper,the roles of toabacco mutants in genes were reviewed,followed by the construction of tobacco mutant pools,the related methods and its future application to tobacco functional genomics.

The effects of seed sterilization were studied by different seed sterilization methods.The results showed that sterilization of seeds soaked in aseptic water for 24 hours was better than that without soaking.Under constant temperature,the sterilization effects of three germicides(10%H2O2,2% sodium hypochlorite and 1‰ HgCl2) on tobacco seeds were studied.The results showed that 10%H2O2 had the greatest sterilization effect.The germ-bearing rate of seeds of K326 treated with 10%H2O2 was 0%.Sterilization of seeds soaked in 75% ethanol before seed sterilization decreased germ-bearing rate.The highest germination rate was observed in treatment with 10%H2O2.