Screening Of Tobacco-specific Gene Expression And The Cloning Of Promoter

Tobacco as a special economic crops which made significant contributions to the country's economic development.In 2007,the tobacco industry's industrial and commercial tax is over 388 billion yuan,an increase of 25 percent.But tobacco production can not avoid the occurrence of many diseases and pests in the course of cultivation。Ralstonia solanacearum in tobacco is a key cause of reduction of tobacco production in southern China. In order to solve bacterial wilt disease in tobacco,we resorted to tobacco molecular breeding by employing the methods of molecular biology,bio-informatics technology and gene engineering,screening and identifying some sequences of tobacco's root-specific expression and obtaining bacteria-inducible plant promoters by homoloc cloning which can be further used to tobacco molecular breeding.The results of this study were as follows:1.DNA array membranes with root differential-expressed genes were checked by comparative hybridization with Root's and leave's cDNA probes labeled with Digoxinin, respectively,and 130 colones were picked up for sequencing.Fifty nine of them could not be found any homologous genes with any interested sequences in NCBI,which were submitted to Genbank,EX465336---EX46527(http://www.ncbi.nlm.nih.gov/sites/entrez),andthe other 71 fragments show high homology with some valuable genes in other plants.Nineteen genes out of the 130 were selected primarily showing expression specifically in root.2.RT-PCR was used to examine the expression patterns in different organs,and it was showed that:gene 3,5,14,16,18 and 19 were specifically expressed in root respectively with gene18 and 19's expressed particularly rich in root and the other's followed by;gene 1, 2,6,11 and 17 were expressed in root,flower and fruit respectively;gene 17's expression in root is more than in flower and fruit,gene 1 and 2's expression is the same in root,flower and fruit,gene 13 's expression is the same in root and stem,gene 12 's expression in stem is more than in root.gene 8 was expressed in root,leaf,flower and fruit,gene 7,9 and 15 were expressed in root,stem,leaf,flower and fruit respectively.3.Two bacteria-inducible plant promoters named as PP1 and PP3,were cloned by PCR from the leaf total DNA of FJJIAN characteristic flue-cured tobacco variety CB-1.PP1 and PP3 were constructed on cloning vector pGEM-T,and then were identified by sequencing. Sequencing results showed:the PP1 promoter is 1291bp with 99%identity with the sequence on GenBank;the PP3 promoter is 222bp with 99%identity with the one on GenBank.Six plant expression vectors(PPPP1.PPGP1.PPCP1.PPPP3.PPCP3.PPGP3)were constructed by resistant genes(Tobacco Chitinase(Chi)gene andβ-1,3-glucanase(Glu)gene,peanut trypsin inhibitor gene)driven under PP1 and PP3.Above all,we obtained six plant expression vectors which were constructed by resistant genes driven under two bacteria-inducible plant promoters and some root's differentially-expressed genes,the paper will provide a good basis for tobacco molecular breeding.