| Tobacco as a special economic crop makes significant contribution to the country's economic development each year. In 2007, Chinese tobacco industrial and commercial tax is over 388 billion yuan, an increase of 25 percent in comparison with the previous year. 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, which has not been effectively solved so far. In order to solve bacterial wilt disease in tobacco, we resorted to tobacco molecular breeding by employing the methods of molecular biology, bio-informatics and gene engineering technology, and we have cloned the tobacco root-specific promoters some of which was identified genetically; cloned resistance genes from the arabidopsis and constructed specific expression vectors drived by the root-specific promoter and induced promoter, respectively, which are under transformation into tobacco. The main results are as follows.1. Five genes previously obtained from a roots SSH library of tobacco were analyzed for their expression specificity in various tissues employing semi-quantitative RT-PCR,the result showed that NtR12, NtR14 , NtR17, NtR18 and NtR19 genes only expressed in tobacco root in material sampled from rapid growth period , but in the mixed materials sampled from tissues of different stages, only NtR19 expressed in roots, and NtR12 did not express in the leaves, so the best candidate specific expression genes are NtR12 and NtR19, and NtR17 and NtR18 expressed more in root than in stem, leave, flower and fruit, showing that the expression of the two genes may be related with the growing stages and environmental stresses.2. Employing total RNA from K326 as template and using modified 5 'RACE, we obtained 5' part of NtR18 sequence and got the full-length sequence of NtR19 by the means of 5 ' RACE and 3 ' RACE. However, we acquired the full length sequences of NtR12, NtR17 and NtR14, prognosticating from the available full-length mRNA of other plants reported on the internet. Therefore, the full-length of the five genes are 798bp, 604bp, 801bp, 1422bp, 867bp, respectively. These sequences could be of helpful for the upstream promoter cloning of these genes.3. We have cloned the upstream promoter sequences of NtR12, NtR17, NtR18 and NtR19, by a modified method of Flanking PCR technique established by our laboratory, the length of them are 956bp, 1037bp, 924bp and 1230bps, respectively. Through bioinformatics analysis, all the sequences contained transcription start sites and some basic promoter elements such as CAAT-box and TATA-box.4. To evaluate the specificity, an expression vetor bearing NtR12 promoter upstream of GUS gene was constructed, and then was transformed into tobacco mediated by Agrobacterium, via kan screening. Root, stem and leave tissues from transgenic seedlings was analyzed using GUS gene activity assay and showed GUS activities only expressed in root, which is basically conformed with the results of semi-quantitative PCR, and therefore initially confirm that NtR12 promoter is root-specific.5. In order to enhance resistance to deseases and stresses of tobacco, NPR1, WRKY60 and CBF2 from Arabidopsis were cloned, and then sequenced, showing that the full length of these genes were 1782bp, 816bp and 651bp, exactly the same with their conterparts online. Then, these genes were constructed on vectors driving by NtR12 promoter and PP1 promere, a bacteria-induced promoter ovailable in the lab. These vectors are being used for tobacco transformation research.Up to the present, only one report has touched on the root-specific promoter which lacks the practical value. ,So the work done on root-specific promoter and resistant genes cloning in the study have provided with a solution to Ralstonia solanacearum desease by genetic engineering, and it is important both theoretically and practically.
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