| The plant pathogen is an important factor on limiting crop yield. Using synthetic fungicides to control plant diseases has not only bad effects on food and possible side-effects on human health, but also has disadvantages such as big investment. So It is important and necessary to breed new anti-disease varieties. But the traditional breeding methods need many years. With the developing of anti-disease genetic engineering, a major effective way against pathogens can be manipulated through introducing resistance genes, signal transduction/regulation genes and pathogenesis-related genes int plants with or without R genes to improve plant disease resistance.On the contract of traditional strategies, it has big improvement in stable genetic.RAR1 and SGT1 genes were identidied as a required component in some signaling pathways mediated by resistance genes(R) in many plant species.In addition, they also involved in plant non-host resistance.In order to know their roles in further level and breed new anti-disease breed, RAR1 and SGT1 which involved in plant disease resistance were isolated from tobacco( Nicotiana tobacum). At the same time, the overexpression vectors and RNAi vectors were constructed. Then vectors were transformed into tobacco by Agrobacterium tumefaciems mediated method. More ever, the transient expression was analysed on transgenic plants. The works laid foundations for the further study of disease resistance of RAR1 and SGT1 and breeding anti-disease tobacco. The results were as follows.1. Construction overexpression vector and RNAi vector of NbRAR1.PCR primers were designed according to the sequence of RAR1 published in GenBank. Then cloning vector of NbRAR1 was successfully constructed. Overexpression vector of NbRAR1 was constructed through double restriction enzyme digestion method. Using the same method, RNAi vector of NbRAR1 was obtained. Then they were verified by PCR and restriction enzyme digestion. The results showed that the vectors were successfully constructed. 2. Construction overexpression vector and RNAi vector of NbSGT1.First, cloning vector of NbSGT1 was obtained from tobacco. Then the target gene was amplied by PCR, next NbSGT1 gene was subcloned into plasmid of pCAMBI1301. Similarly, RNAi interference fragments were respectively subcloned into plasmid of pTCK303. Both the recombinant plasmids were detected by PCR and restriction enzyme digestion.3. Genetic transformation of tobacco.In this study, tobacco leaf was used as explants, and the genes were transferred into tobacco by Agrobacterium EHA105 mediation which has been inserted with the plant expression vector.Then co-cultivation was carried out for 2-3days, after that, the transformants plantlets were obtained by transferring explants to selection medium and differential medium.And its plantlets differentiation rate was, 68% of NbRAR1ox, 70% of NbiRAR, 58% of NbSGT1ox, 66% of NbiSGT1.4. Transgenic plants for idetification and RT-PCR analyzation.In this study, a certain number of transgentic plants was obtained, and the number of NbRAR1ox,NbiRAR1,NbSGT1ox,NbiSGT1 transgentic plants were 32,38,15,26,respectively. Then, two detection methods were used. The PCR testing results showed that the target gene had been integrated into tobacco. The positive rate of NbRAR1ox,NbiRAR1,NbSGT1ox,NbiSGT1,which were detected by PCR assay with hygromycin gene, was 91%, 92%, 87%, 73%, respectively. And the positive rate of target gene were, 88%, 89%, 73%, 78%, and the PCR positive independent plants were: 28,34,11,20 stains, respectively. Secondly, GUS assay were carried out on positive plants, the results showed that GUS gene had been integrated into tobacco acccompanying with the target gene. Additional, the positive plants were picked for RT-PCR analysis. The results showed that the transgenic plants of NbRAR1 and NbSGT1 gene have a certain degree overexpression, and there were interference effects on plants of RNAi interference. At the same time, there were obvious discrepancy among transgentic plants.
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