Isolation And Characterization Of NPR1 Homolog Gene And Gene Fragment Of Retrotransposons Of Wheat And Its Wild Relatives

Blumeria graminis and Rhizoctonia cerealis can cause serious diseases, which often result in enormous economic losses of wheat. As the isolates of B.graminis change frequently, the wheat varieties with a single resistance gene are easy to lose resistance because Blumeria graminis isolate varies. The available resource with resistance to Rcerealis is deficient. Resistance to R.cerealis is controlled by multigene. Traditional breeding for R.cerealis is not successful so far. Therefore, it is one of mainly developmental prospects to study molecular mechanism of host resistance response and isolate broad-spectrum resistance of signal transduction gene. NPRl is a key regulator of SAR (systemic acquired resistance) and ISR (induced systemic resistance), and plays an important role in basic resistance and the response mediated by R (resistance) gene. Overexpression of NPR1 can enhance resistance to multi-diseases. It's reported that NPR1 and its homolog genes exist in some important crops and confer broad-spectrum resistance. Defense response mediated by NPRl is conserved in many plants. So, isolation of NPR1 and its homolog gene from wheat and its relatives will be important significance in wheat breeding for broad-spectrum resistance. As wheat genome being huge and containing many repetitive sequences, isolation of any new resistance gene is quite difficulty. Former researchers found that retrotransposons were activated by defense-related stresses, such as SA, JA and pathogens. In this study, active retrotransposon was isolated from wheat leaf induced by B.graminis. Because active retrotransposon as a gene tagging could facilitate to isolate genes and analyze the gene function, it offers an effective and powerful tool for cloning gene and gene function analysis.This study makes the following progresses:1. RT-PCR was conducted to amplify the ANK domain of NPR1 gene by using primers derived from the conserved peptide motifs of cloned plant NPRl from RNAs of Thinopyrum intermedium and Aegliops squarrosa. The NPRl homologs were named as TiNH1 and AsNH2 from Th. intermedium and Ae.squarrosa, respectively. The cDNA full-length of TiNHl was obtained by using RACE (rapid amplification of cDNA ends) method. At the same time, the cDNA full-length of AsNH1 and TaNH1 were cloned from Ae.squarrosa and wheat. The identity of proteins encoded by TiNH1 , TaNH1 and AsNH1 is more than 98%, implying that these gene are orthologous. While AsNH2 encoding protein shares 84% identity with the proteins of TiNH1, TaNH1 and AsNH1, indicated that TiNH1, TaNH1, AsNH1 and AsNH2 are different types of NPR1 homlog gene. These implied that other NPR1 homlog genes existed in Th. intermedium and Ae.squarrosa genome.2. Comparing the protein structure of TiNH, TaNH1 and AsNH1, the proteins include two domains of the POP/BTB and ANK domain. The ailgement of TiNH1, TaNH1 and AsNH1 with the NPR1 proteins of Arabidopsis, tobacco, wheat and maize, showed that amino acids crucial for the NPR1 function, such as nprl-l(H), nprl-2(C), nim1-4(R), are conserved. GC content of 5 ' end of TiNH1, TaNH1 and AsNH1 is more than 70%. Analysis of phylogenetic tree indicate that TiNH1, TaNH1 and AsNH1 are more closely related with rice NPR1 (Genbank accession: NM₁89701.1) anddistant from wheat NPR1 (Patent number: WO0070069), suggesting that there are many NPR1 homlog genes in wheat genome.3. Northern blot was carried out, in which total RNAs from Th. intermedium leaves treated with different pathogen were hybridized with TiNHl probe. Northern result showed that the expression level of TiNHl was very low under normal conditions, but reached the highest from 10 h to 24 h after inocatlation of B.graminis. The expression of TiNHl is also induced by infection of Rhizoctonia cerealis. The TiNHl expression level reached the highest amount at 5 hour after infection of Rxerealis, then, reduced again. These results indicated that the TiNHl expression induced by the above pathogen is different in dynamics.4. In order to clarify copy number of TiNHl in Th. intermedium genome, genomic DNA from Th. intermedium digested with Dar\ and Bam\\\ were hybridized with TiNHl. Southern blot showed that TiNHl was a signal copy in Th.intermedium genome.5. Transient expression system was used to analyze the function of TiNHl. A plant expression vector was constructed, in which the TiNHl gene was controlled by the maize ubiquitin promoter. The vector containing TiNHl gene was bombarded into leaf epidermal cells of 5gmw/??-susceptible wheat variety zhong 8601 by biolistic particle delivery. At same time, GUS gene was co-transformed with TiNHl gene to mark the transformed cells. After incubation of 4 hours at 20 °C, leaf surface was inoculated with conidiospores of B.graminis. After 44 hours post inoculation, the penetration of the fungus and formation of haustoria in the transformed cells were observed to evaluate the effect of the TiNHl expressing protein on the invasion of B.graminis. The results showed that the TiNHl expressing protein can inhibit to some extent the penetration of conidiophores and formation of haustoria of B.graminis, so that increase the host resistance to B.graminis.6. An activation mode of wheat retrotransposons was studied. It was reported that wheat retrotransposons was activated by defense-related stresses, such as SA, JA and pathogen (B.graminis). Fifty-one clones of the reverse transcriptase (RT) domain of retrotransposons were obtained from the seedlings of wheat line Pm97034 treated with B.graminis. Three new families, designated family 8 to family 10, were identified. Matsuoka and Tsunewaki (1996) inferred that family 4 of retrotransposons in wheat and its related species had transcriptional activity. In order to understand the transcriptional activity of family 4, total RNAs from Pm97034 leaves treated with defense-related stresses such as SA, JA and B.graminis were hybridized with an RT20 probe selected randomly from family 4. The expression of RT20 was very low under normal conditions, but up-regulated when treated by SA, JA and B.graminis. The highest expression level was observed at 24h after the treatments and recovered to normal level at 48h post treatment. This indicated that the retrotransposons of family 4 had transcriptional activity. Southern blot showed that RT20 was present with multiple copies in genomes of wheat and its relatives. This similar stress-responsiveness seems to be the common feature of the wheat retrotransposon families.In this thesis, NPR1 homolog genes and reverse transcriptase domain of retrotransposons inducedby B.graminis were isolated from wheat and its relatives, and their molecular characterizations and functions were analyzed. To some extent, TiNHl gene can increase the resistance to B.graminis. It is important to isolate NPR1 homolog genes from wheat and its relatives for understanding disease resistance mechanism and biotechnology breeding for broad-spectrum resistance. Isolation of the activate retrotransposon as a gene tagging will be a powerful tool for other gene cloning and assay on gene function in wheat.