抗条锈病小麦N95175抗性基因的遗传图谱与克隆
摘要
Stripe rust caused by Puccinia striiformis f. sp. tritici is one of the most devastating diseases of wheat in China as well as in Pakistan. In the present studies F2 population was established by crossing N95175 resistant to stripe rust race CYR32 with two susceptible lines Huixianhong and Abbondanza to molecularly tag resistance gene existing in wheat line N95175. The segregation of phenotype was accorded with an expected 3:1 ratio in both combinations studied and fit the model of a single dominant gene controlling stripe rust resistance in N95175. Thirty five SSR primer pairs were screened on the parents and bulks and also on individuals since resistance gene to be located in chromosome 1B. The result indicated that most of resistant plants amplified same band as resistant parent while susceptible plants amplified same as susceptible parents studied and considered that markers co-segregated with resistant loci in N95175. This yellow rust resistance gene was considered to be Yr26 originally thought to be also located in chromosome arm IBS linked to marker loci Xgwm273 and Xgwmll with genetic distances ranging from 1.075cM to 2.74cM in both combinations studied. However, the closest loci were observed 2.67cM for Xgwm273 and 1.075cM for Xgwmll in HuixianhongXN95175 and AbbondanzaXN95175 crosses respectively. Hence, it has been concluded that the PCR-based micro satellite markers Xgwm273 and Xgwmll located in chromosome 1B were shown to be very effective for the detection of Yr26 gene in segregating population and can be applied in future wheat breeding strategies.
Wheat TaCOPⅡgene was isolated from EST sequence of wheat leaves infected by Puccinia striiformis f. sp. tritici, initially using RT-PCR a primer pair, because available EST sequence was not enough to design two GSP sequences. Following 3'RACE analyses, cDNA fragments around 1865 bp including poly-A signal region at 3'-UTR, was detected by two round RACE-PCR aplications including nested PCR using two sense GSP primers on 1% agarose gel. CAP3 software was used to combine the two fragments into an 1865 bp consensus sequence including a poly-A signal region at 3'-UTR. The sequencing results showed that the 1865 bp sequence shared high similarity to COPII in Arabidopsis thaliana. The region from 35 to 1450 nucleotides was the open reading frame (ORF) encoding a polypeptide of 486 amino acids. The deduced molecular mass of the peptide was 53.378 KDa with a predicted pI of 8.14.
The Real Time PCR observation revealed that maximum induction occurred at 24 hpi, among all time points, followed by a slightly increase at 48hpi, while from 72 to 120hpi, the accumulation of transcripts decreased steadily. However, the magnitude of transcripts at 24hpi time point was much higher than that of control Ohpi. It was also observed that all the candidate genes studied were expressed at basal level before Pst inoculation. These results demonstrated that the transcription of TaCOPII gene was expressed at the early stage of stripe rust inoculation, hence suggesting that this gene may participate in host defense response through different regulatory mechanism specialty at early stage of inoculation.
Analysis using nulli-tetrasomic Chinese Spring lines further confirmed that the candidate gene TaCOPII was located on chromosome 3A. On the basis of the above results, it is reasonable to assume that the candidate gene TaCOPII was located on the chromosome 3A in wheat line N95175, and its comparison with the other resistance genes located on 3A most of plants suggested that TaCOPII may be involved indirectly to resistance mechanism against stripe rust PST in wheat line N95175. Exploration and utilization of resources of disease resistance genes in native wheat varieties will be helpful both to diversify the resistance genes and to amend the situation of resistance gene simplification in the commercial wheat cultivars.
Wheat TaHSP70 fragment, was detected by two RACE-PCR applications including nested PCR using two gene specific sense primers (GSP). Following plasmid construction, transformation and cDNA clones sequencing,3'RACE fragment was obtained by removing vector and adapter sequences. The rest of about 1203 bp sequence was obtained by RT-PCR using a primer pair. Furthermore, CAP3 software was used to combine the two fragments into an 1810 bp consensus sequence including a poly-A signal region in the 3'-UTR. BLAST analysis showed that the 1810 bp sequence shared high similarity (94%) to Arbidopsis thaliana. The region from 43 to 960 nucleotides was the open reading frame (ORF) encoding a polypeptide of 305 amino acids. The deduced molecular mass of the peptide was 31.31 KDa with a predicted pI of 5.29. Based on the strong identity to A. thaliana, hence putatively designated the wheat 1810bp sequence as TaHSP70. Nulli-tetrasomic Analysis using Chinese Spring lines further confirmed that the candidate gene TaHSP70 was located on chromosome 4A. The Real Time PCR results demonstrated that the transcription of TaHSP70 gene was down regulated at the early stage of stripe rust inoculation hence concluded that this gene may not participate in host defence regulatory mechanism specialty at early stage of inoculation.
引文
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