Cloning And Localization Of Root-knot Nematode Resistance Relative MYB Transcription Factor Genes In Honggengansutao Peach(prunus Kansuensis L.)

Root-knot nematode is a kind of serious disease in peach cultivation in the world. Breeding of peach rootstock resistant to root-knot nematode is one of key important means to solve this problem. Honggengansutao peach (Prunus kansuensis L.) is a special wild germplasm which immunizes to root-knot nematodes in China. As one of the largest plant transcription factor families, MYB transcription factors play important roles in regulation of phenylpropanoid pathway and response of biotic and abiotic stresses. In order to identify the molecular resistance mechanism of Honggengansutao peach, myb genes were isolated from root cDNA of Honggengansutao peach by the homologous cloning strategy and then were localizated on peach chromosomes. Myb genes related to root-knot nematode resistance in Honggengansutao peach were predicted by expression analysis and phylogenetic analysis. Then myb genes function in molecular mechanism of resistance in Honggengansutao peach.The results were as following:1. Established a method suitable for extracting RNA from peach root based on Trizol method, CTAB method and SDS method.2. Fifteen myb genes fragments were isolated from root cDNA of Honggengansutao peach by RT-PCR. By alignment, it was found that their inferred amino acid sequences have high identity with their counterparts from other plants in Gene Bank. By alignment with peach genome sequence, it was found that PkMYBs have one or two introns in their DNA binding domin. The result of alignment and phylogenetic analysis showed that PkMYB1-9, 11, 13, 14, 15 belong to R2R3-MYB transcription factors.3. The result of phylogenetic analysis showed that PkMYB4, 8, 9 were clustered with MYBs with function of regulation of phenylpropanoid pathway from Arabidopsis, tomato, poplar and persimmon in different subgroups. PkMYB8, 9 probabily defense root-knot nematode infection by increase proanthocyanidins. PkMYB1, 2, 3, 5, 6, 7, 11, 12, 14, 15 were clustered with MYBs with function of response of biotic and abiotic stresses in different subgroups. According to above results, we predicted that PkMYBs have similar functions with other MYB proteins.4. Two different myb genes: PkMYB14, 15 were isolated from seedling with infection of root-knot nematode. The results of RT-PCR suggested that expression of PkMYB7, 10, 11 were induced by infection of root-knot nematode. Further, PkMYB7 was clustered with disease responsive AtMYBs in same subgroup, so that it may regulation expression of defence genes through signal transduction pathway to resistant root-knot nematodes.5. The all 15 PkMYBs were located on peach chromosome. It was found that there were a plenty of disease resistance protein genes, wound-responsive family protein genes, pathogenesis-related protein genes, anthocyanin biosynthetic genes and other myb genes nearby these PkMYBs genes and suggest that they were related to resistance. PkMYB12 was located on the second chromosome which contain root-knot nematodes resistant marker and resistance gene analogue. There were also anthocyanin biosynthetic genes nearby PkMYB12. For these reason, we predicted that PkMYB12 defense root-knot nematode infection by increase anthocyanin and flavonoid.6. According to results of this study, the effects of MYB transcription factors in root-knot nematode resistance probabliy have two pathway. One was defensed root-knot nematode infection by increase anthocyanin and flavonoid as PkMYB8,9 and PkMYB12. The other was regulated expression of defence genes through signal transduction pathway to resistant root-knot nematodes as PkMYB7.