Cloning And Analysis Of The Genes Related To The Resistance Of Jujube Witches' Broom Disease

Chinese jujube (Ziziphus jujuba Mill.), a precious germplasm resources originated from China and a kind of important and specific fruit trees in China, belongs to family Rhamnaceae. Jujube witches' broom disease (JWB), caused by phytoplasma, is one of the most serious and destructive diseases of Chinese jujube. It is important to clone the resistant gene by using molecular biology from resistant resources. In preliminary studies, our group had found that there were resistant and sensitive strains in Junzao. By the aid of AFLP analysis and SON-PCR, resistant and sensitive strains in Junzao were employed in this study. The differential bands related to the resistance of JWB on the level of DNA were screened by AFLP and then the flanking sequence of one differential band was extented successfully by SON-PCR. The main results obtained were as following:1 11 differential expression bands were screened by AFLP among the resistant and sensitive strains in Junzao. Only 10 out of 56 pair primers were screened for AFLP analysis and 11 polymorphic bands were amplified by using those 10 pair primers. 11 differential bands included 6 bands with high-resistance to JWB and 5 bands with sensitive to JWB.2 An optimized SON-PCR system and the PCR program suitable for amplification of the flanking regions of gene of Chinese jujube were established. In this research, the efficient PCR amplification could not achieved by traditional SON-PCR system, so it must be improved and optimized for suitable to Chinese jujube. The main changes were that second and third rounds of the improved SON-PCR system were divided into two steps instead of only one step in traditional system. The linear extension of the second and three rounds of nested amplification had been listed separately in improved system, it would increase the specificity and efficiency of PCR reaction. The ideal result of the improved system was verified in this paper.3 A resistance-related fragment (165bp) was extended to 1119bp by the improved SON-PCR system.The R1 fragment had been extended 390bp in upstream and 564bp in downstream by the improved SON-PCR system. Those sequences were spliced to 1119bp in length. Using NCBI database and the analysis of Blast, the genetic similarity between the sequence of 1119bp and the mRNA sequence of coding SAUR family protein (SAUR3) from Populus trichocarpa was 80%. The analysis of open reading frame showed that this sequence contains an incomplete open reading frame which included a number of start codon and no exit stop codon. 161 amino acids encoded by this incomplete open reading frame which in the first 24 amino acids and 121 amino acids exited the conserved domain of the SAUR (Small auxin-up RNAs) superfamily protein.