Cloning And Analysis Of Resistance Gene Analogs From Grape And Peach

According to conserved domain of NBS-LRR and STK classes of resistance genes and by using a method of candidate gene based on analogs of resistance genes, this research carried out the cloning and analysis of resistance gene analogs. The results were as follows: 1 .Comparison of the methods of plant genomic DNA extrationIn the experiment of extraction of peach genomic DNA, according to Qiao Yushan improved-SDS of micro-method, the micro-method was modified to mid-method in which the eppendorf-tube of 1. 5ml had been replaced by that of 5ml and the amount of plant material accordingly added more. And in order to get over the negative effect of impurity thereout, times of purif iction was increased and 0. 3mM NaAC was added into the solution before precipitating DNA; super-purified water instead of TE buffer was used to dissolve DNA. Genomic DNA of peach that had higher concentration and better quality was obtained finally, which provided a good foundation for next PCR reaction.In the research of extraction of grape genomic DNA with SDS method, much of pigment (saccharide) was found, so CTAB method that is useful to get rid of saccharide was adopted. And also, micro-method was modified to mid-method. At last, disturbance of pigment was eliminated, and moreover, genomic DNA of grape that had higher concentration and better quality was obtained.In the process of extraction of peach genomic DNA, RNA was eliminated absolutely with RNase concentration 10mg/ml, while RNA still existed with its concentration 0. lmg/ml.2. Designation of primers, optimization of PCR condition and filtration of primersIn this research, two sets of degenerate oligo-nucleotide primers were designed according to amino acid conserved regions of reported disease resistance genes, based on P-loop (GGVGKTT) and trancmembrane domin which encode proteins that contain nucleotide-binding site and leucine-rich repeats (NBS-LRR), and up conserved region (FG (K/V/I/S) VY(K/R)G) and down conserved region(D(V/I)YS(F/Y)G(V/I/M) which encode serine/ threonine protein kinase(STK) . They were in total up to 13 pairs of primers, thereinto 9 pairs of NBS-LRR classes and 4 pairs of STK classes.Using separately genomic DNA of grape and peach as template of PCR reaction, 8 grads of Mg2- from 1. 0mM-4.5mM and 0.5mM of each grads. Based on the grads, optimization of general PCR condition had been carried through: plant genomic DNA denatured at 99-100*C for 10 minutes, and then put on ice quickly; the temperature of warm boot improved to 95*C and the time of it prolonged to 5 minutes; annealing temperature below 5'C primer' s Tm and the amount of primers whose Tm is lower wasUnder the optimized condition of PCR and among 13 pairs of primers, the results showed that clearly characteristic strips were obtained from genomic DNA of 5 grape species based on primers WN3/WN5 of NBS-LRR classes under the final concentration of Mg2+ 3mM; that some of faint strips were found on the display of agarose gel electrophoresis analysis of PCR product amplified by primers WS2/WS4 from the 4 peach species under Mg2+ 3mM too.Aiming at STK primers, using separately the their product from peach species as template, and improving annealing temperature at the same time, it was shown that the non-characteristic amplification had been reduced and the expected ideal strips appeared finally at annealing temperature 43*C .3. Analysis of the obtained sequenceThe DNA fragments from amplification were collected separately, then cloned into the pGEM-T vector and transforming into the E. coli DH5 a compatible cell. Tested by PCR-method and sequenced by bio-corps, 44 sequences that were between 300bp and 600bp had been obtained. After the preparatory comparison, 29 sequences different with each other were get out, 18 of them from grape species and 11 from peach species. By the analysis of amino-sequence and further comparison with each other and the reported disease resistance genes,21 desired RGAs were obtained. Except for both end-side, all of them contain more or less, the conserved motifs of NBS-LRR cla...