| Our country keeps on developing Botryosphaeria dothidea resistance breeding of poplar, but the large outbreak harm of Botryosphaeria dothidea still can’t be controlled effectively in ractical production. Therefore, developing Botryosphaeria dothidea resistance studies on molecular mechanism, especially developing studies on thaumatin-Like poteins, will lay the foundation for breeding popular Botryosphaeria dothidea resistance varieties, and controlling poplar Botryosphaeria dothidea raging. In this experiment, leaf, root and bark infected by Botryosphaeria dothidea of Populus trichocarpa were taken as material. The best reference genes were screened from eleven candidate housekeeper genes by real-time quantification PCR technology and then thaumatin-like proteins genes expression characteristics in different times and different locations were also monitored by real-time quantification PCR technology. In order to determining the Botryosphaeria dothidea resistance relevant TLPs genes, the relationship TLPs genes with Botryosphaeria dothidea was analysised. Meanwhile, types and distribution of cis-acting elements in TLPs upstream regulatory region were forecasted by biological software. The relationship was discussed between cis-acting element and popular Botryosphaeria dothidea resistance. The experimental results are as follows:1. Five common housekeeping genes UBQ-L, CYP, EF1β, TUB and 5.8srRNA from UBQ, UBQ-L, ACT2, EF1α, ACT11, 5.8S, CYP, EF1β, EIF4B-L, TUA, TUB expressed high stability in bark of Populus trichocarpa. UBQ-L and CYP were the most stable housekeeping genes, and ACT11, EF1αwere the worst stable housekeeping genes.2. The expression stability of UBQ-L, CYP, EF1-β, TUB and 5.8srRNA in popular leaf, root and bark were assessed. The result revealed that UBQ-L was the best reference in root and bark, EF1-βwas the best reference in leaf.3. Pairwise variations V2/3 was 0.101<0.15, indicating two reference genes was the optimal number in the experiment.4. TLPs genes induced expressione in leaf, root and bark. TLPs in leaf and bark expressed quickly, but TLPs in root expressed a little later.5. TLPs genes have three expression profiles. profileâ… : After infectde by popular Botryosphaeria dothidea, TLPs genes expression were rising rapidly ; profileâ…¡: After infected by popular Botryosphaeria dothidea, TLPs genes expression amount decreased slightly, and then increased gradually; profileâ…¢: After infected by popular Botryosphaeria dothidea, TLPs genes expression steady declined. The three profiles all existed in leaf and bark ,but only profileâ… and profileâ…¡existed in root.6. One TLPs gene had different expression profiles in different locations.7. P01, P02, P23, P31, P38, P42, P45 and P47 TLPs genes were rising rapidly after inoculated with Botryosphaeria dothidea. They maybe relate to resistance reaction.8. There are 26 types cis-acting element in TLPs upstream regulatory region. They are ABRE(abscisic acid responsiveness),ARE( anaerobic induction responsiveness),AT1 motif, box 4, box I, GT1-motif, I-box, MRE, G-Box, box II, G-box, GATA-box and TCT motif(light responsiveness),Box-W1(fungal elicitor responsiveness),ERE(ethylene-responsiveness),GARE and P-box (gibberellin responsiveness), HSE(heat stress responsiveness), LTR(low-temperature responsiveness) ,MBS(drought-inducibility),TC rich(defense and stress responsiveness) ,TCA-element( salicylic acid responsiveness) , TGACG-motifå'ŒCGTCA-motif(MeJA responsiveness) , TGA-elementå'ŒGC-motif(auxin responsiveness).9. ABRE, ARE, Box-W1, ERE, GARE, P-box, HSE, LTR, MBS, TC rich and CGTCA-motif are related with disease resisitence. TC rich is the most widely distributed. P01, P07, P10, P13, P26, P31 upstream control genes have fungus cis-acting element Box-W1, and expressed quickly after Botryosphaeria dothidea inoculated. Which indicates they are closely related to Botryosphaeria dothidea infected. |
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