Establishment Of Transformation System And Genetic Mechanism Of Paraquat Resistance In Eleusine Indica L.

Goosegrass(Eleusine indica L.), is a Poaceae weed, which had become one of the world's ten malignant weeds. As a long-time used of paraquat, goosegrass has developed resistant to it. In this study, the Agrobacterium-mediated genetic transpormation system of goosegrass was established. On the other hand, based on the results of the high-throughput sequencing, studied the differential expression of Pq E?Pq TS1?Pq TS2 and Pq TS3 genes by q RT-PCR. Then, the effect of changes of the endogenous polyamines on goosegrass resistance to paraquat was studied by HPLC. Finally, the effects of the differential expression of Pq E, Pq TS1, Pq TS2 and Pq TS3 on polyamine metabolism and goosegrass resistance to paraquat were discussed. The main results were as follows:1. Using sensitive goosegrass seeds as explant, studied the seeds treatment, callus induction and the condition of differentiation. The results as follows:(1) The best way to lift the seeds dormancy was that-20 ? treatment for 20~30 d.(2) The optimum concentration and of seed disinfection was 0.05% Hg Cl2, and the best time was 10 min.(3)The best callus induction medium was: N6 + 0.5 mg·L-1 2,4-D + 0.5 g·L-1 proline + 0.6g·L-1 casein hydrolysates 30 g·L-1 sucrose + 3 g·L-1 gelling agents, and the maximum frequency of callus induction was 75%.(4) The best hormones of differentiation media was:0.5 mg·L-1 6-BA + 0.5 mg·L-1 KT + 0.05 mg·L-1 NAA + 0.05 mg·L-1 IAA, and differentiation rate was up to 83%. Futher more, a new type of callus related to concentration of 2,4-D was found for the first time, which increased the callus derivation rate obviously, improved of embryogenic callus, reduced the period of plant regeneration,which laid the foundation for the establishment of genetic transformation system.2. Using callus of sensitive goosegrass as acceptors, the Agrobacterium-mediatedgenetic transpormation system of goosegrass was established by selecting the appropriate concentration of hygromycin, carbenicillin, microbial, acetosyringone etc. The results as follows:(1) The best concentration of hygromycin was 30 mg·L-1.(2) The best concentration of carbenicillin was 300 mg·L-1.(3) The best conditions of genetic transformation was that OD600=0.5, the infection of 30 min which contained 10 min of vacuum and intermittent 5min of intermittent, then 10 min of vacuum and 15 min of oscillation infect.(4) The best time of cocultivation was 3 d.(5) The best concentration of acetosyringone was 300 ?mol·L-1.(6) After rooting of screening, and in combination with GUS staining and PCR amplification, 6 transgenic goosegrasses were obtained.3. The q RT- PCR results show: After spraied paraquat 30~90 min,the expression of these genes are different. The expression of Pq E was lower than sensitive type in resistance type, then increased after 90 min which similar to Pq TS2 and Pq TS3. After paraquat treatment, the expression of Pq TS1 increased and achieved the highest level at 90 min, but had no difference in sensitive type.4. The HPLC results show:(1) No obvious change of putrescine(PUT) between R type and S type after spraied paraquat, so PUT has no effect on reducing toxicity.(2) The content of spermidine(SPD) increased by 400-fold after 60 min of paraquat treatment in resistance type, and then returned to the same level as the initial period; The content of SPD was very few and had no difference in sensitive type during the period of paraquat treatment; That is, SPD reduced paraquat toxicity.(3) The content of spermin(SPM)gradually increased after 90 min of paraquat treatment in resistance type, and decreased thereafter in resistance type; The content of SPM had no obvious difference in sensitive type after paraquat treatment; That is, SPM involved in the mechanism of goosegrass resistance to paraquat and could inhibit the chlorophyll loss.