| Plant disease is one of the serious problems affecting plant growth and output of crops etc. It is difficult to control because pathogens mutated frequently, and this leading to the disease resistant plants broke down their resistance. The excessive application of chemical pesticides is not only leading to the pathogen resistance against the chemicals, but also harmful to the environment, and certainly to the health of human beings. Therefore, biological control agents (BCA) are considered as the possible replacement of chemical pesticides, and as an environmental friendly agent for further plant disease control.Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical representation of plant defense reactions. Once the establishment of SAR, the plants exhibit a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins for example elicitins and hatpins, which elicit plant defense reactions. It would be feasible to explore biological control pesticides base on the characteristics of plant SAR.Trichoderma viride is an ubiquitous soil saprophyte biological control microbe having the action modes with pathogens of competition for nutrient resource, antibiosis, and mycoparasitism. If T. viride can be used as a producer and carrier of an elicitor protein, it would be applicable for the development of plant specific BCA. To test this idea, we used cryptogein, a proteinous elicitor secreted from Phytophthora cryptogea for the development of bioengineering T. viride. The plasmid containing Crypt or its mutant was introduced into T. viride by the method of restriction enzyme mediated integration (REMI). Our study indicated that T. viride acts as the producer and carrier of Crypt, and at the same time the transgenic lines enhanced disease resistance when applied on tobacco plants. The summary of our study as follows:1. Construction of pCSNTCC and pCSNTCCm plasmidsFirstly, Crypt gene was mutated by change the K at the position 13 of Crypt into V (the mutant named CryK13V) as described earlier. In order to secrete the produced protein out of T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5' of Crypt and CryK13V. The chimeric genes were constructed under the control of trpC promoter in vector pCSN43 respectively. Then, a hygromycin resistant gene was introduced into the vectors, and plasmids of pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) were obtained.2. Establishment of T. viride transformation systemThe conditions for protoplasts isolation and regeneration from T. viride were studied. The optimum condition for protoplast isolation was the 24-hours-cultured hypha of T. viride digested with 4 mg/mL Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 , the yield of the protoplasts was 4.7 107colony forming unit/ mg. In this study, the maximum regeneration rate (14.5%) was obtained in the CM medium containing osmolutes of 0.3 mol/L KC1 and 0.3 mol/L inositol. Further, plasmids of pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by restriction enzyme Xho I mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The hygromycin-resistant transformants were determined by polymerase chain reactions. The elicitor protein was detected in the culture media but not in the cells by western blot analysis. The result indicated that the exogenous gene was expressed in T. viride and the produced protein was secreted into the culture media of transformants as expected.3. Expression of Crypt in T. viride enhanced plant disease resistanceTobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride in the soil. Ten days after the treatment, the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria lalternata, Pseudomonas syringae tabaci (Pst), and...
|
PDF Full Text Request