Gene Cloning Of Root-knot Nematodes Resistance, Genetic Transformation And Rgeneration Of Cucurbita Ficifolia

Root-knot nematodes are comprised of the most agronomic important plant pathogenic nematode species. These plant pathogens are obligate sedentary endoparasites. Juvenile animals invade the roots of the plant and establish specialized feeding structures within the root system. Once established, the animals, particularly the females, persist in the root system for many weeks, feeding continually. This has dramatic, deleterious consequences on plant health and crop yield. These microscopic organisms penetrate the roots of 2000 plant species and cause 10%-20% products damages, especially reduced 75%. The products value are 100 billion dollar every year. The use of biotechnology as part of an integrated pest management strategy to provide nematode control offers a solution with benefits to the producer, the consumer, and the environment in both developed and developing countries. So, we researched the gene engineering of root knot nematodes resistance. The main results are as followes:1 We constructed the clone vector of pGEM-T- RKNIP through analyzing the sequence of TobRB7 promoter. We have applied GenBank, and the accession number is DQ486886. Then we have analyzed the motifs of the RKNIP500 and RKNIP300. To the RKNIP500, we have predicted which have promoter region on forward strand in 56 to 306, and have promoter region predicted on reverse strand in 385 to 135. To RKNIP300, it have not promoter region, but it has many elements been relative to promoter, so we predicted that the RKNIP300 have the potential functional of the promoter during induced. RKNIP was expressed in the root-knot during affected by root-knot nematodes. It is important to deduce the damage caused by root-knot nematodes and increase the biosafety.2 We synthesized the OC-I-ΔD86 by overlap extension PCR method with 7 oligonucleitides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IΔD86. 2 oligonucleitides fragments were disigned, and annealed to synthesize interference sequence. The fragment was inserted into pGEM-T-RKNIP vector and the recombined plasmid was named pGEM-T-RKNIP -16D10-33(pTT33). 3 We have constructed the expression vector of pRTO, pRO, pRT33, pRTOT33. pRTO is TobRB7Δ0.3:OC-I-ΔD86; pRO is CaMV35S:OC-I-ΔD86; pRT33 is TobRB7Δ0.3:16D10-33; pRTOT33 is CaMV35S:OC-I-ΔD86 and TobΔ0.3: 16D10-33. We have prepared the material for system study of gene tranfer.4 OC-IΔD86 gene was being cloned into the corresponded sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IΔD86. OC-IΔD86 gene was expressed in E.coli (BL21(DE3)plysS) after 5 hours′IPTG(Isopropylβ- D -1-thiogalactopyranoside) inducement. The fusion protein of OC-IΔD86:6His gene accounts for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. We have got the high effective antibody (>10000 times) by immunized mice.5 We have got the transgenic tobacco plants of pRTO, pRT33, pRO By Agrobacterium- mediated. The plants of pRTO, pRO developed normal plants, but the plants of pRT33 can develop the abnormal plants. Because the pRT33 is interference vector, the expression probably is induced by wound. The transgenic plants of pRTO were detected by the methods of PCR, SOUTHERN, ELISA, Character resistance and expressed the protein of OC-IΔD86. In the experiment of root knot nematode resistance, the damage of root knot nematode can be reduced greatly by three stubbles.6 Cucurbita ficifolia is an important plant with the extensive adaptability. A protocol is outlined for adventitious bud regeneration from cotyledon explants of Cucurbita ficifolia. Adventitious buds were induced from the cotyledon of in vitro plants. Explants were collected from seven-day-old seedlings, supplemented with BA (2.0mg/l), TDZ (1.0mg/l) and ZT (2.0mg/l). The data obtained showed that in vitro organogenesis of Cucurbita ficifolia occurred with higher efficiency, 91.67%, 98.33% and 91.67% respectively. On MS medium supplemented with 0.25 mg/l KT, adventitious buds grew quickly and 80-100% of buds developed into shoots. The shoots rooted successfully with MS medium supplemented with 0.1 mg/l IAA, and the data is 100%. The survival rate of transplantation of tissue culture plantlets reached 85.0%. This system of adventitious bud regeneration from cotyledon explants could be useful for the genetic transformation and rapid clonal propagation of Cucurbita ficifolia. We have formed the regeneration of mature embryo of Cucurbita ficifolia. The proper cultural medium is MS+TDZ0.5mg/L.