| Cucumber (Cucumis sativus) is the mainly vegetable in China. With the adjustment of planting method, the root-knot nematodes(Meloidogyne spp.) have become the major problem in cucumber production. The cucumber mainly infected by four nematodes of Meloidogyne incognite, M. javanica, M. arenaria and M. hapla. Several sources of resistance to root-knot nematode have been identified in wild cucumber sources. The cucumber inbred NC-46 was developed from the improved North Calinahardwickii 1(NCH 1) cucumber popularion and had LJ90430 germplasm in its background. NC-46 habours resistence to M. javanica 1,M. arenaria race 2 and M. arenaria race 2.In this study, a cDNA library of NC-46 transgenic hairy root was constructed and analysis. A novel method for propagation root-knot nematode using cucmber plantlets and hairy root were established. And a new method of studing root gene function using composite cucumber plantlet with hairy root was developed. The results are as follows:1.The transgenic hairy roots of NC-46 were induced from cotyledon by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 76.7%. Besides, cDNA library of the transgenic hairy roots was constructed successfully. The percentage of recombinant clones in the cDNA library was 98.3%. The original storage capacity reached 2.02×106 cfu, while the ratio of full-length gene and unigene were 47.6% and 66.7% respectively. The cDNA library is useful for screening resistant genes, and cloning of developing gene and founding the developing mechanism of hair roots.2. Root-knot nematode of Meloidogyne incognite, M. javanica and M. arenaria can host in cucumber seedlings for propagation in transparent glass bottles. In addition, the Meloidogyne incognite could grow on hairy roots normally. It is a novel method for efficient separation of root-knot nematode. This method not only enriches the host of root-knot nematode reproduction, but also is convenient to observe the root-knot formation and prevent nematode to escape to the surrounding soil.3. A vector pRI 101-AN-gfp was constructed with plasmids of pRI 101-AN and pGFP. After the cucumber plantlets infected by K599 with the recombinant plasmid pRI101-AN-gfp at 0.2-0.5 cm location below coteledon, the hairy root was induced. The comopsite plant was developted when the hairy root grown to length 5-10 cm. The comoposite plant could be used to study the root gene function. The utility of the procedure made possible analyses in wide gene function rapidly and easily.The above-results provide the important foundation for cloning the candidate resistant gene to nematode from cucumber inbred line NC-46 and identification gene function rapidly and effectively. |
