| Root-knot nematode,an important pathogenic nematode,are able to infest more than3000 plant species,causes huge economic losses to agricultural production.As a result,RKNs bring about an estimated $1000 billion annual loss worldwide. The second-stage juveniles of root knot nematode invade root of host plants, then inject secretions of esophageal gland through stylet, which stimulate the formation of giant cells and thus root knots. Some research results show that the proteins synthesized in the esophageal glands play an important role in the interaction of root-knot nematode and host plant. The induced giant cells were found vacuolation and the density of the giant cells contents were reduced gradually after nearly a month of root-knot nematode infecting tomato, so it is thought that females inject proteins causing the intrinsic apoptotic pathway in the host. In this study, we use RNAi and VIGS to analyse the function of Mi PDCD6 and Le MYB330 in order to lay the foundation of revealing the signal pathways how M. incognita regulates the giant cells of its host plant and resistance breeding.In this paper, the innovative results were as follows:1. Revealing the RNA Interference Effect of Mi PDCD6 of M. incognita in vitro. J2 of M.incognita was soaked in 2mg/m L Mi PDCD6 ds RNA for four hours, induced by 1%resorcinum, while the same treatments were done for GFP ds RNA solution, M9 buffer as CKs. After soaking, the J2 of M. incognita of treatment group and CKs were in a state of rigidity. After recovering in water for 4h, the J2 of M. incognita of CKs return to normal activities, but the J2 of M. incognita of treatment group appears irregular contortions and convulsions,the percentage of rigidity have a significant difference between the treatment and CK at statistical level. Semi-quantitative RT-PCR analysis showed lower transcript abundance for the targeted m RNAs than that of control. The J2 of M. incognita of treatment was used to inoculate Ipomoea aquatica Forsk. 28 days after inoculation, the numbers of root-knots was 46.3% less than CK. Those results indicated that Mi PDCD6 plays an important role in nematode's development and parasitism.2. Revealing the RNA Interference Effect of Mi PDCD6 by VIGS tcchnique in vivo. The silencing vector p TV-Mi PDCD6, mediated by Tobacco rattle virus(TRV), was firstly constructed, then the Mi PDCD6 of M.incognita was silenced after agrobacterium-mediated transformation, the amounts of root knot were also investigated on the tomato plants inoculated with M. incognita. 35 days after inoculation, the Mi PDCD6 expression level was lower than that of control by q RT-PCR. The number of root knot and eggmass number on the p TV-Mi PDCD6 treated tomato plants were reduced 62.8% and 60% than the water control. Statistical analysis showed that the amounts of root knot and eggmass t number on p TV-Mi PDCD6 tomato plantlets decreased significantly compared with the control. The study revealed the Mi PDCD6 silencing had a significant effect on root-knot nematode disease caused by M. incognita. It also showed that Mi PDCD6 gene may be involved in the pathogenesis of nematode, and play an important role in it.3. Revealing the RNA Interference Effect of Le MYB330 of tomato by VIGS tcchnique in vivo. The silencing vector p TV-Le MYB330, mediated by Tobacco rattle virus(TRV), was firstly constructed.Then the Le MYB330 of tomato was silenced after agrobacterium-mediated transformation, the amounts of root knot were also investigated on the tomato plants inoculated with M. incognita. 35 days after inoculation, the Le MYB330 expression was lower than that of control by q RT-PCR. The number of root knot and eggmass number on the p TV-Le MYB330 treated tomato plants were increased 6.9% and15.3% than the water control, without significant difference. It showed that Le MYB330 silencing may reduce the resistence for M. incognita. |
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