| Cassava(Manihot esculenta Crantz) is one of the most important root crops in the tropics. It’s not only the main source of dietary carbohydrates for almost750million people worldwide but also provide a large amount of easily extractible starch for industry material, fodder, biomass energy and emergency crop. It’s the fifth crop after rice, corn, sweet potato and sugarcane. Tetranychus cinnabarinus is an important pest mite around the world. In China, it is one of the most important cassava pests with serious damage and loss and difficult to control. Until now, acaricides are the main control method against T.cinnabarinus. Howerver, T.cinnabarinus often outbreaks after cassava planted for about6-8monthes when it is very difficult to spray pesticedes, which will result in uneffective utilization of pesticides, increase of frequency and dose and serious "3R" problem. Seeking new strategies and methods to effectively control T.cinnabarinus become an important issue to be solved in current cassava industry development. Development of host-plant resistance varieties is the most economic, effective and the simplest method to control cassava pest mites. In our study, on the basis of the screened stable resistant and susceptible cassava germplasms and the biological and enzymatic defensive foundmental in cassava mite-resistant, we further cloned the protective enzyme gene MeCu/ZnSOD and MeCATl and produced transgenic cassava. Then, from the aspect of developmental biology and enzymatic defense effection, we verificated the function of MeCu/ZnSOD and MeCATl gene in cassava mite-resistant. It will provide basic information, experimental material, and technical support for the molecular design breeding in mite-resistant cassava germplasms. The main results are as follows:1. Four germplasms steadily developed resistance to T. cinnabarinus, C1115, Myanmar, Colombia-4, PI1167and four susceptible germplasms, CM1210-10, WT (C3), Swiss-F21and BRA900, were screened from228cassava germplsms on the basis of the established mite-resistant evaluation standard of cassava germplasms. The mite damage index of four resistant germplasms and four susceptible germplasms were0.6,1.8,25.8,25.9and98.8,99.2,99.2,99.2, respectively. It provided experimental material for the mite-resistance mechanism of cassava germplasms.2. The development and reproduction of T. cinnabarinus fed on four resistant germplasms and four susceptible germplasms were differences significantly (P<0.05). In compared with the susceptible germplasms, after feeding on the resistant germplasms, the eggs production, female adults lifespan and the hatchability were 7-16eggs,7-13d and25%-36%decreased significantly than the susceptible cassava germplasms’39-49eggs,18-21d and.77%-100%. The developmental duration in each stage of mite after feeding on the resistant germplasms were5-6d、4-4.5d、4-6d、4.5-5d and18.5-21d respectively, significantly prolonged than the susceptible cassava germplasms’4d、2d、2.5-3d、2.5-3d and11-12d. The results preliminarily clarified the resistance mechanism of cassava germplasms to the T. cinnabarinus from the aspect of develomental biology and it aslo provided the basic information to support the mite-resistance mechanism studies of cassava germplasms.3. The activity of protective enzymes SOD, CAT, POD and PPO increased after the leaves were damaged by T. cinnabarinus, however, more significant increasing were detected in the resistant cassava germplasms than those of in the susceptible cassava germplasms (P<0.05). Compared with those before damaged, the activity of SOD, CAT, POD and PPO in the resistance cassava leaves increased2.07-2.21and2.06-2.42times,2.04-2.15and2.08-2.19times,2.00-2.30and2.12-2.17times,2.07-2.33and2.19-2.35times, respectively after they were damaged1day and8days by the mite. However, in the susceptible cassava leaves, those of them only increased1.01-1.08and1.02-1.17times,1.06-1.18and1.11-1.23times,1.01-1.15and1.04-1.15times,1.18-1.20and1.11-1.39times. Correlation analysis showed that the cassava germplasms on the T.cinnabarinus resistance and activity of the protective enzymes SOD, CAT, POD,and PPO increased after the leaves were damaged by T. cinnabarinus was a significant positive correlation (P<0.05, correlation coefficients were0.9435and0.9462,0.9387and0.9323,0.9345and0.9356,0.9348and0.9402) results laid a theoretical foundation for the mite-resistant gene mining and innovative use of resistance cassava germplasms to spider mites.4. Combining bioinformatics and molecular biology, the gene of MeCu/ZnSOD and MeCATl were cloned and used to produce transgenic cassava. The mite-resistance of transgenic cassava and the function of MeCu/ZnSOD and MeCATl gene was identified and evaluated. The results shows that in compared with the no-transgenic cassava, the higher expression of MeCu/ZnSOD and MeCATl gene in transgenic cassava SC2, SC4and SC11leaves has enhanced tolerance to oxidative stresses. The eggs production, female adults lifespan and the hatchability of T. cinnabarinus fed on the transgenic cassava were15eggs,13eggs and13eggs,12d,10d and10d,36%,32%and31%, respectively, significantly decreased than the no-transgenic cassava germplasms’46eggs,22d and100%(P<0.01).But both of these data have no significant difference with the mite-resistant germplasm C1115’s 7eggs.7d and25%.. After feeding on the transgenic cassava SC2, SC4and SC11, the developmental duration in each stage of mite was5d、3d、4d、4-5d and16-17d, significantly prolonged than the no-transgenic cassava germplasms’4d,2d,2d,2d and10d, these data also have no significant difference with the mite-resistant germplasm C1115’s6d,4d,6d,5d and21d (P<0.05). Meanwhile, the activity of SOD and CAT in each stage of the mite fed on transgenic cassava was siginificantly decreased compared with those of them fed on WT (C3), only0.337-0.648times of them fed on WT (C3). The results showed that the transgenic cassava on T. cinnabarinus resistance have a significant positive correlation with the T. cinnabarinus body protective enzymes SOD and CAT activities.(P<0.05, correlatin coefficients were0.9342and0.9435,0.9454and0.9423,0.9356and0.9346,0.9436and0.9462). And the mite damage index of transgenic cassava was19.8,18.2and20.9respectivly, showed a resistance performance. In a word, our study demonstrated that the transgenic cassava can significantly inhibited the development, reproduction and protective enzyem activities of T. cinnabarinus, and showed up the same mite-resistance level as the mite-resistant cassava germplasms C1115. Therefore, we verificated the function of MeCu/ZnSOD and MeCATl gene in the cassava mite-resistant directly and broadened the content of plant anti-insect gene engineering, the theory and utilization ways of SOD and CAT, it will provide a theoretical basis for new germplasm creation and molecular-designed breeding of cassava resistance to T. cinnabarinus. |
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