| Cotton Gossypium Spp is one of the most important economic crops all over the world. Cotton bollworm Helicoverpa armigera (Hiibner) is a serious agricultural insect pest worldwide, especially to cotton. Bt is the most important and successful biological pesticide so far, which is used to prevent and control agricultural pests widely. Vast planting of Bt transgenic cotton (Bt-Cryl Ac cotton) has been high-effective for keeping several major insect pests, including H. armigera, under control and reducing the need for insecticide sprays. However, as the growing area of Bt cotton has been increasing rapidly, it is not clear whether H. armigera has resistant to Bt and how it happens at present.In this study, by using Cryl Ac protein to screen H. armigera resistant strains 196 and 105 generations respectively,we choose resistant strains(BtR and LFR) whose relative resistance coefficient reaches 3937 and 4448 times compared with control sensitive strains(96s and LFS) as research materials,three main receptors, CAD、APN1、ALP1 and the resistant mechanism of H. armigera to Bt were compared and analysed. At the same time,we compared the transcriptome changes of Gossypium Spp after induction of H. armigera feeding at different time points.The main results were as follows:1.The EST sequences of CAD, APN1 and ALP1 of resistant strain and sensitive strain cotton bollworm were cloned and compared. The results showed that, CAD toxin binding sites bases of resistant strains have obvious mutations in coding regions that result in changes to the amino acid sequence of proteins; however, both APN1 and ALP1 had only a few silent mutations. Based on the results above, the mutation of CAD gene might be associated with the resistance of resistant strain cotton bollworm to Bt.2.By comparing the expression level of CAD、APN1 and ALP1 in different instars of resistant strain and sensitive strain cotton bollworm, we found that the expression quantity of CAD and APN1 in resistant strain were lower than that in sensitive strain in the whole generation cycle. However, ALP1 expressed more in resistant strain than in sensitive strain cotton bollworm. In the sensitive strain, the highest expression level of CAD, APN1 and ALP1 were in the 2nd,2nd and 1st instars respectively; however, to resistant strain cotton bollworm, their highest expression level appeared in the 3rd,3rd and 2nd instars respectively. It was clear that the maximum expression of three receptor gene in sensitive strain presented earlier than that in resistant strain. To confirm the function of the three genes, the sensitive larvae which were feed on artificial food containing 240μg/μl Cry1Ac proteins were silenced ALP1, CAD and APN1 respectively. Bioassay results showed that the larvae which were injected siRNA of CAD or APN1 had a lower mortality, higher pupation rate and eclosion rate than control. However, after injecting siRNA of ALP1, the mortality rate of the larvae was significantly increased and the larvae can not pupate and eclosion.3.The non-transgenic conventional cotton (ShiYuan321) were feeding induced at the seven true leaf stage by 4th instar cotton bollworm for 8,24 and 72 hours respectively, and the leaves which were not affected by the pests in the corresponding period were control (CK). Then these four samples were analyzed by high-throughput transcriptome sequencing technology RNA-Seq (RNA sequencing). Solexa sequencing results showed that when the average sequence length was 200 bp, the Reads numbers of CK, SYC8, SYC24 and SYC72 were 43,567,864,42,320,029,44,243,652 and 42,676,507 respectively. 141,396 EST cluster (contigs) with average length of 527bp were got through EST assembled by trinity.4.GO analysis by GoPipe was carried out. Firstly, matched the forecast proteins with Swiss-Prot and TrEMBL database by blastp under the E value< 1 e-5. Then comparing the results by GoPipe program according to gene2go to get the GO information of the predicting protein. A total of 14,146 predicted protein, matching 110,649 GO terms. Matching the predicted proteins in KEGG database, under the conditions of two-way blast, E value<1 e -5, predict protein KO number was got. According to KO number, predicting proteins involved in the metabolic pathways can be known. Totally,3,249 proteins obtained KO number and their metabolic pathways were mapped.5.The significant different genes compared with CK in samples showed that:the up regulated genes numbers (p< 0.001) of SYC8, SYC24 and SYC72 were 2,591,3,652 and 8,667 respectively; the down regulated genes numbers (p< 0.001) of SYC8, SYC24 and SYC72 were 1,362,4,615 and 2,819 respectively. Besides, there were 677 genes showed up regulated and 826 genes down regulated in all SYC8, SYC24 and SYC72.The results have important reference value for the optimal use of Bt, prolonging the life of Bt cotton varieties and the development of new insect-resistant gene. Morever it laid a foundation for the analysis of the interaction mechanism of Bt cotton and H. armigera. |
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