| The cotton bollworm Helicoverpa armigera has been successfully controlled by transgenic cotton(Bt cotton) engineered to express cry toxin from Bacillus thuringiensis which has been planted in Chinasince1997. However, under the selection pressure of Bt protein which persistently expressed in Btcotton, the resistance of H. armigera to Bt has been the key factor for sustainable utilization of Bt cotton.The fitness cost of resistance to Cry1Ac occurred after the resistance appeared in H. armigera, but theintrinsic reasons caused its fitness cost is still unknown. The synthesis and uptake of Vitellogenin (Vg)plays a crucial role in maturate ovary of insect and could directly affects the reproduction ability ofinsect. In this study, the ovary development and the relative expression level of Vg in different strainsare compared after H. armigera had resistance to Bt. The results could not only contribute to understandthe molecular mechanism of pest reproduction, the internal mechanism of fitness, but also provide thetheoretical basis to explore the resistance evolution mode and optimize the resistance managementstrategy. The main results are as follows:1. The ovaries of H. armigera in Cry1Ac-susceptible strain (96S) were dissected using lightmicroscope and transmission electron microscopy. The ovaries was classified which simplified theprevious classification standard. The oogenesis process was classified for the first time, thedevelopment of oocyte in ultrastructure was analyzed and the accumlation of yolk granules duringoocyte formation was cleared. The ovarian development between susceptible strain (96S) andCry1Ac-resistant strain (BtR) was compared. The results showed that the length of ovariole, the ratio ofmature eggs and the proportion of ovarian in resistant strain were significant lower than those insusceptible strain. It was also found that the egg internal nucleus, mitochondria, yolk deposition,microvilli in the resistant strain were changed compared with the susceptible strain.2. Vg cDNA from H. armigera was cloned and sequenced, which was named Helicoverpaarmigera vitellogenin gene (HaVg). HaVg cDNA was5704bp with the open reading frame (ORF) of5265bp (GenBank Accession No. JX504706), which encoded a protein of1765amino acids. HaVgcomprised3conserved domains which included the Vitellogenin–N domain, the DUF1943domain anda von Willebrand factor type D domain (VWD), a protein cleavage sites and RARR period ofpoly-serine zone. The highly conserved GL/ICG motif, the DGXR motif and cysteine residues werefound in the C-terminus of the Vg. HaVg was an outer membrane protein through analysis online. Thepredicted molecular weight of the mature protein was197.28kDa with an isoelectric point (pI) of8.74.HaVg mRNA was specific expressed in the female and was highly expressed in fat body. Themaximum level of HaVg mRNA appeared in48-h-old adults, and then declined.3. The expression levels of the Vg mRNA in different strains along with development of H.armigera were compared using q-RT PCR method. It was found that the expression trend of Vg mRNAin different strain was varied. The Vg mRNA expression level of BtR strains was significantly delayedcompared with the96S strain. The expression level of HaVg gene fluctuated significantly during the developmental stages and detected maximally value was on day3of adult emergence in the96S strain.In the BtR strains, however, the maximum expression level of HaVg occurred on day7of adultemergence and it account was only approximately50%of that in96S strain. The expression level of VgmRNA in the CK1which eliminate resistance pressure for38generations was similar to the96S strain.While BtR off spring fed a non-Bt-toxin artificial diet (CK2) followed similar trends as the BtR strain. Itwas indicated that the Vg gene had obviously changed in transcriptional level after H. armigera the hadresistance to Bt.In summary, there was the fitness cost which performance in reduced of egg hatchability andsurvival rate of larvae after the H.armigera had resistance to Cry1Ac, while the length of ovariole,ovaries ratio and proportion of ovarian mature eggs reduced significantly, and the expression level ofHaVg also reduced significantly with the expression peak. Whereas the susceptibility to Bt, the fitnessand Vg expression level of H.armigera were gradually restored after they were eliminated the selectionpressure. Therefore, it was suggested that there was a close correlation between the fitness cost ofresistance to Cry1Ac in H.armigera and the development of ovaries, the expression of HaVg. |
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