| The secretion of cotton bollworm(Helicoverpa armigera.Hubner)during feeding on cotton plays an important role in the study of the interaction mechanism between cotton and bollworm.There are many small protein molecules with unknown functions in the oral secretions,such as the activator of inducing plant defense response and the effector of inhibiting plant defense response.By studying the effects of effectors in oral secretions of cotton bollworm on the control of antiinsect defense response in cotton and the interaction between target proteins in cotton,we can provide targets for the creation of cotton resistant to bollworm,and also provide theoretical basis for the cultivation of cotton insect-resistant germplasm.In this study,19 candidate effectors were selected from the EST database(PRJNA388211)of salivary glands of cotton bollworm by bioinformatics method,and the effects of candidate effectors on plants were further screened and verified by transient expression experiments of tobacco and genetic transformation experiments of cotton.The results are as follows:1.Among the 19 candidate effectors,there were 11 known functional proteins,including a lipid-associated protein MD-2 family 5481(XM-021325481),an insect pheromone associated protein 4835(XM-021334835),two serine-like trypsin0600(XM-021340600)and 3673(XM-021343673),and four serine-like proteinase inhibitor proteins 6086(XM-021346086)、 2397(XM-021342397)、 2398(XM-021342398)and 2399(XM-021342399).One insulin-like growth factor 6326(XM-021326326),one insulin-like growth factor binding protein 0252(XM-021340252).2.The results showed that 7997(XM-021327997)induced the cell death reaction in tobacco,while the other proteins did not cause tobacco necrosis.Four effector protein transgenic materials were successfully obtained by stably expressing the 19 candidate effectors in cotton,namely 24 effector proteins 0600,20 effector proteins 2191(XM-021332191),14 effector proteins 5481 and 4 effector proteins4835.Further positive tests of these four transgenic materials showed that 12 plants were 0600 positive,8 plants were 5481 positive and 11 plants were 2191 positive.The expression level of the positive material was detected,and the results showed that 0600-1,0600-10,0600-34,0600-54,0600-43,0600-53;2191-2,2191-8,2191-25,2191-35;The expression of target gene was high in 5481-9,5481-24,5481-31,5481-33,5481-40,5481-46,5481-57 plants.These materials will be the focus of subsequent research.3.Transgenic lines with high expression levels were selected for the resistance experiment of cotton bollworm,and it was found that the average body weight of cotton bollworm fed on transgenic materials with 0600-10,0600-34 and 0600-54 was 0.06g-0.12 g higher than that of the control material group on the 8th day,and the body length of cotton bollworm was also significantly increased by about 2mm.The cotton bollworms fed on transgenic cotton lines 2191-27,2191-30 and 2191-25 increased by 0.04g-0.15 g compared with the control group,and their body length was 2mm-3mm longer than that of the control group.Compared with the control group,cotton bollworms fed on transgenic lines 5481-9,5481-40 and 5481-31 increased by 0.02g-0.09 g and body length by 2mm-3mm.The results indicated that the expression of bollworm effector in cotton may reduce the defense response in cotton.4.The expression levels of JA and SA-related genes in the transgenic materials were detected,and it was found that the transcription levels of JA-related genes LOX1,PDF1.2 and JAZ10 in the transgenic materials were significantly decreased.The SA synthesis genes ICS was significantly increased,and the regulatory genes NPR1 and PR2 in the SA-signal pathway were significantly decreased,compared with the control materials.The data showed that the stable expression of0600,2191,5481 in cotton triggered hormone-related pathways,which increased the sensitivity of cotton bollworm to transgenic materials,and further confirmed that the expression of cotton bollworm 2191,5481,0600 in cotton reduced the defense of cotton. |
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