Study On Aphid-Resistance Of Cotton Gene GhRac6

Aphis gossypii is one of the important economic pests of cotton in the world.They not only suck plant sap,plunder plant nutrients,secrete honeydew that causes coal pollution,but also spread a variety of plant virus diseases,and make serious damage to crops.The aphids are not only small in size,but also have a short life cycle and have amazing reproductive capacity.They can breed for about 20 generations a year.These characteristics lead to aggravation of frequent and unreasonable use of pesticides,resulting in the emergence of highly resistant varieties resistant to various pesticides.Therefore,the prevention and control of aphids is more difficult,and still has a long way to go.It is always the goal of the majority of plant protection workers to find an effective,long-lasting and environmentally friendly method for controlling aphids.Using the resistance to aphids of plant itself to control aphids is considered to be a highly efficient and environmentally friendly way to prevent and control aphids,and get the general attention of researchers.However,with the widespread use of anti-caries genes,some plant anti-caries genes lose resistance to aphids,suggesting that the prevention and control of aphids will face more serious tests sooner or later.Thus,continue to explore new anti-caries genes,understand the molecular mechanism of interaction between plants and aphids,and make full use of crop resistance,which is of great significance for the effective management of aphids in a long-lasting production system.Plant ROP/RAC protein belongs to the subfamily of the Rho family GTPase,which is considered to be a versatile molecular switch that modulates various plant developmental processes and produces defensive response to multifarious abiotic and biotic stresses.However,whether they have insect resistance has not been reported.Herein,we cloned a new member of ROP/RAC family GhRac6 based on the suppression subtractive hybridization cDNA library of cotton leaf induced by cotton aphid and used the transgenic technology to verified its function and mechanism in the defense response to aphid.The results are as follows:(1)The cotton gene GhRac6 was cloned from red-chicken-feet-leaf cotton,and its ORF is 636 bp in length and encodes a 211 amino acid.Its isoelectric point is 9.029 and the predicted protein molecular weight is 23,338.97 Da.(2)Homologous sequence alignment analysis showed that GhRac6 has a high degree of sequence similarity(93.25%)to ROPs of other plant species.Phylogenetic analysis of GhRac6 and other plant ROP proteins indicated that GhRac6 is most closely related to GhRac2,and its relationship with PeARAC8 and cARAC8 is more distant.(3)The transcriptional level of GhRac6 in cotton leaves was inhibited after cotton aphid feeding,and the transcriptional level of GhRac9 was only inhibited in the leaves of cotton.The transcription levels of GhRac6 and GhRac9 were significantly up-regulated in the leaves of cotton after induction by aphid saliva.The transcription levels of GhRac1 and GhRac2 were also significantly inhibited in the pre-feeding stage of aphids,but there was no significant change after the induction of aphid saliva.(4)Transgenic Arabidopsis plants of GhRac6 were obtained by Agrobacterium-mediated transformation.The lines of Line1,Line2 and Line3 were selected for insect resistance experiments.The number of aphids on transgenic Arabidopsis was less than that of wild-type Arabidopsis in both selective and non-selective experiments.In the experiment of measuring the secretion of aphid honeydew,the amount of honeydew secreted by the aphids on the transgenic Arabidopsis Line3 was significantly lower than that of the wild type Arabidopsis.These results indicate that overexpression of GhRac6 can increase Arabidopsis resistance to Myzus persicae(5)In order to elucidate the reason why GhRac6 enhances the resistance of Arabidopsis to aphids,soluble sugar and proline content,phenylalanine ammonia-lyase and catalase activity,and leaf callose accumulation in transgenic Arabidopsis were determined.The results showed that the soluble sugar content and the proline content were respectively higher in transgenic Arabidopsis than those in wild type.The activities of PAL and CAT in transgenic Arabidopsis were also higher than those in wild type.Callose depositions were more in transgenic Arabidopsis than those in wild type.(6)Th insect-resistant experiments were carried out on cotton plants that transiently expressed GhRac6 and GhRac9.The results showed that the number of aphids on the cotton leaves overexpressing GhRac6 and GhRac9 was lower than that of the control plants,and reached a significant level.(7)To elucidate the reasons for GhRac6 and GhRac9 to increase the resistance of cotton,the transcription levels of CAT,PPO,POD,PAL,NPR1 and EDS1 genes were detected.The transcription levels of POD,PPO,PAL and EDS1 in transiently overexpressed GhRac6 cotton leaves were significantly up-regulated,and POD and PAL reached extremely significant levels;in the transient overexpressing GhRac9 cotton leaves,the transcription levels of CAT,POD,PPO and PAL were significantly up-regulated,and CAT,PPO and PAL reached extremely significant levels.