Cloning And Functional Verification Of Several Candidate Insect-Resistant Genes In Cotton For Whitefly Control

As an important natural textile resource,Cotton is always faced with the feeding of various kinds of herbivorous insects,which can cause a massive reduced production.With the widespread adoption of BT Cotton,the BT non-target piercing sucking insects such as Whitefly(Bemisia tabaci)and Apolygus lucorμm,have gradually from the secondary pests to main pests.Therefore,it’s meaningful to study the interaction mechanism of Whitefly and Cotton with transgenic technology.In our study,we have selected 8 genes from the transcriptome data of cotton-whitefiy interactions.These 8 genes are GhWRKY18,GhWRKY21,GhWRKY32,GhWRKY40,GhWRKY46,GhWRKY70,GhERF105 and Gh6PGL2.We want to verification the function of this 8 genes in Cotton insect-resistant signal path by creating the RNAi and Overexpression transgenic materials.1.By constructing phylogenetic tree for grouping WRKY transcription factors,the results show that GhWRKY 32 belongs to the group I;GhWRKY 18 and GhWRKY 40 belongs to the group II-a;GhWRKY 21 belongs to the group Ⅱ-d;GhWRKY 46 and GhWRKY 70 belongs to the group III.Based on analyzing the expression patterns of the WRKY transcription factors under Whitefly induced.The results showed that these 6 WRKY genes may respond positively to the stress of the Whitefly in Upland Cotton.We have constructed 6 RNAi and 4 Overexpression vectors of WRKY transcription factors.Through genetic transformation tecnology,a total of 50 successful positive transgenic lines has been created,and 21 of them were single copy.By qRT-PCR and Southern blotting,we have selected 5 Ri-GhWRKY18 and 4 Ri-GhWRKY 70 low-copy transgenic lines,which the number is Ri-GhWRKY18-1/2/3/4/6 and Ri-GhWRKY70-1/2/3/4.2.Based on analyzing the expression patterns of the GhERF105 and Gh6PGL2 under Whitefly induced.The results showed that GhERF105 and Gh6PGL2 may respond positively to the stress of the Whitefly in Upland Cotton.In this study,26 positive transgenic lines were obtained by using RNAi technology.By qRT-PCR and Southern blotting,we have selected six low-copy transgenic lines of Ri-GhERF105 and eight low-copy transgenic lines of Ri-Gh6PGL2,which the number is Ri-Gh6PGL2-1/2/4/5/7/8/9/11 and Ri-GhERF105-7/8/11/12/13/14.This selected lines will be the point of follow-up insect resistance identification.By statistics the field characters of low copy lines,we found that compared with the control,the height of transgenic plants were significantly lower than control,and most transgenic lines are lower resistant to stinging insects than controls,In particular,the data of Ri-GhWRKY18 and Ri-GhWRKY70 were significantly higher than the control group in the hazard classes of phytophagous insects and the damaged number of blade byphytophagous insects.Speculated that after the interference of GhWRKY18,GhWRKY70,GhERF105 and Gh6PGL2 respective in Cotton,the resistance of Cotton to the piercing sucking insects was weakened and the basic growth of Cotton was affected.