Mining And Functional Verification Of Apolygus Lucorum(Meyer-D(?)r)-resistance Gene In Gossypium Hirsutum L.

Cotton is an important economic and fiber crop,providing an essential irreplaceable raw material for the textile industry.Before the development and commercialization of transgenic cotton,the cotton aphid,bollworm,leaf mite and pink bollworm were the plant's most serious pests.With the widespread planting of transgenic Bacillus thuringiensis cotton,and the reduction of pesticide doses in the field,the damage caused by cotton bollworm has been effectively controlled.However,the numbers of non-target pests,such as cotton aphid,cotton mirid and whitefly,in cotton fields have increased significantly,and they have gradually become the main insect pests,causing great losses to cotton production.In particular,the harmful cotton mirid Apolygus lucorum(Meyer-Dür)is the main mirid found in cotton fields.Owing to the lack of suitable resistant species,the main solution to Apolygus lucorum(Meyer-Dür)infestations at present is to continuously spray chemical insecticides during cotton cultivation.This not only increases the planting cost but also has adverse environmental impacts.In this study,the Apolygus lucorum(Meyer-Dür)-resistance gene Cry51Aa1.C006 was obtained by mutagenizing the Cry51Aa1 gene.Then,Cry51Aa1.C006's function was verified in transgenic cotton,and homozygous lines of Apolygus lucorum(Meyer-Dür)-resistant cotton were selected.The main results are as follows:1.The Cry51Aa1 gene underwent site-directed mutagenesis,and six novel mutated genes,Cry51Aa1.C005,Cry51Aa1.C006,Cry51Aa1.C007,Cry51Aa1.C008,Cry51Aa1.C009 and Cry51Aa1.C010,were obtained.Then,prokaryotic expression vectors individually harboring Cry51Aa1 and the six mutated genes were constructed,and the seven proteins were each expressed and isolated.The in vitro virulence levels of these proteins against Apolygus lucorum(Meyer-Dür)were determined using a feeding experiment.The mutant protein Cry51Aa1.C006 had a strong killing effect on Apolygus lucorum(MeyerDür),while,in comparison,no obvious effects of the other five mutant proteins were identified.2.The Cry51Aa1 and Cry51Aa1.C006 genes were independently transformed into cotton variety Zhongmiansuo 24 using an Agrobacterium tumefaciens-mediated method,and 11 homozygous transformants were identified by kanamycin selection and PCR amplification.Then,total RNA was extracted from different transgenic tissues(leaves,stems,stem ends and buds)for a qRT-PCR analysis.Cry51Aa1.C006's expression was up-regulated in all the tissues of the 11 transformants.Among the transformants,Cry51Aa1.C006-10-13 and Cry51Aa1.C006-10-61 had the strongest resistance levels against Apolygus lucorum(Meyer-Dür).An agronomic and quality trait analysis showed that the transgenic materials expressing Cry51Aa1.C006 had significantly greater plant heights,boll numbers and the numbers of fruiting branches,while there were no significant differences in the lengths of the first fruiting branch,boll weights,lint percentages and fiber qualities(upper half mean length,regularity degree,fiber strength,micronaire value and elongation ratio)compared with the recipient material Zhongmiansuo 24.3.The two highly resistant transformants Cry51Aa1.C006-10-13 and Cry51Aa1.C006-10-61 were analyzed by Southern blot,which revealed that they both had single copy insertions.The identification of the flanking sequences by genome walking showed that the insertion site of Cry51Aa1.C006-10-13 was at 63,328,845-63,329,028 of chromosome D12,replacing the original 182-bp fragment,while the insertion site of Cry51Aa1.C006-10-61 was at 32,939,476-32,939,524 of chromosome A13,replacing the original 47-bp fragment.