Creating Low-gossypol Cotton Material By Regulating CAD1 Gene

Cotton is an important economic crop,while cottonseeds are rich in nutrients such as protein and fatty acids.Gossypol is a phytoalexin of cotton itself,but it is also toxic to monogastric animals and humans,which makes the use of cottonseed products extremely limited.Breeding the cotton that the plants have gossypol while the seed is gossypol free has always been the goal of scientific and technical personnel.This study expected to reduce the gossypol content of seeds through two ways,the first way is using RNAi technology combined with a seed-specific promoter to reduce the expression of CAD1 gene in seeds,the second way is using CRISPR/cas9 system to edit CAD1 gene which plays a key role in the gossypol synthesis pathway.Thus the gossypol content will decrease and this study hoped that some new materils with low gossypol in seeds could be obtained.The main research results of this experiment are as follows:(1)The αGP-CAD1-RNAi vector was transferred into upland cotton J668 and 52 transgenic regenerated positive plants were obtained.Eight strains of the lower-ranking gossypol-containing lines were initially screened.(2)The gossypol content of buds,bells,sepals and seed tissues of different individuals in the T1 generation of line 4 was determined.The content of gossypol in buds,bells,and sepals was not significantly different among the individuals.However,in the seeds of different individuals,the gossypol content has a significant difference,which achieved the purpose of reducing the gossypol content of seed.(3)From the T1 generation plants,13 individuals with significantly reduced CAD1 gene expression were screened.Their seed gossypol contents ranged from 1.58 μg/mg to2.61 μg/mg,and compared to wild-type materials,the gossypol content was reduced by38.4%-62.7%.The microscopic observation of the seeds revealed that the pigment glands were less visible in the interference materials than in the wild-type controls.(4)The agronomic traits of 13 individuals were examined.Compared with the control,their plant height,100-grain weight and fiber quality did not change significantly.It was proved that the intervention of CAD1 gene had no significant effect on the normal development of the plant.(5)The CRISPR-CAD1-A/C vector was constructed and transformed into upland cotton J668.Eight positive transgenic CRISPR-CAD1-A cotton plants have been obtained.The seed’s gossypol content has been reduced by 25.7%.