Manipulation of biosynthesis of aliphatic glucosinolates in Brassica crops and Arabidopsis through gene replacement and RNA interference

The hydrolysis products of glucosinolates can inhibit iodine uptake by the thyroid gland, reduce thyroid hormone circulation and cause goiter. Overall, total glucosinolate concentration in Brassica seed meal has to be reduced to a very low level for animal feeding purposes. On the other hand, some specific glucosinolates such as glucoraphanin are reported to block the initiation of tumors in many mammalian tissues by inducing Phase I and Phase II enzymes, so glucoraphanin concentration needs to be increased to add nutritional quality in selected Brassica vegetables.;Turning to Arabidopsis, four MAM-like genes in the elongated amino acid pathway of glucosinolate biosynthesis could potentially control total glucosinolate concentration in Arabidopsis thaliana. Using RNAi techniques, several MAM genes were simultaneously silenced and the total aliphatic glucosinolate concentration in the RNAi lines of A. thaliana were reduced greatly in leaves, and moderately reduced in seeds compared to wild type A. thaliana Columbia (Col-0). It appears that RNAi can effectively overcome gene function redundancy and can therefore be used to study gene function in polyploidy species.;To study gene silencing efficiency in A. thaliana, six different RNAi constructs were developed with the sense/antisense arms ranging from 112 to 898 nt. All the constructs efficiently silenced MAM genes. It was demonstrated that in order to obtain efficient gene silencing, the sequence identity between the source gene for the insert fragment and the target gene of RNAi constructs could be as low as 56% and the effective insert fragment size could be as small as 59 bp. It was also demonstrated that the RNAi transgene was genetically and functionally stable in RNAi hybrids. It appears that RNAi lines could be used in Brassica breeding programs to develop desired varieties with reduced concentration of aliphatic glucosinolates.;It was demonstrated that the GSL-ALK gene in the glucosinolate modification/biosynthesis pathway regulates glucoraphanin concentration in Brassica crops. Broccoli (B. oleracea) with a non-functional GSL-ALK (GSL-ALK-) gene enriches glucoraphanin, white Chinese cabbage (B. rapa) with a functional GSL-ALK (GSL-ALK+) gene does not. Replacement of the functional GSL-ALK+ gene in Chinese cabbage with the nonfunctional GSL-ALK- gene from broccoli could improve glucoraphanin concentration in Chinese cabbage using a non-GMO approach. Introgression of the GSL-ALK - gene from broccoli into Chinese cabbage is required for gene replacement. Two GSL-ALK- gene introgression lines of B. rapa without additional C-genome chromosomes were developed through marker assisted selection and cytogenetic analysis. These gene introgression lines can be used for further gene replacement.