Creating Colored Cotton Germplasm With Indigoidine Synthesis In Fiber

Naturally-colored cotton has problems such as poor fiber quality,low productivity,and limited fiber color.The color limitation of colored cotton fiber is the main bottleneck for the development of colored cotton industry.Different fiber colors and superior fiber quality have always been an important subject in the study of colored cotton.To get new fiber color,a new metabolic pathway must be introduced into the fiber.Indigoidine is a natural blue pigment,which is formed by the cyclization of L-glutamine to form lactam and then spontaneous dimer.The dual expression plasmids of BpsA and Svp genes have been successfully used to synthesize and accumulate indigo in E.coli,yeast,and rose petals,and the corresponding colony and tissues appear blue.This provides new ideas to introduce indigoidine synthesis pathways into cotton fibers.In this paper,the fusion gene of GoBpsA and GoSvp is expressed in cotton fiber to synthesize and accumulate indigoidine in the fiber to create a new blue fiber cotton material.Considering that the expression of GoBpsA and GoSvp genes may be strongly toxic to plant cells,and may inhibit the development of cotton fibers,we use a fiber elongation stage-specific promoter to drive Svp RNAi elements to inhibit the GoBpsA:GoSvp expression in the elongation stage(1-30 days after flowering),and to delay indigoidine synthesis to the late stage of secondary wall synthesis.The main results are as followings:1.Functions of GoBpsA and GoSvp genesBased on the protein coding sequence of BpsA of Streptomyces lavendulae and S.verticillus Svp,and codon optimized according to cotton codon usage frequency,GoBpsA and GoSvp genes were synthesized and fused with the guide peptide sequence of small subunit of the bean Ru Bis CO.About 31% and 32% nuleotides were optimized in the gene GoBpsA and GoSvp,respectively.Simultanous expression of GoBpsA and GoSvp in tobacco leaves and cotton cytolydens lead to wilting,showing that co-expression of GoBpsA and GoSvp genes can synthesize indigo,but at the same time induce cytotoxicity.2.Expressiing GoBpsA: GoSvp fusion gene in cotton fiberThe fusion gene GoBpsA: GoSvp was constructed downstream to a fiber secondary cell wall thickening stage-specific promoter p Fbl2 A.The expression vector FblBpsA-2A-Svp was transformed into cotton and 22 positive transgenic lines(referred to as BAS)were obtained.The gene expression analysis showed that the GoBpsA: GoSvp fusion gene had the highest expression in the 14-DPA(days post anthesis)fibers.Compared with the acceptor cotton,the transgenic cotton had light blue fiber,indicating that simultaneous expression of GoBpsA: GoSvp could promote the synthesis of indigoidine in cotton fibers.However,the fiber development is significantly inhibited,and the cotton fiber is film-like attached to the surface of the seed and cannot form a long and fluffy mature cotton fiber.Furthermore,the lint percentage and lint index of BAS transgenic plants were significantly lower than those of non-transgenic regenerated plants,indicating that fiber development was significantly inhibited in GoBpsA: GoSvp expressing cottons.It can be inferred that expression of GoBpsA:GoSvp fusion gene has a biological function,which promote indigoidine synthesis and make the fiber appear blue,but simultaneously cause serious cytotoxicity,inhibiting fiber development in the early stage of secondary wall synthesis.3.E6-Svp RNAi provides new ideas for improving cotton genetic qualityIn order to alleviate the inhibitory effect of GoBpsA: GoSvp expression on cotton fiber development,the Svp RNAi element,driven by a promoter specific in fiber elongation stage(1-30 DPA),was enmployed to inhibit the expression of GoBpsA:GoSvp in the early stage of secondary wall synthesis(15-30 DPA).The Svp RNAi element was constructed downstream to p E6.The final specific expression vector E6-Svp RNAi was transformed into cotton and a total of 21 positive transgenic lines(abbreviated as ESI)were obtained.ESI lines were pollinated with BAS01 homozygous plants,and the offsprings were planted to test genotypes,gene expression and fiber phenotypes.Expression analysis showed that,compared with B+E-lines(containing only BAS),the expression level of GoBpsA: GoSvp was significantly reduced in15-DPA fibers of B+E+ lines(containing both BASand ESI),but GoBpsA: GoSvp expression level increased relatively in 18-DPA fibers.Phenotypic analysis showed that fiber development has been significantly improved in B+E+ lines compared with the B+E-line.These result suggested that E6-Svp RNAi can significantly down-regulate the expression level of the GoBpsA: GoSvp in the early stage of secondary wall synthesis,delay the expression of the GoBpsA: GoSvp in the developing fibers,and consequently relieved the cytotoxicity of GoBpsA: GoSvp expression,thereby improving the development of cotton fibers.