The Preliminary Study Of Molecular Mechalism Of Red Mutant By T-DNA Insertion In Upland Cotton

Cotton has long been the world’s most important source of natural textile fiber, over90%of economic value resides in the cotton fiber. Howerver, the development of cotton genomics is very slow because of its tetraploidy and complex genome. The mutation of T-DNA insertion for functional genomic research hasn’t been reported now.The molecular mechanism of the red mutation of T-DNA inserted into Upland cotton via Agrobacterium tumefaciens is studied primarily. The mutant plants grow normally with normal phenotypes except red colour in the whole plant with strong insect resistance, the fibre yield almost was at the same level of that in background cotton. Single copy of foreigh T-DNA inserted in red mutant has been confirmed by Southern Blot. The characteristics of the red mutant show red colour during the whole growth period of seedling, flowering and fruiting under the condition of natural light. However, under the artificial light in plant culture room, the new leaves and stem tips turn green, then they are moved to cotton fields under natural light, the whole plants appear red again in one day. Tail-PCR techonology was used to confirm the locus of T-DNA insertion firstly. T-DNA was located at3’terminal of a silent gypsy retrotransposon. cDNA-AFLP analysis was used to indentify differentially expressed genes between red mutants and background plant. Most differentially expressed gene fragments belonged to gypsy retrotransposon groups and genes flanked. The flank sequences of T-DNA insertion were sequenced as flavonol3-O-methyltransferase and photosystem Ⅱ. The promoters and other regulation elements of two genes haven’t been scanned in their upstreams. The red mutant was perhaps resulted from the overexpression of GhFOMT and GhPⅡ drived by the CaMV::35S of foreigh T-DNA. GhFOMT and GhPⅡ genes were cloned and overexpression vectors were constructed with pB7WG2D. The two genes were transformed into Arabidopsis thaliana via Agrobacterium tumefaciens LB4404to verify genes’functions, and then will be transformed into cotton Coker312.The chlorophyll and anthocyanin were measured, the content of chlorophyll in red mutant is slightly lower than background Coker312, but the content of anthocyanins is extremely significantly higher than background. The levels of these two genes expression were obviously higher than their backgrounds which were analysised by Real time PCR. From these data, the red mutant was perhaps further confirmed to be resulted from GhFOMT and GhPⅡ overexpression. By bioinformatics, FOMT has the key role in the process of anthocyanins synthesis, one of the third key enzymes for modifying colourless anthocyanins into coloured anthocyanins. PⅡ plays an important role in light absorption which is involved in chlorophyll a/b binding protein superfamily. Therefore, GhPⅡ may be interacted with GhFOMT to regulate the synthesis of anthocyanin under different lights. The prilimary study on the two genes was the basis for further study the molecular mechanism of red mutation.