RNAi Vector Construction And Expression Analyse Of Pigment Genes In Brown Cotton

Taking RT-white(white),Brown 1(brown) and Brown 1-61(dark brown) as test materials, chemical chromogenic reaction and ultraviolet spectrophotometry were conducted to study the pigment components and the variation trends of relative pigment contents at different fiber developmental stages of natural colored cotton. The ihpRNA interference vectors and amiRNA interference vectors target to CHS gene and F3'5'H were constructed and transformed to brown cotton respectively through pollen-tube pathway to identify the genes'function and to facilitate the improvement of natural colored cotton. To elucidate the relationship between fiber quantity and pigment development on moleculer level, the expression differences of transcription factor genes CPC,TT8 and TTG1 at different fiber development stages in lines of RT-white and Brown 1-61 were analysed by real-time fluorescent quantitative PCR. Main conclusion were as follows:1.The methanol extract of fibers ,leaves,flowers and buds at different developmental stages in RT-white,Brown 1 and Brown 1-61were analyzed by NaOH staining method, HCl-Mg reaction method and ultraviolet spectrophotometry scanning techniques. The evidences suggested that immature fibers in these 3 lines contained flavonol or flavanonol,and the mature fibers didn't;The pigment content of the fibers at different developmental stages in these 3 lines showed a general trend:Brown 1-61>Brown 1>RT-white. The pigment content of white and brown cotton kept almost the same after +28DPA, and the pigment content of brown1-61 was higher than that in RT-white and Brown 1 only at +60DPA. The differences of pigment content of white fibers at different developmental stages was comparatively lower, the change in pigment content of Brown 1-61 fibers at different developmental stages were similar to that of Brown 1. They all reached the maximum at +17DPA,and Brown 1-61 reached the minimum at +60DPA,Brown 1 reached the mininmum at +28DPA.2. The full-length mRNA sequences of CHS and F3'5'H genes were obtained through PCR reaction by using specific primers designed based on the sequence information of NCBI. The ihpRNA vectors driven by constitutive promoter CMW35S and fiber specific promoter SCFP targeted CHS and F3'5'H were constructed by using pHANNIBAL and pART27, and these ihpRNA were transformed to Brown cotton by pollen-tube pathway.3.AmiRNA sequences targeted CHS and F3'5'H genes were obtained through WMD3-web (http://wmd3.weigelworld.org)microRNA designer. The amiRNA interference vector target CHS and F3'5'H were constructed by taking cotton microRNA Ghr-MIR156d as backbone and replacing its core region with designed sequence , Constructed vectors were transformed to brown cotton through pollen-tube pathway.4.Gbpolyubiqutin gene was selected as a proper and stable intermal reference gene in QRT-PCR for testing gene expression level during fiber development from 3 housekeeping genes including the Histone gene,Actin gene and Gbpolyubiqutin gene.5.The expression level of 3 transcription factor genes CPC,TT8 and TTG1 in flavonoid biosynthetic pathway was analysed by QRT-PCR. The results showed that the expression level of CPC and TTG1 in white cotton fibers at different development stages was higher than that in brown cotton; the expression level of TT8 in brown cotton fibers at different development stages was higher than that in white cotton. To sum up,CPC,TT8 and TTG1 might work as a bHLH-MYB-WD40 complex to control gene expression and the development of fibers of natural colored cotton.The CPC and TTG1 might control the yield character and TT8 might control the pigmentation during brown fiber development.