| Rice Black-Streaked Dwarf Virus Disease(RBSDV)is an important rice virus disease,which will cause huge yield loss when it occurs severely.Due to the lack of highly resistant resources and highly effective identification methods for disease resistance,the traditional breeding method is hard to cultivate the high disease resistant variety.Our lab confirmed that the interference of the black dwarf virus gene S6(encoding a RNA silencing suppressor)obtained high resistance transgenic lines in rice.Based on it,we further use the marker-free transformation strategy to create new transgenic rice materials that have a high resistance to RBSDV.The main progresses obtained are as follows:(1)We constructed the double T-DNA plant expression RNAivector of gene S6(56RNAi)and transfered it into the susceptible japonica rice cultivar Wuling No.1(WLJ1)by the Agrobacterium-mediated method.We got 164 plants carrying S6RNAi gene and 171 plants carrying HPT gene respectively.Our results prove that the co-conversion ratio is more than 90%.Southern hybridization reveals that the percentage of S6RNAi single copy is less than 5%and more than 90%transgenic plants carried three or more copies in S6RNAi transgenic plants.Through molecular detection of successive generations,25 single and double copy transgenic plants were retained for later experiments.The result of Northern-Page hybridization experiment shows that the difference of expression level of small RNA in different transformants or the different plants of same transformant is obvious.(2)We analyzed singlećdouble and multiple copy transformants in natural inoculation locations tested.Found that the incidence rate of the susceptible control was varied between 30%and 50%,and the general difference between replicates or years was small through the natural inoculation identification of single copy,double copy and a small number of multi copy transgenic lines.In progeny lines of 25 transfonnants tested successively,We obtained 9 high resistance transgenic lines of which incidence rates are within 10%,some even within 3%,and 5 transgenic lines with middle resistance of which incidence rates are lower than the half of that of the control.We performed artificial inoculation identification to part of them and found that the incidence rates of the susceptible control was more than 80%,while there are 3 transgenic lines of which incidence rates were lower than 10%,and 2 lines of which incidence rates were lower than 3%displayed high resistance to RBSDV.Our results reveal that lines having high expression level of small RNA show high resistance phenotype and lines having weak level expression display medium resistance phenotype.So far,combined with molecular detection information,we obtain 4 label-free single copy transformation lines with high resistance and 2 with medium resistance to RBSDV.(3)We primarily measured the plant height,panicle length and flag leaf length of the offspring of 25 transformants and found that there was one or more traits difference between most of plants and the control plant and the main sources of variation was the tissue culture variation in the transformation process.It is primarily found that the agronomic phenotypes of 3 disease resistant transgenic lines with single copy are almost identical with that of the control plant.(4)We analyzed the insertion location of exogenous gene in 9 single copy transgenic lines by inverse-PCR method and obtained genomic flanking sequencesof three S6RNAi insertion lines.The position of this three insertion lines is in the sixth,first,and thirdc hromosome,respectively.It is proved that the location of S6RNAi in 7-9 transgenic line s is in the 7.79Mb of the sixth chromosome by designing specific PCR primer.Analysi of insertion positions of other transformants with single copy is still goingon. |
