| Self-incompatibility (Self-incompatibility, SI) is an important reproductive mechanism for phanerogam that is developed in long-term species evolution. Species with self-incompatibility show pistil stigma or style can distinguish autologous and allogeneic pollen, and the pistil stigma can prevent autologous pollination but accept allogeneic pollen poliination, this feature is beneficial to maintain a high degree of heterozygosity. SI is a very common phenomenon in phanerogam, which is used widely to produce hybrid by assembling different incompatible haplotypes, and it is also an important hybrid breeding approach of Brassica plants.Cabbage(Brassica oleracea L.) is an important cruciferous crop in the world and play an important roles in autumn and winter leaf vegetable cultivation in China. Self-incompatibility is an important approach for cabbage hybrid breeding,, strong heterosis, easy breeding procedure, short breeding time are the features for this breeding approach. At present, artificial bud pollination is the main method for cabbage self-incompatibility lines raising, propagation. Undoubtedly, this method is operations trouble, inefficiency, time-consuming and labor-intensiv, and higher cost, that reduces the economic benefits of cabbage hybrid production greatly. Therefore, how to propagate SI lines efficiently is concerned by cabage breeders all the time. Incuding chemical, physical, biological and mechanical methods are used to overcome the self-incompatibility. Such as spraying Nacl solution and boric acid after flowering, electrical stimulation, high temperature treatment, mixed pollens pollination, CO2 treatment, y-ray radiation, pollination by steel brushes and so on. Although these methods can increase the self-pollination seed number to a certain extent for cabbage, but these methods are operation complex, easy to make stigma injured, and difficulty to achieve in field condition.In present, the molecular and genetic mechanisms of self-incompatibility are more and more clear, that provide an approach to solve regulation of the self-incompatibility by molecular biology technology. For further study of the impact of key self-incompatibility genes in cabbage self-incompatibility and the development of artificial regulation biotechnology method of cabbage self-incompatibility, MLPK antisense gene driven by stigma-specific SLR prmoter and SLGi gene controlled by SLG13 stigma-specific promoter were transformed into self-incompatible cabbage material’TF’by agrobacterium-mediated transformation. The aim is to explore the function of MLPK and SLG genes in the process of self-incompatibility signal transduction in cabbage by antisense interference MLPK and RNA interference SLG genes expression, and a few direct or indirect evidence that MLPK and SLG genes involve in the process of SI signal transduction in Brassica expect to obtain. Furthermore, the Cre/loxp recombination system and AlcR/alcA ethanol inducible gene expression systems were combined to constructed a self-incompatibility artificial regulation system to achieve the simple and convenient propagation of self-incompatibile lines as well as the hybrid seed production of cabbage. The mainly results are as follows:1、pCABarMLPK plant expression vector with a Bar maker gene was transformed into cabbage self-incompatibile line ’TF’ by agrobacterium-mediated transformation. The endogenous MLPK mRNA accumulation in stigma were significantly lower than non-transgenic wild-type plants after flowering. Fluorescence microscopy observation showed that a large number of pollen adsorption and germination on the stigma, and significant increase pollen tubes through the stigma and style of transgenic cabbage plant after self-pollination during flowering stage, lead to increased seed number. Both the self-compatible index of flowering stage and bud stage of transgenic cabbage plants were significantly higher than the wild-type control plants. The results showed that the self-incompatibility can be partially broken by reducing MLPK gene expression in cabbage, and improve the self-pollination seed capacity during flowering stage.2. pCABarSLGi vector was introduced into highly self-incompatibile cabbage material ’TF’ by agrobacterium-mediated transformation method. Fluorescence microscopy observation showed that a lot of pollen grains are adsorbed on the surface of the stigma and germination, and pollen tubes can be seen clearly through the stigma in transgenic cabbage plants after self-pollination at flowering stage, resulting in increased number of self-pollination seed,1.03～13.96 flowering affinity index were observed, that was significantly higher than the wild-type control plants with 0.06 flowering affinity index. Quantitative PCR analysis showed that the mRNA accumulation of SLG gene in stigma of transgenic cabbage plants at flowering stage was significantly lower than that of non-transgenic wild-type plants. The results showed that the interference expression of SLG gene has a positive effect on self-incompatibility break in cabbage, and lead to increased self-pollination seed capacity significantly at flowering stage.3、pCABaralcRSLGi plant expression vector that assemble Cre/loxp recombination system, AlcR/alcA ethanol-induced gene expression system and SLG-RNAi gene cassette was transformed into self-incompatibile cabbage material ’TF’ by agrobacterium-mediated transformation. Fluorescence microscopy observation showed that a lot of pollen grains are adsorbed on the stigma surface and a large number of germinated pollen, tubes through the stigma in transgenic plants after self-pollination during flowering stage. The flowering affinity index were 0.66～14.7 of transgenic plants, that was significantly higher than those of wild-type control plants. Quantitative PCR analysis showed that the SLG mRNA accumulation in flowering stigma of transgenic cabbage plants was significantly lower than that of wild-type plants, and alcR gene expression can be also detected in the stigma. A 5% concentration of ethanol treatment on pCABaralcRSLGi-5 T1 transgenic plants could remove the SLG-RNAi gene expression cassette efficiently based on 1%-5% concentration of ethanol root-irrigation inducing. Progenies from five different pCABaralcRSLGi transgenic plants were used to investigated the gene deletion efficiency after three times 5% ethanol treatment, the results showed that complete gene deletion occurred in 60% T1 generation plants from No.17 transgenic plant. But less efficient gene deletion were observed in T1 generation plants from else transgenic plants under the same treatment conditions, lead to chimeras generation caused by incomplete gene deletion. The expression of the Bar, alcR and Cre genes in offspring of pCABaralcRSLGi-5 transgenic cabbage plant were further investigated after ethanol-induced. the results showed that with time expand after ethanol-induced, Bar gene expression increased gradually, but due to the continuous alcR gene deletion, mRNA accumulation of alcR gene decreased gradually, at the same time, mRNA accumulation of Cre gene increased in early stage after ethanol applied, but with time expand, mRNA accumulation of Cre gene decreases gradually. |
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