Study On Molecular Mechanism Of Male Sterility In Xanthoceras Sorbifolia Bunge

As the proper oil plant to north China, Xanthoceras sorbifolia Bunge has great officinal and ornamental value as well, but it belongs only one species in one genus. The flower of Xanthoceras sorbifolia Bunge is polygamous. There are fertile stamen and sterile ones in male flower and hermaphrodite flower respectively, so the anther of Xanthoceras sorbifolia Bunge is perfect stuff to research the molecular mechanism of male sterility and the temporal and spatial gene expression for plants. Male sterility has great value in utilizing heterosis and population improvement. But it's impercipient for the molecule mechanism of male sterility in plants. In order to study the mechanism of Xanthoceras sorbifolia Bunge male sterility on molecule level, this dissertation deal with the method of isolating total RNA from the anthers and how to improve the DDRT-PCR system suitable for the differential mRNAs in anthers of Xanthoceras sorbifolia Bunge, selecting and cloning the cDNAs correlative with male fertility or sterility, and analyzing their sequence and specific function. The main research and the results of experiment were showed as following:1. Total RNA from anthers of Xanthoceras sorbifolia Bunge were isolated by the methods of TRIZOL reagent, guanidine thiocyanate and CTAB, and the rate of yield, purity and electrophoresis of RNA were analyzed. The results show that the RNA isolated by CTAB presents three clear bands- 28S rRNA, 18S rRNA and 5S rRNA respectively,and its purity is higher than that of the two other methods though its yield is lower slightly, the ratio of A260/A280 is 1.944 and the value of A260/A230 is 2.165; RNAs isolated by the methods of TRIZOL reagent and guanidine thiocyanate decompose seriously and disperse in the pattern of small molecular instead of bands. Data of mRNA differential display (DDRT-PCR) show that there are ideal amplified bands using the RNA isolated by the method of CTAB reagent. So the method of CTAB is an ideal ones suitable for total RNA isolation from anthers of Xanthoceras sorbifolia Bunge.2. It establishes the DDRT-PCR system that can be applied to show the mRNA differences among the different kinds of anthers in Xanthoceras sorbifolia Bunge. Using three anchored primers including 5 ' -T13A-3 ' , 5 ' -T13C-3 ' , 5 ' -T13G-3 ' and different 10mer arbitrary primers, it can get perfect differentially expressed cDNA strips. The ideal different cDNA fragments were obtained using 2% Agarose Gel.3. Using forty random primers and three anchored primers, the twenty-seven different cDNA fragments which were from the male-flower and hermaphrodite flower anthers of Xanthoceras sorbifolia Bunge were obtained and sequenced. The different fragment is detected by northern bloting. Four fragments, which were named as N15, N17, N24 and N26, were specifically expressed in monosexual male-flower anther. Two fragments named as N12 and N14 were specifically expressed in the hermaphrodite flower anther. Four fragments, which were named as N16, N18, N19 and N27, were higher expressed in the male-flower anther than expressed in hermaphrodite flower anther. The N22 fragment was higher expressed in the hermaphrodite flower anther.4. Intermediate vector was constructed by inserting antisense N15 fragment into vector pBI121 and transgenic Arabidopsis thaliana plants resisting to kanamycin were obtained preliminarily.