The Constraction And Analysis Of Male Flower SSH Library In Asparagus Officinalis

Asparagus officinalis L. is a dioecious plant. Asparagus sex chromosomes are in the early stages ofchromosome evolution, and the sexual dimorphism in asparagus is under the control of M-locus regionlocated on a pair of homomorphic L5chromosomes. The genetic control of sex determination in this plantis based on a model in which sex determination genes control the differention from hermaphrodite flowerto unisexual flower. So asparagus is a model plant to study the evolution of chromosome, the origin ofgender plants and the sex determination and differentiation of dioecious plants. As an edible vegetable, it iscultivated worldwide for its succulent stems, which is delicious and nutritious. The yield of male plants is25%higher than the female, what’more, resistance and vitality of male asparagus plant are stronger thanthat of female plant. So male plants especially of supermale plants are considered to be advantageous in thebreeding and cultivation of asparagus.In this paper, the differences in male asparagus (UC309) flowers were studied by SuppressionSubtraction Hybridisation (SSH). The subtractive hybrization library of the male flowers in asparagus wasconstructed, and a large of specific male genes were cloned and sequenced. After Blast analysis and GOclassification, several genes differentially expressed in male bud and flower were screened withsemi-quantitative PCR and Q-PCR. Genes cloned in this paper are deem to provid the theory assurance forthe mechanism of sex determination and differentiation and lay a foundation for cloning the sex linked gene.The main results are as follows:1.107effective ESTs have been obtained in the suppression subtractive hybridization library of maleflower. The sequence fragment is about100-1200bp, and mostly concentrated in the200-500bp. It isconsistent with PCR results.2. Blast ESTs with the Blastx database,35ESTs have the homology with proteins or putative proteins.103ESTs, of which39ESTs were related to flower development, find the similar ESTs except for4ESTsby tBlastx. However,2ESTs, which may be the new gene, can not find the similar sequence by Blastndatabase.34ESTs had been annotated by Blast2GO,6molecular function,10biological process and9cellular component included.3. Part of ESTs correlation with flower development and new gene were tested by semi-quantitative RT-PCR.7ESTs, which were respectively named AO12-40(lipid transfer protein of nsLTP family), AO4-5(SGNH hydrolase superfamily), AO13-38(FA hydroxylase super family), AO13-87(photosystem IIstability/assembly factor HCF136of PLN00033family), AO13-92(Rab protein, belonging to theP-loop_NTPase superfamily), AO13-40(putative PCD protein of MA3superfamily) and AO18-87(new gene)showed good repeatability and different expression patterns in male bud or flower. Then the results wereinvestigated in various tissues by Q-PCR. AO12-40, AO4-5, AO13-92, AO13-40, AO13-87,AO18-87and AO13-38showed the higher expression level at the male flower stage. The difference (P<0.01) is significantcompared to the same female stage. AO13-87and AO18-87(P<0.05) were striking in the male flower bud.4. Analysis of differential genes which might be involved in the sex determination and staminatedevelopment, can contribute to understanding of the possible mechanisms of sex determination and earlysex identification.