Study On The Anther Culture And Male Sterility Differential Expression Analysis Of Alfalfa

Research on the formation mechanism of male sterility in alfalfa is important for obtaining the male sterility pure lines and improving the heterosis utilization of alfalfa. In this study, the bud of male sterile and fertile plants of alfalfa was considered as the experimental materils. The anther culture technique was used to cultivate alfalfa haploid plants. And the cDNA-AFLP differential display technique were used to research on the level of gene regulation in different growth period of alfalfa bud, and to discuss its gene differential expression patterns of alfalfa male sterility and analyse the amino acid sequence and protein structure of different genes, in order to analyse the formation mechanism of male sterility. The main results were as follows:(1) The study on male sterile and fertile line of alfalfa anther culture showed that the good callus induction can get under the condition of sampling materials in the bud stage, without per-treatment with low temperature, and the highest callus induction rate was66.7%. MS+2,4-D0.5mg/L+6-BA0.25mg/L+NAA0.2mg/L+KT3mg/L+sucrose3%+ager0.7%was suited for callus induction. MS+KT lmg/L+NAA0.2mg/L+sucrose2%+ager0.7%and MS+KT4mg/L+NAA0.2mg/L+sucrose2%+ager0.7%was suited for sterile and fertile callus differentiation respectively.1/2MS+NAA0.1mg/L+sucrose2%+ager0.9%was suited for rooting. Furthermore, the vitreous shoots could be rooted by MS.(2) DNA contents and ploidy of alfalfa anther culture plants were detected by flow-cytometric analysis. The proper cell nucleus suspensions could be obtained by cutting the fresh leaves in the suitable buffer solution (0.1mol/L citric acid and0.2%TritonX-100) and centrifuging twice (800rpm,5min). Moreover, the most appropriate dyeing effects to cell nucleus were realized by PI (100～200μL,50mg/L). In a word, the results of flow-cytometric indicated that16%plants were haploid,78%plants were tetraploid, and6%plants were mixoploid.(3) The buds gene differential expression of afalfa male sterile and fertile lines were analyzed by cDNA-AFLP technology. The results showed that cDNA-AFLP could be applicable to the research on differential gene expression of different alfalfa buds stages. In addition, its had a high reproducibility and good polymorphism.1746stable bands were detected with12primers, and proportion of differentially expressed genes was12.37%which including3differential gene expression patterns (sterility specific expression, sterility expression, and fertility specific expression).(4) Through identification of repeated tests,4alfalfa buds gene fragments were randomly cloned and analysed by BlastN and BlastX from NCBI. The retrieval results were indicated that3gene fragments had a high homologous with alfalfa (93%～100%). Especially, No.3differential fragment had98%～99%nucleotide similarity to1,5-bisphosphate carboxylase/oxygenase large subunit gene of broad bean chloroplast. What’s more, the CMS was controlled by the1,5-bisphosphate carboxylase, which involved in anther development, programmed cell death, and material energy metabolism of wheat. After sequence analysis, it found that the No.3differential fragment was maybe related to male sterility in alfalfa.