以中国罗田板栗品种"玫瑰红"的幼叶和花为试材,采用EST数据库分析,结合RACE技术从板栗中分离到MADS基因的cDNA全长序列并构建了其RNA干扰载体,研究开花关键基因对板栗花芽分化的影响。结果表明:CmMADS基因全长为922bp的CmMADS的cDNA序列,该序列含有1个681bp的可读框,编码227个氨基酸序列。生物信息学预测CmMADS蛋白的分子质量为25.87kDa,理论等电点为6.27,N端具有M盒保守序列,其二级结构主要由α?螺旋和无规则卷曲组成。蛋白质同源分析表明,CmMADS含有M盒和K盒2个特征性序列区域。同源建模分析显示CmMADS序列与苹果MADS蛋白的三维结构及活性位点高度相似。系统进化分析表明,板栗MADS蛋白归属植物进化分支,且与太行花的MADS蛋白归为一支。将CmMADS基因2段相同长度(283bp)的反向互补片段RMADS和FMADS连入载体pBluescript SK plus,构成中间载体pBluescript SK plus-FR。用BanHI和KpnI同时酶切中间载体pBluescript SK plus-FR和植物表达载体pCl301-ubi,回收pBluescript SK plus-FR的酶切小片段,连入pC1301-ubi大片段中,构成植物表达载体pC1301-ubi-CmMADS-RNAi。下一步拟用构建好的RNA干扰载体转化农杆菌并由其介导将重组质粒转入烟草,为深入研究该干扰载体的功能及CmMADS基因的功能提供参考。
为板栗FLC同源基因控制开花的相关功能及春化作用的相关分子机理提供研究基础,以罗田板栗为研究材料,分离并鉴定板栗花芽调控相关基因FLC,依据NCBI数据库提供的EST序列片段,利用RACE技术,从板栗叶片总RNA中分离了与开花相关的基因-CmFLCcDNA的序列,通过在线分析预测其序列同源性及3D结构,以pBluescript SK plus作为过渡载体构建其干扰表达载体。结果表明,该序列全长为968bp,其中有1个编码含227个氨基酸蛋白序列的开放阅读框。CmFLC蛋白的理论等电点为5.80,分子质量为25.62kDa,N端含有M盒保守序列,该蛋白的二级结构大部分由无规则卷曲和α-螺旋构成。CmFLC存在M盒、K盒两个特征性序列区域。CmFLC蛋白与毛白杨FLC蛋白的三维结构存在高度相似性。板栗的FLC蛋白属于植物进化分支,且与山核桃的FLC蛋白同属一支。设计特异引物扩增得到FLC基因的正、反义基因干扰片段,酶切结果显示插入片段大小为320bp,板栗FLC的干扰载体pc1301-ubi-CMFLCRNAi构建成功。结论:板栗中存在FLC同源基因,并可能参与板栗开花相关功能及春化调控。
In order to explore moldy mechanism of chestnut from Luotian County in storage process,the strains of pathogenic fungi were isolated from chestnuts after storage at room temperature for 70d.Six genera of fungi were found in chestnut through experimental identification,which were Ozoniumsp.,Fusarium sp.,Aspergillus sp.,Penicilliumsp.,Rhiopus sp.and Stachybotrys sp.,respectively.The re-inoculation tests had been conducted on pathogenic fungi whose isolating rate was greater than 10%.The result showed that the rest genera of fungi generally had no pathogenicity except Penicilliumsp.could infect non-injured chestnut with a lower moldy rate and lighter symptoms;but the moldy rate of strains was above 60% in injured inoculation and they showed heavy symptoms,among which the moldy rate of Ozoniumsp.and Aspergillus sp.were higher than 80%.The experimental results showed that injured chestnut were more likely to decay.Ozoniumsp.and Aspergillus sp.were important pathogenic fungi causing decay during storage process of chestnut.
Li LinlingLi ZhengHua JuanCheng HuaLiao ZhiqinCheng ShuiyuanTang Qing
