【作者】 周菲；

【导师】 刘雪梅；

【作者基本信息】 东北林业大学 ， 遗传学， 2010， 硕士

【摘要】 通过解剖观察及生物制片技术确定白桦雌花发育的各个时期,以白桦雌花序为试验材料,构建了白桦花期的正反向消减文库。从消减文库中筛选特异基因并进行初步的生物信息学分析。同时在两个文库中选出4个特异基因,通过做实时定量PCR检测这4个基因在不同时期的雌花序,雄花序和叶中的表达情况,并进行基因功能的初步分析。主要研究结果如下：1.确定了白桦雌花序在不同时期的发育情况,根据胚胎学发育特征,确定4月30日至5月11日为受粉、胚珠珠心、珠被分化及大孢子发生时期,确定5月12日至25日为雌配子体形成和受精时期,以这两段时期雌花序的cDNA互为tester和driver构建消减文库BHHQ和BHHZ.2.在BHHQ消减文库中筛选到480个特异基因,文库的冗余度为30.64%,在反向的BHHZ消减文库中筛选到503个特异基因,文库冗余度为29.55%。与数据库中已经登录的基因进行blastn和blastx比对,分析表明BHHQ和BHHZ库中分别有357个特异基因(74.38%)和431个特异基因(85.69%)与已知数据库中的核酸或蛋白序列具有同源性(Blastn E-Value<e-10,Blastx E-Value<e-5),其余的分别为103个和57个特异基因与已知序列无同源性或同源性较低,可以视作新基因。在BHHQ中筛选到与花发育相关的序列中包括WD-重复蛋白、核苷二磷酸激酶、组蛋白H4、质膜H+-ATPase等基因；在BHHZ中筛选到与花发育相关的序列包括开花位点T、锌指蛋白、抗震蛋白MADS8.AP2/ERF包含域转录因子、锚蛋白等基因。3.在消减文库中选择4个与花发育相关的基因,它们分别是MADS-box转录因子SEP3、苯甲醇苯甲酰转移酶、锌指蛋白、铝诱导蛋白,以ACTIN(肌动蛋白基因)为内参基因,做实时定量PCR分析它们的表达。结果表明,这几个基因在花发育不同时期不同部位均有不同程度的表达,并根据其表达模式对其在花发育过程中可能参与的功能做了初步分析。更多还原

【Abstract】 Developmental stages of female flowers in Betula Platyphylla Suk.was determined by anatomical observation and biological slice making technology. As femle inflorescences were test materials, positives and reverse subtraction libraries of Betula platyphylla Suk. in flower phase were constracted.From the two libraries, unique genes were selected and were fundamentally analyzed by bioinformatics methods.Four unique genes from the two libraries were selected and detected expression in female inflorescence, male inflorescence and leaf in different development stage by real-time quantitative PCR, and the gene function was analysised preliminarily. The main results are as follows.1.Developmental stages of female flowers in Betula Platyphylla Suk.was determined. According to embryological development characteristics, the periods from 30 April to 11 May were determined as pollination, ovule nucellus,integument differentiation and Megasporogenesis stage, and the periods from 12 May to 25 May were determined as megagametogenesis and fertilization stage, and the BHHQ subtraction liabrary and the BHHZ subtraction liabrary was constructed as the cDNA of female inflorescences in the two stages used as tester and driver.2.From BHHQ subtraction liabrary,480 unique gene were selected, the redundancy is 30.64%, in reverse BHHZ subtraction library,503 unique gene were selected, the redundancy is 29.55%. Blastx and blastn analysis respectively showed that 357(74.38%) and 431 (85.69%)unique gene were homologous(Blastn E-Value<e-10, Blastx E-Value<e-5)to the nucleic acid or protein sequences already in the database, the other 103 and 57 unique gene had no homology or low homology with sequences already in the database, which could be regarded as new genes.Some genes such as WD-repeat protein, nucleoside diphosphate kinase, histone 4 and plasma membrane H+-ATPase were screened from BHHQ subtraction liabrary involved in flower development; Some genes such as flowering locus T, zinc finger protein, SHATTERPROOF-like protein MADS8,AP2/ERF domain-containing transcription factor and ankyrin were screened from BHHZ subtraction liabrary involved in flower development.3 Four unique gene involved in flower development were selected from the subtractive libraries,which are MADS-box transcription factor SEP3,benzyl alcohol benzoyl transferase, zinc finger protein and aluminum induced protein.As ACTIN was internal reference gene, the fundamental expression analysis was made by real-time quantitative PCR. Result showed that they all had different degree expressions in different parts during different inflorescence development stages,and their probably functions were deduced preliminarily according to their expression modes.更多还原