【作者】 吴梅；

【导师】 李友国；

【作者基本信息】 华中农业大学 ， 微生物学， 2009， 硕士

【摘要】 在本室前期工作中,通过抑制差减杂交,建立了紫云英结瘤固氮过程中的差异表达cDNA文库,本研究将其下调表达cDNA文库进行了全文库测序,共获得180个有效序列,cDNA克隆插入片段在200-1,300 bp之间。将所有序列进行了同源比较,比对条件为匹配区域长度＞100 bp且相似度＞75%。符合要求的共有94个,发现其中4个在数据库中找不到其同源片段,可能代表4个新的基因。将符合条件的基因按分子功能分类,分为10个类群:核糖体蛋白11个,信号转导相关11个,基因表达相关13个,代谢相关22个,膜及转运相关13个,应激防御相关9个,未知蛋白6个,假定蛋白4个,无同源片段4个和其他1个。根据测序分析结果,对其中的6个基因进行了半定量RT-PCR验证分析,结果表明:它们在根瘤中表达水平均比未接种根中要低。运用RACE（rapid-amplification ofcDNA ends）法获得了其中2个基因（AsA8和AsD5）的全长cDNA和编码区。序列分析表明:AsA8编码的多肽含379个氨基酸,分子量为42 kDa,等电点为5.78。与豌豆中的12-氧代植二烯酸-10,11-还原酶同源性较高,不含信号肽。AsD5编码191个氨基酸的多肽,含有一个22个氨基酸的信号肽,信号肽可以被切除,成熟的多肽分子量为19 kDa,等电点为8.78。与拟南芥质体蓝素相似。另外4个基因:AsB3编码的蛋白与拟南芥C₃HC₄型锌指家族蛋白相似,AsG6编码的蛋白与拟南芥过氧化物酶相似,AsT6编码的蛋白与杨毛果预测蛋白相似,AsC2编码的蛋白与热休克蛋白（heat shock proteins,HSPs）相似。进一步通过实时荧光定量PCR,研究了AsA8和AsD5的时空和组织表达特征。结果显示:从不同植物器官中基因的表达情况来看,AsA8和AsD5在根瘤中的表达水平都较未接种根中低,均在紫云英根瘤中呈下调表达特征。AsA8在植物茎中几乎不表达,根瘤和叶片中的表达差异不大,去瘤根中的表达量最高。AsD5则在所有检测组织中均有表达,根瘤、茎和叶中表达相当,去瘤根中表达量最高。从接种后不同时段的基因的表达情况来看,AsA8在成熟根中的转录水平远高于幼根,主要在21-25 d表达。相反,AsD5在成熟根中的转录水平远低于幼根。接种后6d,可以检测到一个较高的表达量,随后一直到接种后30d,其表达量都很低（P＜0.05）。由此推测,AsA8主要在成熟根中参与茉莉酸的合成,调控根瘤菌的侵染和根瘤形成,而AsD5主要在接种早期幼根中发挥生理功能,具体作用尚不明确。以上实验结果为进一步通过超表达深入研究目标（下调表达）基因在紫云英结瘤固氮过程中的功能打下了良好工作基础。更多还原

【Abstract】 In previous work,a cDNA library of Astragalus sinicus L.genes specifically or enhanced expressed in uninfected roots has been generated by using a PCR-based suppressive subtractive hybridization(SSH) technique in this laboratory.A total number of 180 expression-down-regulated clones was obtained and sequenced,the length of inseted segments were between 200 to1,300 bp.Based on homology analysis(indentity＞75%,length＞100 bp) by Blast,there were 94 clones with significant matches and were sorted into 10 broad functional categories as following:11 clones belong to ribosomal protein,11 clones belong to signal transduction,13 clones belong to gene expression,22 clones belong to metabolism,13 clones belong to membrane and transporter,9 clones belong to stress defense,6 clones belong to unknown functional proteins,4 clones belong to hypothetical protein,4 clones belong to no homologous fragment and 1 clones belong to others.semi-quantitative RT-PCR was performed with 6 clones selected according to the analysis.The results showed that the 6 clones examined were expressed lower in the nodules than in the uninfected roots.Further more,the full-length or open reading frame of cDNA sequences of 2 clones(AsA8 and AsD5) were obtained by RACE(rapid-amplification of cDNA ends) and analyzed by bioinformatical software.The homology analysis indicated that AsA8 encodes a small polypeptide with 379 amino acids with homology to 12-oxophytodienoic acid 10,11-reductase.Its mature protein has an evaluated molecular weight of 42 kDa and isoelectric point of 5.78.For AsD5,it is similar to plastocyanin.The putative amino acid sequence of AsD5 contains 191 amino acids, which contains a signal peptide with 22 amino acids and a putative cleavage site.Its mature protein has an evaluated molecular weight of 19 kDa and isoelectric point of 8.78. Turning to other 4 genes:AsB3,AsG6,AsT6 and AsC2 are found homologous to zinc finger,peroxidase,putative proteins from Populus trichocarpa,heat shock proteins(HSPs),respectively.Fluorescence quantitative real-time PCR analysis indicated that the expressions of AsA8 and AsD5 were down-regulated in nodules.The expression levels of AsA8 in infected leaves and nodules were similar,they were both lower than its in uninfected roots. The expression level in infected roots with nodule removed was highest,and there was nearly no expression of AsA8 in infected stems.The expression patterns of AsD5 in leaves,stems and nodules were similar,they were all lower than its in uninfected roots. The expression of AsA8 in mature infected roots is much higher than that in young infected roots.It mainly expressed during 21 to 25 d after inoculated with rhizobium.In contrast,the expression of AsD5 in mature infected roots was much lower than that in young infected roots,which mainly expressed at 6 d after inoculation(P＜0.05).It is presumed that the function of AsA8 is possibly involved in the metabolism in the mature root to produce jasmonic acid.Meanwhile,AsD5 is thought to play an important role on the nodule formation in the earlier stage after rhizobium inoculated,the function was unknown..The results above have built up a solid basis for the function identification of those down-regulated-expression genes,associated with symbiotic nitrogen fixation in Astragalus sinicus L root nodules,by gene-over-expression technique.更多还原