【作者】 杨玲；

【导师】 陶勤南； 吴平；

【作者基本信息】 浙江大学 ， 植物营养学， 2003， 博士

【摘要】 本研究采用自然排水72h模拟水分亏缺处理，比较旱稻(Azucena)幼苗在水分亏缺处理与正常淹水培养条件下根系生长，利用cDNA-AFLP技术对不同供水条件下的种子根尖、侧根区、不定根原基区进行了差异显示分析，结果如下： 1．水分亏缺处理明显地影响了旱稻根的构型。同一根系中不同类型的根对水分亏缺胁迫的反应不同。短期水分亏缺促进种子根的伸长及侧根的发生和伸长；而不利于不定根的生长——伸长速率下降和数目减少。水分亏缺促进根系总根数和长度的增加，导致根干重和根冠比的上升。 2．旱稻Azucena种子根伸长区成熟皮层细胞的长度在处理4h至16h内迅速增大，随后逐渐下降。采用cDNA-AFLP技术分析了种子根尖(1cm)基因在水分亏缺处理期间4个时间点(4h、16h、48h和72h)的差异表达，获得了106个诱导增强表达的克隆。从不同表达类型中选择了21个克隆进行Northern杂交分析，其中16个(76％)与cDNA-AFLP表达谱一致。 在106个表达增强的基因中，60个编码已知功能蛋白，28个编码未知功能蛋白，其余的18个仅与未注释的基因组序列相似，或者在核苷酸数据库无同源序列。60个已知功能的基因可以被分为7类：运输，代谢和能量，胁迫和防御相关蛋白，细胞组织及细胞壁生成，信号转导，表达调节子及转座元件。说明种子根尖为适应水分亏缺发生了复杂的代谢变化。 22个基因的表达在水分亏缺处理16h前达到高峰，它们可能在转录水平上与水分亏缺促进种子根的生长有关。包括水孔蛋白(PIP2a)、糖酵解作用中的磷酸丙酮酸水合酶、S-同化途径中的O-乙酰丝氨酸(巯基)裂解酶(OASTL)、肌动蛋白解聚因子(ADF)、腺嘌呤主要补救途径中的腺嘌呤磷酸核糖基转移酶(APRT)；5个基因参与信号转导和表达调控——钙调素(CaM)、MAP3K beta 1蛋白激酶(MAP3K)、ABA合成关键酶9-cis-环氧类胡萝卜素双氧酶1(NCED1)、GA信号转导的负调节子(SPY)和剪接体安装必需的SR相关蛋白(SART1)；3个参与细胞壁的松弛——内切葡聚糖酶(EGase)、木葡聚糖内切转糖基酶(XET)、膨胀素(OsEXP2)；5个参与囊泡的运输——VPS33a(vacuolar protein sorting protein)、小G蛋白Rab11b ＿博十学位论文：水分亏缺条件下早稻根系发生发育及相关基冈的表达分析 一（Rabl］b）、2个鸟瞟吟核苦酸交换因子（GEP禾 GEPZ）及APG（autophagocytosis ”protein）。从水分亏缺信号的感知到根的加速伸长，我们提出了一个简单的模型。另 外3个基因分别编码ASR、转座子TNT多蛋白和未知蛋白，还有一未知基因。 ＿3．采用 cDNA．AFLP技术分析了水分亏缺下种子根尖门。m）、侧根区和不定根 一原基区基因的差异表达，克隆了 112个差异条带。58个 TDFS在 3种组织中均差异表 ＿达；32个同时在侧根区和种子根尖差异表达；10个同时在种子根尖和不定根原基区 ＿差异表达；4个同时在侧根区和不定根原基区差异表达；7个仅在种子根尖差异表达： 1个仅在侧根区差异表达。选择了 ZI个克隆做探针进行了 Northern杂交分析，与 CDNA－AFLP结果有较好的可比性。 、112个差异表达的 TDFS中，67个是已知功能基因，涉及到许多代谢途径，根据 ＿功能分为8类一细胞的组织和细胞壁的生成、物质运输、代谢及能量的产生、胁迫＿及防御相关蛋白、基因的表达和调控、信号转导、细胞的生长与分裂和转座元件。29一 个基因编码推断的蛋白，3个与水稻 EST相似。其余的门个仅与未注释的基因组序 列相似，或者在核着酸数据库无同源序列。 、4．根据核着酸序列将 105个基因电子定位到水稻的高密度连锁图谱上，其中 19，个差异表达基因定位在 BalaXAzucena、IR64XAzucena和 IR1552XAzucena中至少 两个群体共同的与根生长相关的QTLs区问，并用Southern杂交将其中的4个定位到，IRI 552 X Azucena群体遗传连锁图谱相应的位点上。19个基因中的5个编码推断的未一＿知功能蛋白质，其余 14个编码己知功能蛋白，分别为膨胀素（OSE 人胚胎后期一，丰富蛋白（LEA）、剪接体安装必需的 SR相关蛋白（SARTI）、自吞噬蛋白（APG）、一 碱性螺旋一环一螺旋（bHLH）转录因子、与 ASR相似的果实成熟蛋白、镍结合蛋白、＿－＿DNA结合蛋白、丙酮酸脱氢酶激酶（PDK入stomatins类蛋白、蔗糖调节蛋白SRI、－－一液泡蛋白分类蛋白（VSP33a）、GA负调节团于（SPY）、逆转座元件。一更多还原

【Abstract】 To investigate the genetic background and genes for rice root growth under water deficit, the natural drain-off system was used to simulate water deficit condition in this experiment. The root growth of upland rice, Azucena, under water deficit was compared with that under flooding condition. cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis was used to examine gene expression profile in different root tissues during 72-h water deficit. The results are summarized as follows:1. Water deficit condition clearly affected root system architecture in upland rice. Different roots showed different responses to the water deficit condition. Short-duration of water deficit stimulated seminal and lateral root growth by accelerating seminal root elongation, promoting lateral root initiation and elongation, while reduced adventitious root elongation and number. Total root dry weight and the root-to-shoot ratio were increased under the water deficit condition.2. The elongation of cortical cells in the elongation zone was rapidly stimulated within 16h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips (1cm) at four time points (4h, 16h, 48h and 72h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of 76% genes were confirmed by Northern blot analysis based on 21 selected genes representing different induced patterns.The 106 upregulated genes were composed of 60 function-known genes, 28 function-unknown genes and 18 novel genes. Sixty genes with known function were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit.Expression of 22 genes reached a maximum within 16 h of water-deficit treatment, which may be related to the root elongation stimulated by the water deficit attranscriptional level. Aquaporin (PIP2a), enolase in glycolysis, O-acetylserine (thiol)-lyase (OASTL) in S-assimilation, actin-depolymerizing factor (ADF), adenine phosphoribosyltransferase in the main adenine salvage pathway (APRT) were included. Five genes encode proteins involved in signal transduction and expression regulation, including calmodulin (CaM), MAP3K beta 1 protein kinase (MAP3K), 9-cis-epoxycarotenoid dioxygenase (NCED1) for ABA biosyntheses, a negative regulator of gibberellin signal transduction (SPY) and,an SR-related protein essential for spliceosome assembly (SART1). There were three genes for cell wall loosening proteins: endo-glucanase (EGase), xyloglucan endotransferase (XET) and expansin (OsEXP2). Five genes encode proteins involved in vesicle traffic, including vacuolar protein sorting protein (VPS33a), GTP-binding protein rabllb (Rab11b), two guanine nucleotide exchange proteins (GEP and GEP2) and autophagocytosis protein (APG). A simple model outlining the sequences of cellular events linking perception of a water-deficit signal to root elongation was proposed.3. cDNA-AFLP analysis was used to identify differentially expressed genes under water deficit condition in three root tissues: seminal root tips, lateral root zones and adventitious root primordial zones. One hundred and twelve specific bands were isolated and cloned. Fifty-eight transcript derived fragments (TDFs) showed differential expression in all three types of root tissues, 31 in both lateral root zones and seminal root tips, 10 in both seminal root tips and adventitious root primordial zones, 4 in both lateral root zones and adventitious root primordial zones, 7 only in seminal root tips and 1 only in lateral root zones. Northern blot analysis was carried out with random 21 clones to confirm their cDNA-AFLP expression patterns. The results obtained by cDNA-AFLP were in good agreement with those found in RNA gel blot analysis.Among the 1更多还原