【作者】 杨杰；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 水稻籼粳杂种具有强大的杂种优势,但籼粳杂种的低温敏感性是限制其利用的因素之一。以17个不同类型的籼、粳、爪哇型品种及其杂种为材料,通过分期播种,研究了温度变化对花粉育性的影响。结果表明,当花粉母细胞减数分裂期的日平均气温低于22-23℃时,亚种间杂种的花粉育性显著降低,不同组合花粉育性存在明显差异,而品种间杂种和常规品种的花粉育性受影响较小;当花粉母细胞减数分裂期的日平均气温低于20℃时,常规品种和品种间杂种的花粉发育也受到不同程度的影响,不同品种与组合间存在差异。花粉母细胞减数分裂期的日平均温度与花粉育性的相关性分析表明,温度与亚种间杂种花粉育性呈显著正相关。说明减数分裂期的亚种间杂种花粉育性受温度影响,温度对亚种间杂种花粉育性的影响较常规品种大.为探明籼粳杂种低温花粉不育的遗传基础,以籼稻品种3037和粳型广亲和品种02428的F₂分离群体进行了低温花粉不育的遗传分析。通过推迟播种调节抽穗期后,F₂群体花粉母细胞减数分裂期的日平均温度为21～23℃,调查了F₂群体各单株的花粉育性。利用108对SSR引物构建了包含157个F₂单株,覆盖12条染色体的分子标记连锁图谱。该连锁图的总长度为1857.8 cM,标记间平均距离为16.26 cM。采用区间作图法对F₂群体花粉不育性进行QTL分析,共检测到2个低温花粉不育QTL,即qLTSPS2和qLTSPS5,分别位于第2、5染色体,其加性效应分别为0.021、0.045,显性效应分别为-0.246、-0.251,显性度分别为11.7和4.8,具有超显性效应,分别解释表型变异的15.6%、11.9%。两因素的方差分析表明这两个QTL之间不存在互作。利用强耐冷品种日本晴和不耐冷品种Kasalath及其衍生的98个（Nipponbare/Kasalath∥Nipponbare）回交重组自交家系（BILs）进行了水稻芽期耐冷性数量性状基因座的检测和遗传效应分析。25℃正常条件下水稻发芽7d,芽长5-10mm,5℃低温处理10d,之后升温至25℃,缓苗10d,调查活苗率,并以活苗率作为芽期耐冷性的表型值,分析亲本和98个BILs的芽期耐冷性表现。采用复合区间作图法,共检测到4个芽期耐冷性QTL,分别位于第3、第7和第12染色体上,命名为qSCT-3-1、qSCT-3-2、qSCT-7和qSCT-12。4个QTL的LOD值分别为3.51,3.68,2.03和3.61,可解释群体表型变异的12.11%,12.66%,6.82%和15.86%。在第3染色体检测到的qSCT-3-1与前人检测到的耐盐QTL在同一区间,qSCT-3-2与前人检测到的低温发芽QTL在同一区间。通过生物信息学的方法发现在第3染色体qSCT-3-1所在区域存在一个KS型脱水蛋白。利用RT-PCR方法,从水稻中克隆了该KS型脱水蛋白基因（Osdhn2,GenBank登录号:D26538）。对克隆的Osdhn2基因的生物信息学分析表明:其亲源关系与茄子的脱水蛋白更近,Osdhn2编码蛋白与茄子的脱水蛋白同源性达86%;而与大麦和小麦的KS型蛋白基因同源性67%。序列分析表明Osdhn2编码蛋白具有两个脱水蛋白保守的结构域,即一个富含赖氨酸片段（K片段）和一个丝氨酸片段（S片段）。进一步分离克隆了Osdhn2的基因组DNA,Osdhn2基因具有1个完整的外元,1个内元位于3’端非编码区。组织表达谱分析表明,Osdhn2在幼苗的根中低表达,在未成熟胚、幼穗、叶片、茎中都有表达,盐胁迫后根中表达增加。定量PCR表明Osdhn2在雌蕊中高表达,而在花药中低表达,开花授精后,在种子中表达迅速降低。定量PCR表明在干旱和低温胁迫处理后,其表达显著增强。利用PCR克隆了9311和Kasalath的Osdhn2基因及其上游约800bp启动子序列,对其进行了序列比较,发现9311在该区域与日本晴不存在序列差异,而Kasalath在启动子上游557bp处存在一个碱基的差异。为进一步研究该启动子组织表达特性,从日本晴克隆了1.3kb的启动子序列,构建了Osdhn2基因启动子GUS转基因表达载体,目前已获得转基因幼苗。将Osdhn2基因克隆到原核表达载体pET32a（+）系统,诱导基因表达,携带有Osdhn2的大肠杆菌在800mmol/LNaCl或700mmol/L KCl或4℃处理后其生长速度明显快于对照。这表明,携带水稻KS型脱水蛋白的大肠杆菌可以提高大肠杆菌耐低温和耐盐能力。为分析Osdhn2基因的功能,我们构建了Osdhn2基因的CaMV35S驱动的过量表达载体,并转化芽期不耐冷的籼稻品种Kasalath,转基因T₁代种子经潮霉素抗性筛选和PCR鉴定后,对阳性株系芽期耐冷性鉴定,发现其芽期耐冷能力明显好于野生型,初步说明KS型脱水蛋白基因与水稻耐冷性有关;在200mM NaCl溶液中发芽和生长3周后,转基因株系耐盐能力也明显提高。为了功能互补分析,我们构建了该基因的RNA干扰载体,并以玉米Ubiquitin启动子驱动,目前已获得转基因幼苗。更多还原

【Abstract】 There existed a number of biological constraints in exploiting the heterosis between indiea and japonica hybrid rice.The low-temperature-sensitive sterility（LTSS） of indica-japoniea hybrid has become one of the major problems in indica-japonica hybrid rice breeding after the solution to the problem of poor fertility of the hybrids with the finding of wide-compatibility gene.The previous studies revealed that the LTSS might be caused by low-temperature-sensitive pollen sterility（LTSPS）.However,the genetic basis of LTSPS remained unclear.Seventeen rice cultivars and hybrids in different types（indica,japonica,javanica,indica hybrid,japonica hybrid and inter-subspecific hybrid） were evaluated to determine the effect of temperature on pollen fertility.Results showed that the pollen fertility of inter-subspecific hybrids was greatly reduced at miosis stage when the average daily temperature dropped to 22.0-23.0℃,and the extent of pollen fertility reduction varied greatly in different hybrids.However,the pollen fertility reduction of indica and japanica hybrids and conventional cultivars was not obvious under the same temperature conditions. When the average daily temperature dropped to 20℃,pollen development of conventional cultivars and hybrids was also affected.Correlation analysis revealed that there existed positive correlation between pollen fertility and average daily temperature.Temperature at miosis stage was a key factor to pollen development,and the pollen fertility of inter-subspecific hybrids was more sensitive to low temperature than that of conventional variety.To explore the genetic basis of LTSPS in indica-japonica hybrid rice,an F₂ genetic population derived from 3037（indica） and 02428（japonica） was developed.At the booting stage,pollen fertility of F₂ population together with parents were surveyed after the treatment with low temperature daily average 21～23℃.The linkage map was constructed containing 108 SSR markers distributed throughout the whole 12 chromosome with average marker interval 16.26 cM.Using software MapMaker/QTL,two putative QTL, namely qLTSPS2 and qLTSPS5 on chromosomes 2 and 5 were detected by interval mapping,which can explain the phenotypie variation 15.6%and 11.9%respectively.The additive effects were 0.021 and 0.045,dominant effects were -0.246 and -0.215,and the degrees of dominance were 11.7 and 4.8,respectively for the two QTL,therefore the mode of gene action in response to low-temperature stress was overdominanee and LTSPS is mainly the result of interaction between the indica and japonica alleles within each locus.In addition,two-way ANOVA showed that the two QTL acted essentially independent of each other in conditioning LTSPS.Low temperature induced retardation of seedling growth is a common problem in temperate dee growing areas.To gather more information regarding cold-tolerance in rice （Oryza sativa L.）,we identified gene loci controlling cold tolerance at early seedling stage. We analyzed the quantitative trait loci（QTL） using 98 backcross-inbred lines（BIL） derived from the backeross between Nipponbare（strongly cold-tolerant rice,as recurrent parent） and Kasalath（cold susceptible dee）.A total of 4 QTL were detected,located on four regions on chromosomes 3,7 and 12,which can explain the phenotypie variation 12.11%, 12.66%,6.82%and 15.86%respectively.The additive effects were 11.16,11.14,-8.8 and -14.59,respectively for the four QTL.Dehydrins（DHNs,LEA D11 family） are among the most commonly observed proteins induced by environmental stress associated with dehydration or low temperature.A novel full-length cDNA of a KS-type dehydrin,designated Osdhn2,has been isolated by the combination of bioinformatics and PCR based approaches.A homology tree of KS-type dehydrin from various species including solanum wheat barley arabidopsis alfalfa ricinus citrus revealed that Osdhn2 had a closer evolutionary relationship with solanum. Meanwhile,the putative peptide of Osdhn2 includes two important Putative conserved domains:lysine segment（K segment） and serine segment（S segment）.Furthermore,the corresponding genomie clone was isolated and sequenced,and it was composed of 1 exons and 1 introns in the 3’UTR.RT-PCR analysis showed that the Osdhn2 transcripts were detected in all tissues including shoot,matured leaves,old leaves,and developing seeds but low levels in root.The Real-Time PCR revealed that the abundance of Osdhn2 transcripts was detected markedly in matured leaves,pistil.Osdhn2 expression improved after drought,salt and low temperature stress.In order to identify the function of Osdhn2,in vitro functional analyses were performed using an Escherichia coli heterologous expression system.Osdhn2 were cloned and expressed in a pET-32a（+） system.E.coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress.Compared with control cells,the E.coli cells expressing Osdhn2 showed a shorter lag period and improved growth when transferred to LB liquid media containing 800 mmol/L NaCl or 700 mmol/L KCl or after 4℃treatment.The results indicate that the E.coli expression system is a simple,useful method to identify the function of some stress-tolerant genes from plants.The overexpression binary vector of Osdhn2 fused with the constitutively expression promoter CaMV35S has been constructed and transformed to indiea rice Kasalath.The cold tolerance at early seedling stage of transgenic Kasalath has been improved compared with the wild-type and salt tolerance of transgenic Kasalath is also improved when germinated in presence of 200mM NaCl solution.The results showed that the function of Osdhn2 is related to confer clod and salt tolerance.To carry out the complementary test,the RNAi binary vector of Osdhn2 has been constructed and transformed.Also the promoter of Osdhn2 fusion GUS binary construct is also underway.更多还原