【作者】 陈炯炯；

【导师】 张启发；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 水稻是重要的粮食作物之一,如何更加有效的提高水稻产量是全世界都关注的重要问题之一。水稻籼粳亚种间的杂种优势很早就被发现,但是籼、粳杂种F₁普遍存在不育或者半不育的现象,这使得籼粳亚种间杂种优势的利用受到了极大的限制。幸好,水稻资源中存在着一类特殊的种质资源,即广亲和品种。它能有效克服籼、粳亚种不育或者半不育现象,给籼、粳杂种利用带来希望。本研究主要是分离克隆控制籼粳广亲和性状的一个主效基因S5基因,该基因位于水稻第6染色体上,编码一个天冬氨酸蛋白酶,通过控制籼粳杂种F₁代雌配子的育性而影响结实率。本研究在前人对S5位点定位工作基础上展开研究,S5定位区段内主要包含三个候选基因为ORF3、ORF4和ORF5。对ORF3、ORF4和ORF5分别构建包含籼稻片段的正义转化载体,以粳稻Balilla为转化受体进行转化。通过对转基因植株小穗育性的考察,发现ORF3和ORF4的T₀代转基因阳性植株和阴性植株的结实率不存在显著的差异,因此排除ORF3和ORF4作为候选基因的可能性。分析ORF5的T₀代转基因植株育性数据,发现转基因阳性植株的平均结实率2.8%,胚囊育性为12.8%,而阴性植株的平均结实率是53.3%,胚囊育性是76.7%,两者的花粉育性都在80%以上。以上结果表明,转基因阳性植株结实率下降的原因主要是胚囊败育,这表明ORF5可能是S5基因。进一步分析ORF5的T₁代转基因植株育性数据,发现ORF5 T₁（B10）代正义转化阳性植株平均胚囊育性为1.3%,阴性植株平均胚囊育性为92.3%;阳性植株平均花粉育性为90.4%,阴性植株平均花粉育性为87.9%;阳性植株平均结实率为0,阴性植株平均结实率为89.4%,这一结果是完全共分离的,因此确定ORF5就是S5基因。为了分析S5基因在广亲和品种中的功能,采用将正义转化载体ORF5-NJ11转入受体广亲和品种02428后,得到的T₁代转基因阳性植株分别同籼稻南京11和粳稻Balilla测交,考察测交后植株的结实率和胚囊育性,其中转基因阳性株系C04T1与粳稻Balilla进行测交后植株的胚囊育性和小穗育性分别是53.6%和53.5%,转基因阴性株系C04T1与粳稻Balilla进行测交后植株的胚囊育性和小穗育性分别是81.9%和72.8%;转基因阳性株系C04T1与籼稻南京11进行测交后植株的胚囊育性和小穗育性分别85%和67.5%,转基因阴性株系C04T1与籼稻南京11进行测交后植株的胚囊育性和小穗育性分别是85%和63.5%。而另一株系C02T1测交后的胚囊育性类似于C04的结果。这一结果说明S5-n是没有功能的。通过原位杂交分析S5基因的表达情况,发现该基因主要在珠心细胞、珠被、孢原细胞、大孢子母细胞中表达,在叶片、根等营养器官中没有表达。本研究还对S5基因编码的天冬氨酸蛋白酶家族在水稻中的分布情况、进化关系、基因与蛋白的结构及天冬氨酸蛋白酶家族在水稻中在水稻中的表达情况进行了详细的分析。通过BLASTp搜索方法在TIGR、KOME、NCBI RefSeq数据库中进行搜索,最终确定水稻基因组中共有96个OsAP基因,它们分布在水稻12条染色体的不同位置。在进化上96个OsAP蛋白分可分为A、B和C三大类型,其中A类基因相比其他两类在进化上更加保守。而C类基因在数量上最多,在基因结构上变异最大。根据CREP数据库中明恢63和珍汕97表达谱芯片数据,分析水稻中OsAP基因家族表达情况。通过聚类分析66个表达的OsAP基因,可以分为三类。第Ⅰ类有分为4个亚类,Ⅰα和Ⅰγ亚类基因表达量都很低,Ⅰβ亚类的特点是在根中的表达相比其他组织表达要高,而Ⅰδ亚类主要是在开花前一天的雄蕊中高表达。第Ⅱ类包含26个基因,该类特点主要在营养组织表达量相对较高,而在幼穗组织中表达量相对较低:第Ⅲ类包含17个基因,该类特点是基因在大部分营养器官表达量比较低,但在幼穗发育时期的表达量比较高。同时,本研究也对OsAP家族在光暗处理及激素处理情况下的差异表达基因也进行了分析,发现对珍汕97和明恢63发芽后48小时的胚芽及胚根进行连续光照处理48小时和连续暗处理48小时处理后共有14个2倍差异表达OsAP基因。在激素（NAA,GA3和KT）处理的条件下的表达情况相比3叶期的苗叶（seedling）,共有9个2倍差异表达OsAP基因。另外,比较分析12对位于大片段复制区的重复基因的表达模式,有7对在进化中出现新功能化,说明这个家族在进化中出现新功能化是在一个相对较高的水平。更多还原

【Abstract】 Rice is one of the important food crops in the world,so one of the important issues is how to improve rice production.F₁ hybrids between indica and japonica usually demonstrate very strong hybrid vigor,but it is the partial sterility or full sterility frequently observed in most indica-japonica crosses.So this is a major difficulty encountered in the development of such inter-subspecific hybrids.However,a special group of rice germplasm,referred to as wide compatibility varieties,is able to produce highly fertile hybrids when crossed to both indica and japonica.The purpose of this study is to clone a gene S5 which is a major locus for indica-japonica hybrid sterility and wide compatibility,and was previously located on chromosome 6.Here,we show that S5 encodes an AP,which conditioned embryo-sac sterility in indica-japonica hybrids.Our researches have been done based on the previously work according to S5 loci,and it included three ORFs（ORF3-5）. Transformation constructs were prepared for ORF3,ORF4 and ORF5 with indica genomic DNA fragments,and they were introduced into the japonica variety Balilla. Examination of the spikelet fertility of the T₀ plants under natural field conditions showed that there was not a statistically significant difference between the transgene-positive and -negative plants of ORF3 or ORF4,indicating that neither of them is a likely candidate for S5.Then,we examed the spikelet fertility,embryo-sac fertility and pollen fertility of transgene-positive and -negative plants of ORF5 construct.It showed that the average spikelet fertility of the positive plants was 2.8%and the average embryo-sac fertility of the positive plants was 12.8%,but the average spikelet fertility of the negative plants was 53.3%and the average embryo-sac fertility of the negative plants was 76.7%.However, the average pollen fertility of the positive and the negative plants were more than 80%. This result showed the low spikelet fertility of transgene-positive plants because of embryo-sac abortion,strongly suggesting ORF5 to be the candidate for S5.Analysis the fertility of T₁ plants,we found the average embryo-sac fertility of ORF5 T₁（B10） transgenic positive and negative plants were 1.3%and 92.3%respectively,and the average pollen fertility of ORF5 T₁（B10） transgenic positive and negative plants were 90.4%and 87.9%respectively,and the average spikelet fertility of ORF5 T₁（B10） transgenic positive and negative plants were 0 and 89.4%respectively,so the results were perfect cosegregation and ORF5 was the S5 gene.To gain insight into the function of S5, the ORF5-NJ11 construct was introduced to 02428,and T₁ progenies were test-crossed with Nanjing 11 and Balilla,were examined for embryo-sac fertility and spikelet fertility. The average embryo-sac and spikelet fertility of C04T1×Balilla transgenic positive plants were 53.6%and 53.5%respectively,and the average embryo-sac and spikelet fertility of C04T1×Balilla transgenic negative plants were 81.9%and 72.8%respectively. The average embryo-sac and spikelet fertility of C04T1×Nanjing 11 transgenic positive plants were 85%and 67.5%respectively,and the average embryo-sac and spikelet fertility of C04T1×Balilla transgenic negative plants were 85%and 63.5%respectively. This results indicate that S5-n is a nonfunctional allele.RNA in situ hybridization showed S5 has a localized expression in the ovule tissues including necellus,integument, megasporocytes and megaspores.It had no detection in leaves and roots.Another,we analyzes the distribution and expression of all genes belonged to the aspartic protease family in rice.We identified 96 OsAP genes by a comprehensive bioinformaitic analysis of rice genome sequence database including TIGR,KOME and NCBI RefSeq.Totally 93 OsAP genes were localized on the 12 rice chromosome pseudomolecules.96 OsAP were divided into three categories in a phylogenetic tree,and the A category was more conserved structure compared to another two categories.The numbers of genes were the largest in the C category,but the gene structure varied much. Expression profile data of OsAP genes in Minghui 63 and Zhenshan 97,using RNA samples from 31 tissues hybridized with Affymetrix rice gene chips were extracted from CREP database（http://crep.ncpgr.cn）.Sixty-six of the genes had "present" call in at least one of the stages analyzed.Expression pattern of the sixty-six genes were grouped into three main classes.ClassⅠwas subdivided into four subclasses.ClassⅠαand classⅠγshowed low expression levels in all tissues analyzed.ClassⅠβand ClassⅠδparticularly showed high expression in root and stamen in one day before flowering respectively, compared to the rest analyzed tissues.ClassⅡshowed low expression in the stages of young panicle development,but they showed relatively high transcript accumulation in vegetative tissues.In contrast,classⅢshowed high expression level during the stages of young panicle development,but with low expression in most vegetative tissues.To investigate the light regulation of OsAP genes,the microarray data were obtained for the tissues of plumule and radicle treated with light and dark for 48 hr at 48 hr after emergence.A total of 14 OsAP genes were differentially expressed between light and dark treatments in the two tissues in two varieties.For phytohormone treatments,nine OsAP genes in two varieties exhibited differential expression in response to NAA,GA3 and KT treatments.All of 12 pairs of the genes located in the segmentally duplicated regions were found to be represented in the Affymetrix GeneChip,which provided opportunity for comparing their expression patterns.The expression patterns of the two members of seven gene pairs were different in most of the tissues tested,indicating one member of the duplicates might have gained new function.更多还原