【作者】 杨谷良；

【导师】 谭晓风；

【作者基本信息】 中南林业科技大学 ， 森林培育， 2007， 博士

【摘要】 自交不亲和性是植物生殖过程的一种普遍现象,它作为防止近亲繁殖和物种退化的异花授粉机制,为物种的生存、发展以及种群的相对独立性提供了保障。梨是一种配子体自交不亲和性植物,其自交不亲和性主要是由S位点的等位基因所控制。在生产栽培中必须配置不同S基因型的授粉品种或通过人工授粉等措施来保证高产稳产。因此确定不同品种的S基因型将为授粉品种的配置及良种选育提供重要的科学依据。本文以冬黄等28个梨品种为实验材料,开展了品种S基因型的鉴定、新S基因的分离克隆等研究；另外,以爱甘水等30个梨为材料,采用AFLP标记技术,开展了不同品种指纹图谱和亲缘关系的相关研究。本论文主要研究结果如下：1.梨不同品种S基因型的确定。采用PCR-RFLP、序列测定和亲缘关系分析等技术方法,确定了宝珠(S22S33)、冰糖(S19S31)、冬黄(S20S34)、黄梨(S22S34)、金秋(S3S9)、库尔勒(S22S34)、中梨一号(S4S35)、大果水晶(S3S9)、马蹄黄(S17S19)、懋功(S12S13)、柠檬黄(S31S32)、新雅(S4S17)、早酥(S22S35)、西子绿(S1S4)、黄花(S1S2)、黄金(S3S4)、清香(S4S7)、雪峰(S4S16)、雪芳(S4S16)、雪英(S3SX)、雪芬(S3SX)等品种的S基因型。2.梨新S基因的分离鉴定。利用梨S基因扩增的"FTTYQ"和"anti-IIWPNV"特异引物组合,并经过克隆测序,从柠檬黄和冰糖梨中分离鉴定了梨S31-RNase新基因,该基因扩增片断大小为346bp,外显子和内含子的分别为201bp和145bp；从柠檬黄梨中分离鉴定了梨S32-RNase新基因,该扩增片断大小为999bp,外显子和内含子分别为213bp和786bp；从宝珠梨中分离鉴定了梨S33-RNase新基因,该基因扩增片断大小为532bp,外显子和内含子分别为195bp和337bp；从冬黄、库尔勒和黄梨中分离鉴定了梨S34-RNase新基因,该基因扩增片断大小为428bp,外显子和内含子分别为201bp和227bp；从早酥和中梨一号梨中分离鉴定了梨S35-RNase新基因,该基因扩增片断的大小为371bp,外显子和内含子分别为204bp和167bp。上述5个新基因在GenBank的登录号分别是：DQ072113、DQ072114、DQ082897、DQ224345和DQ224344。3. S35-RNase基因的cDNA克隆与表达分析。以早酥梨的雌蕊为材料,提取总RNA,采用3’RACE和5’RACE技术,从早酥梨中扩增了一个S基因的cDNA全长。对该序列进行同源性比较,发现该序列为S35-RNase基因的cDNA全长序列。该序列全长681bp,编码227个氨基酸,具有日本梨S-RNase蛋白的序列特征：5个保守区和两个高变区、具有8个保守的半胱氨酸残基以及2个保守的组氨酸残基。该基因的cDNA序列登录到GenBank,登录号为DQ224344。采用Northern杂交技术,对该基因的表达情况进行了分析,结果发现该基因在大铃铛期表达量明显高于花前3d或花后3d,并且只在雌蕊中表达。4.部分梨品种的AFLP分析。以爱甘水等30个梨品种为材料,利用AFLP技术进行研究,除八月酥和冀蜜两个梨品种没有特异谱带外,其它品种至少有一条以上的特异性条带,这些条带可以作为各品种进行分子鉴别的重要依据。通过聚类,发现所有品种聚为4个组：第一组主要是日本梨及与日本梨有亲缘关系的部分品种；第二组为中国砂梨品种；第三组只有2个品种：黄冠和清香梨,它们的一个共同亲本为新世纪；第四组是康德,其亲本中有一个是西洋梨,被单独聚为一类。从聚类结果看,不同的品种之间表现出了一定的地域相关性。更多还原

【Abstract】 The plant self-incompatibility, a universal biological phenomenon in angiosperm, is an important protective mechanism of preventing close breeding and species retrogression which was formed during the long-term evolution. As a kind of rosaceae plants, Pear is a typical fruit tree of gametophytic self-incompatibility (GSI). Because of its self-incompatibility, it has a low fruiting set of self-pollination and it is necessary to arrange varieties with different S-genotype or to perform artificial pollination to ensure a stable and high yield. In this article, some cultivars’S-genotype was identified and 5 new S genes were isolated and identified. It will provide a theoretical reference for studying the self-incompatibility and for parent’s selection when hybridization is carried out. At the same time,30 cultivars such as aiganshuili were studied by fluorescent AFLP. A fingerprinting has been constructed. Relationship and inheritance of the studied cultivars have been analyzed on molecular level. The purpose is to provide basis for pear classification and theoretical references for hybridization parents. Mainly study results are listed as following.1. Identification the S-genotype. After using the method of PCR-RFLP, sequence and genetic relationship analysis etc., some cultivars S-genotype were identified. Such as: baozhuli(S22S33)、bingtangli(S19S31)、donghuangli(S20S34)、huangli(S22S34)、jinqiuli(S3S9)、kuerleli(S22S34)、lvbaoshili(S4S35)、daguoshuijingli(S3S9) matihuangli(S17S19)、maogongli(S12S13)、ningmenghuangli(S31S32)、xinyali(S4S17)、zaosuli(S22S35)、xizilvli(S1S4)、huanghuali(S1S2)、huangjinli(S3S4)、qingxiangli(S4S7)、xuefengli(S4S16)、xuefangli(S4S16)、xueyingli(S3SX)、xuefenli(S3SX)。2. Isolation and identification of new S gene.5 new S-RNase genes were isolated from the cultivars such as donghuangli after PCR and sequence analysis. The isolated new S-RNase genes were S31-RNase(DQ072113), S32-RNase(DQ072114), S33-RNase(DQ082897), S34-RNase(DQ224345) and S35-RNase(DQ224344). The fragment of S31-RNase contains 346 nucleotides; the exon and intron of it contain 201 and 145 nucleotides respectively. The fragment of S32-RNase contains 999 nucleotides; the exon and intron of it contain 213 and 786 nucleotides respectively. The fragment of S33-RNase contains 532 nucleotides; the exon and intron of it contain 195 and 337 nucleotides respectively. The fragment of S34-RNase contains 428 nucleotides; the exon and intron of it contain 201 and 227 nucleotides respectively. There are 2 different nucleotides and only one different deduced amino acid between S28-RNase and S34-RNase. There are 9 different nucleotides and no different deduced amino acid between S8-RNase and S34-RNase. We concluded that S8-RNase, S28-RNase and S34-RNase are the same one. The fragment of S35-RNase contains 371 nucleotides; the exon and intron of it contain 204 and 167 nucleotides respectively.3. The full length cDNA of S35-RNase (genebank accession number:DQ224344) were obtained from the cultivars of Zaosu pear after using RACE technique. The analytical result suggested that S35-RNase contains 681 nucleotides, codes 229 amino acids. Furthermore, there are 8 conservative’Cys’residues forming 4 disulfide bonds,2 conservative’His’residues influencing the S-RNase activity, and 1 conservative ’Asn-Xaa-Ser/Thr’motif within RC4, which plays an important role in the folding of core structure. To study the expression characteristic of S35-RNase, the technique of northern blotting was employed and discovered that it is highly expressed in the pistil of flowers at the stage of big ball and only expressed in pistil.4. AFLP fingerprinting analysis of 30 pear cultivars. AFLP fingerprinting technique were used to study the relationship of 30 cultivars such as aiganshuili et al. there were 28 cultivars have more than 1 characteristic bands, which were 93% among all of the cultivars. All of the cultivars were clustered into 4 groups. The cultivars of the 1st group are the Japanese pear or have genetic relationship with Japanese pear. The cultivars of the 2nd group are mainly the cultivars of Chinese pear. Huangguanli and qingxiangli were clustered to the 3rd group; these two cultivars have a common parent of shinseiki. The 4th group has only 1 cultivar of kangdeli. One of its parents is Pyrus communis.更多还原