【作者】 齐靖；

【导师】 张玉星； 申连英；

【作者基本信息】 河北农业大学 ， 果树学， 2009， 博士

【摘要】 梨和枣均是我国重要的经济树种,在我国栽培面积极广,是我国农业经济的重要组成部分。深化对这两种果树的分子生物学研究,并应用生物技术对其进行遗传改良,对于加速育种进程至关重要。在本研究中,围绕梨和枣分别开展了基因克隆、遗传转化、遗传图谱加密和QTL分析等一系列研究,填补了相关研究的空白,并为今后生物技术在果树育种中的进一步应用增添了新的理论依据。取得的主要成果如下所示:1.根据ACC氧化酶保守序列设计一对引物,以鸭梨果实cDNA为模板扩增得到831bp的鸭梨ACC氧化酶基因cDNA片段,并以此序列为基础RACE扩增该基因cDNA全长,得到了长度的1235bp的鸭梨ACC氧化酶基因cDNA序列,进一步根据得到的cDNA序列开放性读码框设计引物得到鸭梨ACC氧化酶基因DNA序列,结果显示该基因有4个外显子和3个内含子组成,外显子和内含子的长度分别为942bp和634bp。2.对鸭梨ACC氧化酶基因所翻译的氨基酸序列进行生物信息学分析,结果表明该氨基酸序列与其它14种植物的ACC氧化酶高度同源,包含一个92个氨基酸残基的2OG-Fe(II) oxygenase超级家族保守结构域,且与异青霉素-N合酶及相关双脱氧酶(PcbC)的功能区域同源性较高,说明鸭梨ACC氧化酶应属于2OG-Fe(II) oxygenase超级家族的异青霉素合酶(IPNS)家族,该家族酶成员在发挥酶促作用时不需要以2-OG作为辅因子。3.对所获得的鸭梨ACC氧化酶氨基酸序列进行二级结构和三级结构预测,结果发现该蛋白疏水性氨基酸位于分子内部,亲水性氨基酸位于分子表面,具有可溶性亲水蛋白的典型特征,不属于膜内在蛋白;但研究同时发现该蛋白具有3个豆蔻酰化位点,豆蔻酸可以通过与这些位点的甘氨酸残基相连接将鸭梨ACC氧化酶以可逆共价连接的方式锚定在膜上,故推测鸭梨ACC氧化酶应该是具有酰胺-连接豆蔻酰锚钩结构的脂锚定膜蛋白。4.分别用Southern杂交和Northern杂交分析获得的ACC氧化酶基因在鸭梨基因组中的拷贝数及在不同组织中的表达情况,结果发现该基因仅在生殖器官中表达,且在基因组中以单拷贝形式存在,说明该基因主要参与系统Ⅱ型乙烯的内源合成,而系统Ⅰ型乙烯的合成是由与该基因同源性较低的另外ACC氧化酶家族成员控制的。鸭梨上应至少存在两个ACC氧化酶基因家族成员。5.首次构建了鸭梨ACC氧化酶基因的反义表达载体,并在农杆菌LBA4404的介导下对鸭梨组培苗进行了遗传转化,经PCR鉴定证实共有4株鸭梨组培苗中外源基因得到成功转化。Southern杂交显示在这4株转基因鸭梨中除有1株外源基因呈双拷贝外,其余3株中外源基因均以单拷贝形式存在。6.对影响ISSR扩增的因素进行了优化,建立了枣属植物最适ISSR扩增反应体系,即25μL反应液中包含1×PCR Buffer、2.0 mmol·L-1 Mg2+、0.25 mmol·L-1dNTP、1.5U Taq酶、1.25μmol·L-1引物和50ng模板,PCR反应的最适退火温度为58℃。7.应用18条ISSR引物对150株冬枣×临猗梨枣杂交F1代群体进行扩增,共获得ISSR标记44个,其中34个被成功加入到了已有的枣遗传连锁图谱中,使图谱总图距增加了24.41cM,标记间平均距离减少了0.18cM,并填补了原图谱中3个大于10cM的标记空缺,使枣遗传连锁图谱的饱和度得到了进一步提高。8.比较了枣针刺长度相关性状包括中心干上长针刺长度、中心干上短针刺长度、二次枝上长针刺长度和二次枝上短针刺长度QTL在新构建的遗传连锁图谱和原图谱上的定位结果,发现仅有10个QTL位点一致,证实遗传连锁图谱的饱和度是影响QTL定位精确性的重要因素之一,同时也推测这10个QTL位点可信度较高,与其紧密连锁的标记可为日后分子标记辅助育种提供指导。更多还原

【Abstract】 Pear (Pyrus L.) and Chinese jujube (Ziziphus jujuba M.) are both important economic tree species in China, they are widely cultivated in our country and are important parts in China’s agricultural economy. To deepen the molecular biology study and use biotechnology for genetic improvement has important significance in speeding up the breeding process of the two fruit trees. So, in this study, focusing on pear and Chinese jujube, we carried out a series of expriments, such as gene cloning, genetic transformation, density-increasing of genetic linkage map and QTL analysis. The main results as follows:1. The full-length cDNA sequence of ACC oxidase gene was isolated from Yali fruits for the first time. It was 1235 bp long including 65 bp of 5’UTR, and 225 bp of 3’UTR and an ORF of 942 bp, encoding a 35.36 kDa protein with 314 amino acid residues. Base on the ORF, the genomic gene of ACO in Yali was also isolated, it had four extrons and three introns, which located in nt 106-347, nt 575-750 and nt 1085-1297. All the introns had GT at 5’end and AG at 3’end.2. Reaserch the deduce amino acids of ACO genes from Yali by bioinformatic analysis, it showed that this protein contained many conserved domains, and there were few differences among ACOs from different kinds of plants. The ACO in Yali had the homologous functional domain with 2OG-Fe(II) oxygenase super family, and was similar with Isopenicillin N synthase and related dioxygenases(PcbC), so it proved the ACO in Yali was belonged to Isopenicillin synthase family, which does not use 2-OG as the acidic substrate.3. Predicted the obtained Yali ACC oxidase amino acid sequence to secondary structure and tertiary structure, the results showed that the Yali ACO was a hydrophilic soluble protein but had 3 N-myristoylation sites, so it may be a lipid-anchored membrane protein which had a amide - myristic acid anchor hook to connect the membrane.4. Southern and Northern blotting were employed in this study to detect the copys of obtained ACO gene in Yali’s genomic and it’s expression system. The results showed this gene was single copyed in genomic and just expressed in reproductive organs, it is likely this gene only actived in the system II ethylene biosynthesis, but the system I ethylene biosynthesis was controlled by other ACO genes, so we can conclude that there were at least two ACO genes in Yali, and there were very different in nucleotide sequence.5. The anti-sense expression vector for obtained Yali ACO gene was constructed and transfromed in to Yali plants by using Agrobacterium-mediated method. Finally, 29 kanamycin resistant plantlets were obtained, identified them with PCR and Southern blot method, results showed the foreign gene had only been successful integrated into 4 of the 29 kanamycin resistant plants, the copies of the foreign gene in the 4 plant’s genome were between 1~2.6. The ISSR-PCR reaction system of Chinese jujube was estabblished after analysised the effect of 6 main factors in PCR reaction using the single factor method. The optimal ISSR-PCR reaction system was determined as follows: 1×PCR Buffer、2.0 mmol·L-1 Mg2+、0.25 mmol·L-1dNTP、1.5U Taq polymerase、1.25μmol·L-1primer and 50ng template DNA in total 25μL reaction volume. The optimal annealing temperature was 58℃.7. By using 18 ISSR primiers, the research obtained 44 ISSR markers to increase density of the genetic linkage map for Chinese jujube. Finally, a high-density genetic linkage map of Chinese jujube was constructed, which consists of 15 linkage groups, covers 1338.81 cM, includes 388 AFLP markers, 35 RAPD markers and 34 ISSR markers. The average interval distance is 2.92 cM. Compared with the previous linkage map, the total length of map has increased 24.41 cM, the average interval distance has reduced 0.18 cM, and 3 distance >10cM between adjacent marker were filled.8. The putative QTLs controlling needle length in Chinese jujube, including trunk long needle length, trunk short needle length, branch long needle length and branch short needle length were identified based on the newly constructed genetic linkage map. Totally 34 QTLs were obtained, but only 10 QTLs of it were located on the same position as the QTLs analysis result base on previous linkage map, so it proved the density of genetic linkage map has impact on the accuracy of QTLs anlysis, and it also indicated these 10 QTLs have higher credibility that the tightly linked markers of it could be used in marker-assisted breeding.更多还原