【作者】 韩锁义；

【导师】 喻德跃；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 大豆【Glycine max （L.） Merr.】是世界上最重要的经济作物之一,由于基因组结构复杂、遗传转化效率低等原因限制了其功能基因组研究的发展。但随着大豆基因组测序的初步完成,大豆功能基因组研究将成为研究的重点。突变体是基因功能研究的重要材料,也是大豆遗传改良的基础材料,鉴于此,本研究利用叠氮化钠,60Coγ射线,甲基磺酸乙酯等方法处理大豆品种“南农86-4”,“南农87c-38”和“南农94-16”,以初步构建大豆突变体库,并对其中一个子叶突变体开展了较系统的研究。主要结果如下：1、大豆突变体库的构建对3个品种的M2、M3世代的突变体进行了表型性状调查和分析,都获得包括叶、茎、花、种子等性状变异的材料,其中“南农86-4”叶变异120份、茎变异24份、花变异26份、种子变异9份、熟期及育性变异6份,共185份；“南农87c-38”叶变异10份、茎变异4份、花变异3份、种子变异3份、熟期及育性变异42份,共62份；“南农94-16”叶变异84份、茎变异3份、花变异10份、种子变异21份、熟期及育性变异2份,共120份。对部分突变体的蛋白质及油分含量进行了分析,发现“南农86-4”突变系蛋白质含量范围为：39.5%-52.5%,油分含量范围为：15.3%-22.9%,“南农94-16”突变系蛋白质含量范围为：39.1%-48.1%,油分含量范围为：17.3%-22.2%。2、大豆子叶突变体的鉴定与分析1)农艺品质性状对通过NaN3-60Coy射线诱变“南农94-16”获得的子叶折叠突变体进行农艺性状综合调查,发现该突变体表现为子叶向外折叠,种皮部分皱缩,比对照品种晚熟9天左右,而其它表型无明显差异。子叶形态观察结果表明,突变体子叶折叠形态是在种子发育初期由于子叶伸展受到抑制形成。蛋白质测定表明,突变体蛋白质含量比对照高3个百分点。氨基酸组份分析表明,与对照相比,突变体的17种氨基酸中除丝氨酸含量没有明显变化,脯氨酸、苏氨酸、酪氨酸含量有一定的降低外,其它13种氨基酸含量都增加,尤其是蛋氨酸含量增加33%以上。游离氨基酸测定结果表明,突变体比对照含量高74%以上。2)遗传分析通过杂交对子叶折叠突变体进行遗传分析,F2结果分析表明,该性状由两对隐性细胞核基因控制。3)基因组水平分析为了总体了解子叶折叠突变体与对照在基因组水平上的差异,本研究对突变体及对照的基因组DNA进行了SSR标记分析。筛选大豆SSR标记共957对,结果显示有9对标记表现差异,这些标记集中分布在三个连锁群B1、D2、G的三个位点上。据前人研究结果分析,B1连锁群上的标记Satt426附近有蛋白质与油分的QTL; G连锁群上的Satt163、Satt038> Sat₁68. Satt570. AW734137标记附近有种子性状的QTL。为了发现突变体与对照在基因组水平上的差异,本研究运用比较基因组杂交芯片（Affymetrix（?） GeneChip）进行分析,对数据结果以信号值相差1.2倍为标准进行筛选,获得明显差异的19个候选基因,其中突变体比对照杂交信号值增强的有10个基因,突变体比对照杂交信号值减弱的有9个基因,参考其它作物的研究结果,对这些基因进行了初步的功能预测,突变体比对照杂交信号增强的10个基因中有7个基因编码的蛋白为未知蛋白,3个基因编码的蛋白预测为已知蛋白。3个已知蛋白的功能分别为：天冬氨酸-tRNA连接酶,UDP-葡糖基转移酶,EF-hand钙结合蛋白CCD1.突变体比对照杂交信号减弱的9个基因中有3个基因编码的蛋白为未知蛋白,6个基因编码的蛋白预测为已知蛋白。6个已知蛋白的功能分别为：亮氨酸氨基肽酶,翻译起始因子,核糖体蛋白,Dof锌指蛋白,丙酮酸脱氢酶,细胞色素P450单氧酶。为了从中发现可能与子叶形态密切相关的基因,我们用RT-PCR对19个候选基因在开花后30天的种子中进行了差异表达研究。其中突变体比对照杂交信号值增强的10个基因中,Gma.15488的表达结果与CGH芯片结果一致,但Gma.15488编码的蛋白为未知蛋白,突变体比对照杂交信号值减弱的9个基因中,TC208897的表达结果与CGH芯片结果一致,TC208897编码的蛋白为Dof锌指蛋白。19个基因中除了这两个基因外,其它基因或者全部没有表达,或者表达水平一致,说明这两个在子叶中差异表达的基因最有可能与子叶的异常发育相关,但需要进一步验证。4)子叶突变对大豆种子蛋白组成的影响为了研究子叶折叠性状可能对种子贮藏蛋白组成的影响,本研究运用双向电泳技术对由三种不同处理方式获得的表型相似的子叶折叠突变体进行比较研究,获得3个共同的差异蛋白质,其中两个均为球蛋白亚基G3/AlaBlb,在突变体中含量增加；另一个为大豆凝集素,在突变体中含量降低。3、大豆TILLING技术的建立与初步应用本研究还对新发展的基因功能研究方法TILLING技术进行了优化,将粗提取的CEL I酶初步运用于大豆TILLING技术中,在75份与大豆叶形性状相关的突变体材料中检测KNOX基因,结果显示一个突变体在该基因内发生单碱基变化,但编码的蛋白没有变化。更多还原

【Abstract】 Soybean [Glycine max （L.） Merr.] is one of the most economically important crops in the world. However, complex organization of genomic DNA and low efficiency on genetic transformation restricts the advancement of functional genomics research. With the completion of soybean genome sequencing, the research of gene function will become the research hotspot. Mutants are the important materials for the advancement of functional genomics research, and are the basic materials for soybean genetic improvement.Therefore, in this research; soybean cv. "Nannong 86-4", cv. "Nannong 87c-38" and cv. "Nannong 94-16" were treated with NaN3,60Co y rays and EMS to construct the mutant population. Meanwhile, a systematic research for curled cotyledon mutant has been developed. Main results are as follows:1. Construction of mutant populationPhenotypic characters of M2 and M3-generation mutants were investigated for various mutants including morphological characters such as leaf, stem, flower and seed. There are 185 mutants identified from "Nannong 86-4" including 120 for leaf,24 for stem,26 for flower,9 for seed,6 for maturity and fertility, respectively. There are 62 mutants identified from "Nannong 87c-38" including 10 for leaf,4 for stem,3 for flower,3 for seed,42 for maturity and fertility, respectively. There are 120 mutants identified from "Nannong 94-16" including 84 for leaf,3 for stem,10 for flower,21 for seed,2 for maturity and fertility, respectively. Proteins and the oils of partial mutants have been studied. The protein content of mutants in "Nannong 86-4" was in the range of 39.5%-52.5% and the oil content range was 15.3%-22.9%. While in "Nannong 94-16" the protein and oil content range variation was 39.1%-48.1% and 17.3%-22.2%, respectively. 2. Identification and analysis of a curled cotyledon mutant in soybean1) Characters of agronomic and qualityFirst, overall investigation of agronomic characters of curled cotyledon mutant suggested the mutant cotyledons curled outwards and seed coats were partially shrunken. Plants raised from mutant seeds were found to mature 9 days later than the wild type. The result of observation showed significant difference in morphology between the mutant and its wild type at the seed formation stage. Analysis of quality characters indicated a 3% higher protein content in mutant than wild type. Analysis of 17 kinds of amino acids in mutant indicated that serine content was not affected, while, proline, threonine and tyrosine were reduced. The rest however, showed increased, levels especially the methionine which increased by as much as 33% in the mutant. Analysis of free amino acid content in mutant was 74% higher than that of wild type.2) Genetic analysisGenetic analysis of the curled-cotyledon mutant by hybridization indicated that it was a double recessive mutant based on nuclear inheritance.3) Analysis in genome levelTo further address the difference in genomics between curled-cotyledon mutant and wild type, SSR marker research was developed, altogether 957 pairs were screened, and result showed difference on 9 pairs of markers. These markers were mainly concentrated in three linkage groups namely B1, D2, and G. According to the previous findings, QTL for the protein and the oil was located on B1 linkage group nearby marker Satt426. QTL for the seed character was located among cluster markers Satt163, Satt038, Sat₁68, Satt570 and AW734137 on linkage group GFurthermore, in order to search the difference between mutant and wild type in the genome level, CGH array was used. Regarding a certain specific value of 1.2 times as the threshold,19 different genes were obtained. The ten genes with stronger signal in mutant than that in wild type, among 7 genes encode unknown protein, and 3 genes encode known protein. Functions of the known protein are showed as follows:aspartate-tRNA ligase, UDP-glycosyltransferase, EF-hand Ca2+-binding protein CCD1. The nine genes with stronger signal in mutant than that in wild type, among 3 genes encode unknown protein, and 6 genes encode known protein. Functions of the 6 known proteins are as follows:leucine amino peptidase, translation initiation factor, structural constituent of ribosome, Dof zinc finger protein, pyruvate dehydrogenase, and cytochrome P450 monooxygenase. To discover the possible gene that closely related to cotyledon, the validation of RT-PCR with seeds obtained within 30 days after flower indicates that among the 10 genes with stronger signal there is only one gene complied with the result of CGH. It is Gma.15488. Among the 9 genes with weaker signal there is only one gene complied with the result of CGH. It is TC208897. The result of research indicates that there are intimate relationship between 2 genes above and the cotyledon shape. This viewpoint also needs to carry on further confirmation, such as to obtain full length gene and transgene so on.4) Influence of curled cotyledon on seed storage protein in soybeanIn order to study the influence of cotyledon shape difference to the mature seed storage protein components in soybean, three kinds of curled-cotyledon mutants was searched by 2-D electrophoresis. The research results showed that there were 3 common proteins which are different in mutant and wild type. The increased two protein spots in mutants are both globulin subunit G3/AlaBlb, at meanwhile, the protein spot that decrease is lectin.3. The establishment and application of TILLING in soybeanStudies of optimizing the new method of TILLING have been conducted. CEL I enzyme that extracted from celery were used in TILLING The KNOX gene was studied using 75 mutants of leaf; as a result, one base change was found in this gene in one mutant.更多还原