【作者】 杨超；

【导师】 盖钧镒；

【作者基本信息】 南京农业大学 ， 遗传学， 2009， 博士

【摘要】 大豆[Glycine max (L.) Merr.]起源于我国,在中国已有五千年左右的栽培历史,它也是世界上重要的粮食和油料作物之一,是人类食物中植物蛋白质和油脂的主要来源,与人们的日常生活息息相关。鉴于大豆的重要性,人们一直利用传统育种手段来提高大豆的产量。由于受物种间杂交不亲和性及不良连锁等因素影响,使常规育种方法的应用受到限制。近年来,随着生物技术的飞速发展,尤其是植物基因工程技术的日趋成熟,为大豆的品种改良提供了新的途径,大豆再生和遗传转化技术体系成为当前研究的热点和重点。大豆的组织培养再生主要有器官发生和体细胞胚发生两种诱导途径,两种发生途径具有不同的遗传基础,同时需要与不同的遗传转化方法结合进行转基因工作。本研究拟对大豆器官发生和体细胞胚发生两种再生途径分别进行植株再生技术、再生特性遗传基础以及相应遗传转化方法的研究,由于时间和条件的限制,部分实验未取得成功结果。目前所获的主要内容包括大豆的器官发生、体细胞胚胎发生和植株再生；大豆再生能力相关性状的QTL定位；大豆体细胞胚胎受体蛋白激酶基因的克隆和功能鉴定；农杆菌介导的大豆子叶节遗传转化体系的优化和应用。取得的主要研究结果如下：1.大豆植株再生技术体系方面：(1)以大豆子叶节为外植体对25份资源进行再生能力和丛生芽诱导培养基激素组合进行筛选,结果发现不同品种问再生频率有很大差异,其中N23676、N25277和N00060相对较高,分别是90.35%、86.53%和84.48%；添加不同激素种类(6-BA、2,4-D和TDZ)的丛生芽诱导培养基对大豆子叶节的丛生芽诱导率也不同,只添加1.67mg/L 6-BA就可以得到较高的丛生芽诱导率。(2)以大豆未成熟子叶为外植体对98份中国大豆资源进行了体细胞胚胎发生能力的筛选,同时研究了不同甘露醇浓度、不同ABA浓度和不同外植体大小等三个因子对大豆体细胞胚胎发生能力和植株再生能力的影响,结果发现不同基因型之间体细胞胚胎发生能力具有明显差异,基因型N25281、N25263和N06499具有较高的体细胞胚发生能力；选取4-5mm大小的外植体,基本培养基添加5mg/L ABA和3.0%甘露醇的组合可以提高大豆的体细胞胚胎发生能力,基因型N25281表现出较高的再生率(82.95%),平均每个外植体出苗数为1.35。2.大豆再生特性的遗传基础：(1)利用国家大豆改良中心提供的重组自交家系群体(NJRIKY),开展了大豆幼胚培养再生能力的QTL遗传定位研究。该群体的遗传图谱该群体的遗传图谱共整合了834个标记,分布在24个连锁群上,覆盖大豆基因组2307.83cM,每个连锁群上平均34.75个标记,标记间的平均距离为2.77cM。选用大豆愈伤组织诱导率和体细胞胚诱导率作为反映大豆幼胚培养再生能力的评价指标,采用复合区间作图法(CIM)进行QTL分析,结果共检测到3个与出愈率有关的QTL,位于B2和D2连锁群上,可解释5.84%-16.60%的表型变异；检测到4个与体细胞胚诱导率有关的QTL,全部位于G连锁群上,对表型性状变异的解释率为7.79%-14.16%。大豆幼胚培养再生性状的QTL定位研究为大豆再生性状的改良提供了标记辅助选择的依据。(2)从大豆中克隆了1个编码体细胞胚发生相关的类受体蛋白激酶(GmSERK1)基因,该基因编码624个氨基酸,与其它的SERK蛋白具有很高的同源性,其中包括有SP、ZIP、SPP、TM、LRR、胞内激酶活性结构域以及C端结构域,同时该基因也具有与其它SERK家族基因相似的内含子/外显子结构特征。系统发育分析表明植物SERK蛋白有三个分支,GmSERK1蛋白所在的分支主要由双子叶植物组成,该蛋白与豆科模式植物蒺藜苜蓿中的MtSERK1蛋白亲缘关系最近。Southern blot分析表明GmSERK1基因在大豆基因组中有1个拷贝。组织表达谱分析表明GmSERK1基因在叶、花芽和未成熟子叶中有表达,但是在根和茎中基本上不表达。在体细胞胚发生过程中,GmSERK1基因在早期培养时就有表达,在15天时达到最高点,15天后该基因的表达量反而开始降低,同时该基因在胚状物中有很高的表达量,而在非胚状物中检测不到表达。诱导表达分析表明在受到甘露醇、ABA.JA和SA等处理后,GmSERK1基因的表达方式不尽相同。采用Gateway技术构建了GmSERK1基因与GFP融合的过量表达载体并利用洋葱表皮细胞瞬时表达方法对GmSERK1蛋白进行了的定位研究,结果表明该蛋白被定位到细胞膜上,属于膜结合蛋白。3.大豆遗传转化技术体系：对影响农杆菌介导大豆子叶节遗传转化体系的主要因素进行了研究,包括外植体侵染时间、共培养时间、超声波处理以及AgNO3处理等,结果表明30分钟侵染时间、共培养3天和10秒超声波处理可以提高转化效率,在丛生芽诱导培养基中加入2mg/LAgNO3可以提高丛生芽诱导率；利用该遗传转化方法,将编码逆向转运蛋白的小麦TaNHX2基因导入大豆,PCR检测结果显示TaNHX2基因已经整合到了大豆基因组中,在250mmol/L NaCl溶液处理下发现非转化大豆植株表现出叶片变黄迹象逐渐死亡,而转基因植株则生长正常,以上结果表明TaNHX2基因可以提高了大豆的耐盐性。同时,利用Gateway技术构建了GmFAD2-3基因的RNAi表达载体,为利用转基因技术改良大豆脂肪酸品质奠定了基础。更多还原

【Abstract】 Cultivated soybean [Glycine max (L.) Merr.] has its origin from wild soybean [Glycine soja Sieb. et Zucc.] in China and has been a major source of edible vegetable protein and oil supplement for livestock for several thousand years. The importance of the crop has led efforts to improve its production through conventional breeding, and more recently it has integrated with modern genetic engineering technologies to avoid some of the limitations in traditional breeding procedures. Application of biotechnology is effective way for soybean improvement, for example the establishment of highly efficient regeneration system and transformation system. In the past, the scientists have paid more attention to study how to develop and optimize the methods on plant regeneration and transformation procedures, but the molecular mechanism and genetic basis of tissue culture response has not been well examined. Genetic studies of tissue-culture traits, such as callus growth, embryogenesis and differentiation, will make it possible to transfer genes controlling desirable tissue-culture traits into recalcitrant cultivars or species. With the development of molecular biology, the traditional quantitative genetics has run to the stage of molecular quantitative genetics, which provides a new route to study the complicated quantitative traits. Therefore, detailed genetic studies are required to identify the genes or QTLs associated with the tissue culture response. The present research was intended to study the plant regeneration system, genetic basis and genetic transformation system of organogenesis and somatic embryogenesis, but there was no good results obtained for partial experiments because of the restriction of time and energy. Our studies involved organogenesis, somatic embryogenesis and plant regeneration in soybean; optimization of Agrobacterium-mediated cotyledonary nodes transformation system and its utilization on transgenic soybean; isolation and functional characterization of a novel SERK gene (somatic embryogenesis receptor kinase) from soybean; mapping QTLs for tissue culture response in soybean. The main results of this research were listed as follows:1. Plant regeneration system:(1) Twenty five varieties mainly from Jiangsu province were screened for their regeneration ability. Significant differences in regeneration frequency of these soybean varieties were found. Among them, the regeneration frequencies of N23676, N25277 and N00060 were relatively higher with 90.35%,86.53% and 84.48%, respectively. High shoot induction frequency was found when the cotyledonary node explants were incubated in the SIM only supplemented with 1.67 mg/L 6-BA.(2) Ninety eight Chinese soybean varieties were screened for their capacity to generate somatic embryos. From these 12 varieties were selected for further study to enhance the efficiency of somatic embryogenesis and plant regeneration. The effects of different mannitol concentrations, abscisic acid (ABA) and embryo explant age size were investigated. The results showed that all three factors were relevant for raising rates of callus initiation and somatic embryogenesis, but with differential responses among the genotypes. The three soybean varieties N25281, N25263 and N06499, all agronomically important to the Lower and Middle Changjiang Valleys, were found to have a high somatic embryogenic capacity and therefore recommended in cultivar development through genetic engineering. The treatment of 3.0% w/v mannitol,5 mg/L ABA and a 4-5 mm sized explant was found to be optimal for somatic embryogenesis generating the highest regeneration rate at 82.95%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. The above results provide a basis for efficient regeneration of soybean, and are informative for the development of genetic transformation systems in Chinese soybean germplasms.2. Genetic basis of the regeneration trait:(1) Using a RIL population (NJRIKY) provided by the National Center for Soybean Improvement, the identification of QTLs for tissue culture response in soybean were conducted. The linkage map of NJRIKY contained 834 molecular markers on 24 molecular linkage groups and spaned 2307.83 cM of the soybean genome with an average interval distance of 2.77 cM, the average markers per group of 34.75. Two characters (callus induction and somatic embryogenesis ability) were chosen to evaluate the tissue culture response of soybean. With the method of composite interval mapping (CIM) described in Windows QTL Cartographer Version 2.5,3 quantitative trait loci (QTL) were identified for the frequency of callus induction, on chromosomes B2 and D2, accounting for phenotypic variation from 5.84% to 16.60%; 4 QTLs located on chromosome G were revealed for the frequency of somatic embryo initiation and explained the phenotypic variation from 7.79%-14.16%. The present results will be contributing for genetic improvement for regeneration traits with marker-assisted selection (MAS) in soybean.(2) A novel gene designated GmSERK1 was isolated from soybean [Glycine max (L.) Merr.]. Sequence and structural analysis determined that the GmSERK1 protein, which encodes 624 amino acids, belongs to the SERK gene family. GmSERK1 shared all the characteristic domains of the SERK family, including five LRRs, a SPP motif, TM, and kinase domains. GmSERK1 showed very high homology to rice OsSERK1 (87%), maize ZmSERK1 (85%), alfalfa MtSERKl (94%), and Arabisopsis AtSERKl (88%). As expected from the multiple deduced amino-acid sequences alignment, the result from phylogenetic analysis also revealed that GmSERK1 is closest to MtSERK1 from Medicago, the model plant of legume. DNA gel blot analysis indicated that a single copy of the GmSERKl gene resides in the soybean genome. We also explored GmSERK1 tissue-specific and induced expression patterns using quantitative real-time PCR. The analysis revealed dissimilar expression levels in various tissues and under different treatments. It is obviously that GmSERK1 shows lower expression level in roots and stems, but higher level in leaves and floral buds. For the immature zygotic embryos, there was no significant difference among immature zygotic embryos of <3mm,4-5mm and 6-8mm, while the visible decline occurred for immature zygotic embryos of>8mm in length. An increase in the transcript accumulation could be observed at the early period. The expression level of GmSERK1 reached the maximum at 15d, when the globular-stage embryos firstly occurred. Past 15d of culture, the GmSERK1 transcript level decreased. Meanwhile, GmSERK1 was highly expressed in embryogenic cultures, but no expression was seen in the non-embryogenic cultures. In addition, transient expression experiments in onion epidermal cells indicated that the GmSERK1 protein was located on the plasma membrane. The results of this study suggested that GmSERK1,a member of the SERK gene family, exhibits a broader role in various aspects of plant development and function, in addition to its basic functions in somatic embryogenesis.3. Genetic transformation system:The factors influencing the transformation frequency were investigated, such as the infection time on T-DNA delivery into explants, the co-culture time, the sonication-assisted treatment. The results indicated that infection time of 30 min, co-cultivation time of 3 days, the sonication-assisted treatment can enhance the transformation frequency.2 mg/L AgNO3 supplemented to SIM enhanced the shoot induction frequency of soybean explants. The TaNHX2 gene (GenBank Accession No. AY040246) encoding a Na+/H+ antiporter was transferred to soybean using the above transformation system. The results of PCR detection showed that the transgenic soybeans were obtained. At the same time, the salt tolerant character of transgenic soybean was determined under NaCl treatment. Transgenic soybean could normally grow under the treatment of 250 mmol/L NaCl, while the growth of control soybean was seriously inhibited. From the results, it concluded that transgenic soybean plants were acquired by transformation of a vacuolar Na+/H+ antiporter gene, and TaNHX2 confers soybean resistant against salt stress. Meanwhile, the vector GmFAD2-3-RNAi was produced using the Gateway Technology.更多还原