球等鞭金藻的遗传多样性与高DHA和EPA品系的定向选育

发布时间：2018-04-15 00:17

本文选题：球等鞭金藻 + 脂肪酸　；参考：《上海海洋大学》2017年硕士论文

【摘要】：球等鞭金藻是一种重要的水产养殖饵料微藻,广泛应用于贝类和海参的育苗过程中,其显著的营养价值在于不仅具有较高的DHA含量,而且也能合成少量的EPA,研究表明,多数海水养殖动物以DHA和EPA作为必需脂肪酸,适当的DHA/EPA比率能显著提高水产动物幼苗的孵化率,增强成体的免疫能力。然而,球等鞭金藻藻种经过多年的使用,往往存在着种质退化的现象,表现为生长速度下降,DHA和EPA含量减少,因而有必要进行适当地选择育种,筛选出生长速度快,DHA和EPA含量高的品系供养殖户育苗使用。为了更好地进行球等鞭金藻的纯化培养,首先需要进行琼脂固体培养基的优化。采用3因素3水平的正交设计,研究了琼脂浓度、培养基和氮源对球等鞭金藻固体平板生长的影响,并且利用单因素优化分析比较了维生素和抗生素对其藻落形成的影响。结果表明,琼脂浓度可影响球等鞭金藻藻落的形成,球等鞭金藻(OA-3011)在0.6-1.0%琼脂浓度的固体平板生长最合适,而培养基和氮源对其影响较小,维生素B1和B12对于其固体平板生长不是必需的,抗生素对其固体培养有明显抑制作用,建议在球等鞭金藻(OA-3011)固体平板分离、纯化过程中,使用1%的琼脂平板和不加维生素的F/2培养基。目前我国水产养殖上使用的大多数球等鞭金藻品系都来源于同一株藻Isochrysis galbana O A-3011,经过多年的地理隔离形成了不同的品系,为了揭示地理隔离对球等鞭金藻表型性状的影响,从而为球等鞭金藻的选育工作提供依据,我们对5个品系的球等鞭金藻(I.galbana FACHB-861,OA3011-QD,OA3011-LK,OA3011-AY和OA3011-LZ)的种属关系、生长和脂肪酸组成进行了比较分析。实验在相同条件下对5个品系球等鞭金藻培养6 d,提取基因组DNA并PCR扩增测定18S rDNA基因序列,每天定时记录各品系的OD660,绘制生长曲线,在进入稳定期后收获并进行生物量的测定及脂肪酸的气相色谱分析。18S rDNA基因序列的进化分析表明,OA3011-LK、OA3011-AY和OA3011-LZ具有一致的序列,而OA3011-QD因1个碱基的差异与其它OA3011出现了分离,FACHB-861与OA3011品系亲缘关系较远;对生长和脂肪酸进行对比分析后,结果显示,5个品系的球等鞭金藻在不同的时间达到稳定期,其中FACHB-861表现出了与其他品系明显不同的生长模式。FACHB-861和OA-3011品系的生物量具有显著差异,OA3011-LK、OA3011-AY和OA3011-LZ之间也有显著差异(P0.05),但特异生长率各品系间无显著差异(P0.05)。FACHB-861和OA3011-LZ的总脂肪酸(TFA)含量显著低于OA3011-LK、OA3011-QD和OA3011-AY(P0.05)。FACHB-861的脂肪酸各成分与OA-3011各品系明显不同。然而,在OA-3011各品系间脂肪酸组成也有明显差异,这表明球等鞭金藻存在广泛和频繁的数量性状表型差异。总之,地理隔离造成的这些差异为球等鞭金藻的选育提供了可行性。选择来自山东烟台(OA3011-YT)以及海南省(OA3011-HN)的球等鞭金藻进行胶群相细胞模拟杂交,OA3011-YT品系进行胶群相模拟自交,利用优化后的琼脂平板涂布技术获得单克隆并在F/2液体基中培养出F1代个体,作为模拟自交系F1及模拟杂交系F1,通过测定生长指标、脂肪含量和脂肪酸组分筛选出优势藻株,对优势藻株进一步进行胶群相细胞杂交、自交,再重复上述步骤获得模拟自交系和模拟杂交系的F2代优势藻株,最后对F2代藻株进行脂肪酸组分的分析。比较F1及F2代藻株脂肪酸组分后发现,通过定向选育,F1代中DHA和EPA含量较高的藻株在F2代中得到了保留,并且F2代最低DHA、EPA、PUFA含量水平得到了提高;模拟杂交系在生长、脂肪酸含量以及提高DHA、PUFA含量的能力上均优于模拟自交系,最终筛选获得4株DHA含量高并且生长速度快的藻株,以及2株EPA含量高并且生长速度快的藻株进行保种,未来将对其进行进一步的开发、研究和利用。综上所述,球等鞭金藻作为一种重要的饵料微藻,具有进行高DHA和EPA含量品系的选育的必要性以及可行性,而选择育种作为一项基础性科学研究是很有必要的,不仅可以初步获得球等鞭金藻的数量性状遗传规律,增加对球等鞭金藻遗传多样性的了解,而且可以为进一步的诱变育种和转基因育种提供优良的藻种,优化球等鞭金藻的种质资源。
[Abstract]:I.galbana is one of the important aquatic microalgae, the breeding process is widely used in shellfish and sea cucumber, its nutritional value is significant not only has high content of DHA, but also can synthesize a small amount of EPA, research shows that most animal aquaculture based on DHA and EPA as essential fatty acids, as appropriate the ratio of DHA/EPA can significantly improve the hatching rate of aquatic animal seedlings, enhance the body's immunity. However, Isochrysis Zaozao after years of use, there is a genetic degradation phenomenon, showed decreased growth speed, reduce DHA and EPA content, so it is necessary to choose appropriate breeding, screening a fast growth rate, DHA and high content of EPA strains for farmers. In order to use seedling cultured better of i.galbana, first need to optimize solid agar culture. Using 3 factors and 3 levels The orthogonal design of agar concentration, effect of medium and nitrogen source on the growth of i.galbana solid plate, and the use of single factor optimization analysis has compared the effects of vitamins and antibiotics on the algae colony formation. The results showed that agar concentration can affect the formation of gold Zaozao falling ball whip, Isochrysis algae (OA-3011) in the solid plate 0.6-1.0% agar medium and the growth of the most appropriate, and the nitrogen source has little influence on the vitamin B1, and B12 for the growth of solid plate is not required, antibiotics have obvious inhibitory effect on the solid culture, suggestions on i.galbana (OA-3011) solid plate separation. In the purification process, the use of 1% agar plate and without vitamin F/2 medium. The use of aquaculture in China on the majority of Isochrysis Zaopin lines are derived from the same strain galbana O alga Isochrysis A-3011, after many years of geographical isolation From the formation of different strains, in order to reveal the geographical isolation of i.galbana phenotypic traits, so as to provide the basis for breeding of i.galbana, we studied 5 strains of Isochrysis galbana (I.galbana FACHB-861, OA3011-QD, OA3011-LK, OA3011-AY and OA3011-LZ) of the species relationship that growth and fatty acid composition were analyzed. The experiment under the same condition, 5 strains of i.galbana culture 6 D, extraction of genomic DNA and PCR amplification of 18S rDNA gene sequence determination, every time to record the strains of OD660, draw the growth curve, in stable harvest and determination the biomass and gas chromatographic analysis of fatty acid of the evolution of.18S rDNA gene sequence analysis showed that OA3011-LK, OA3011-AY and OA3011-LZ have the same sequence, and OA3011-QD due to the difference of 1 bases and the other OA3011 appeared from FACHB-861, and OA3011 The Department of distantly related; the comparative analysis of the growth and fatty acid. The results showed that 5 strains of i.galbana reached stable at different time, which showed the FACHB-861 biomass growth model of.FACHB-861 and OA-3011 strains were significantly different from other strains had significant differences, OA3011-LK also, there is a significant difference between OA3011-AY and OA3011-LZ (P0.05), but there was no significant difference between the specific growth rate among the lines (P0.05) of total fatty acids.FACHB-861 and OA3011-LZ (TFA) were significantly lower than those of OA3011-LK, OA3011-QD and OA3011-AY (P0.05).FACHB-861 fatty acid components and OA-3011 strains were significantly different. However, in the OA-3011 strains between the fatty acid composition was also significantly different, indicating the presence of quantitative trait phenotypes extensive and frequent Isochrysis galbana. In short, these differences caused by geographical isolation for the breeding of i.galbana. For the feasibility. Choose from Shandong Yantai (OA3011-YT) and Hainan province (OA3011-HN) of Isochrysis glue group cell hybrid simulation, strain OA3011-YT are simulated using self adhesive group, agar plate after optimization to obtain monoclonal and cultivate F1 generation individuals in F/2 liquid medium. As the simulation and Simulation of hybrid F1 inbred line F1, by measuring the growth index, fat content and fatty acid composition of selected Dominant Algae strains of Dominant Algae strains further glue group cell hybridization, selfing, Heterosis of F2 generation strains and then repeat the above steps for simulation of inbred lines and hybrid simulation, finally analysis of fatty acid composition of F2 strain. F1 and F2 strains of fatty acid composition found by directional selection, DHA and EPA were higher in F1 generation of algae strains in the F2 generation and F2 generation have been retained, the lowest DHA, EPA, PUFA content The level has been improved; simulation of hybrid lines in growth, fatty acid content and improve the ability of DHA, the content of PUFA was better than that of simulated inbred lines, 4 strains were obtained with high DHA content and fast growth of algae strains, and 2 strains of high EPA content and fast growth of algae strains in conservation, will further development in the future, research and utilization. In summary, isochrysisgalbana as an important kind of microalgae, with high DHA and EPA content in breeding strains of the necessity and feasibility, and selection as a basic scientific research is very necessary, not only can be obtained the number of genetic rules of i.galbana, increase of i.galbana genetic diversity of knowledge, but also can provide algae excellent for further breeding and transgenic breeding, optimization of i.galbana germplasm resources.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.3

【参考文献】