本文選題：球等鞭金藻 + 脂肪酸�。� 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：球等鞭金藻是一種重要的水產(chǎn)養(yǎng)殖餌料微藻,廣泛應(yīng)用于貝類(lèi)和海參的育苗過(guò)程中,其顯著的營(yíng)養(yǎng)價(jià)值在于不僅具有較高的DHA含量,而且也能合成少量的EPA,研究表明,多數(shù)海水養(yǎng)殖動(dòng)物以DHA和EPA作為必需脂肪酸,適當(dāng)?shù)腄HA/EPA比率能顯著提高水產(chǎn)動(dòng)物幼苗的孵化率,增強(qiáng)成體的免疫能力。然而,球等鞭金藻藻種經(jīng)過(guò)多年的使用,往往存在著種質(zhì)退化的現(xiàn)象,表現(xiàn)為生長(zhǎng)速度下降,DHA和EPA含量減少,因而有必要進(jìn)行適當(dāng)?shù)剡x擇育種,篩選出生長(zhǎng)速度快,DHA和EPA含量高的品系供養(yǎng)殖戶(hù)育苗使用。為了更好地進(jìn)行球等鞭金藻的純化培養(yǎng),首先需要進(jìn)行瓊脂固體培養(yǎng)基的優(yōu)化。采用3因素3水平的正交設(shè)計(jì),研究了瓊脂濃度、培養(yǎng)基和氮源對(duì)球等鞭金藻固體平板生長(zhǎng)的影響,并且利用單因素優(yōu)化分析比較了維生素和抗生素對(duì)其藻落形成的影響。結(jié)果表明,瓊脂濃度可影響球等鞭金藻藻落的形成,球等鞭金藻(OA-3011)在0.6-1.0%瓊脂濃度的固體平板生長(zhǎng)最合適,而培養(yǎng)基和氮源對(duì)其影響較小,維生素B1和B12對(duì)于其固體平板生長(zhǎng)不是必需的,抗生素對(duì)其固體培養(yǎng)有明顯抑制作用,建議在球等鞭金藻(OA-3011)固體平板分離、純化過(guò)程中,使用1%的瓊脂平板和不加維生素的F/2培養(yǎng)基。目前我國(guó)水產(chǎn)養(yǎng)殖上使用的大多數(shù)球等鞭金藻品系都來(lái)源于同一株藻Isochrysis galbana O A-3011,經(jīng)過(guò)多年的地理隔離形成了不同的品系,為了揭示地理隔離對(duì)球等鞭金藻表型性狀的影響,從而為球等鞭金藻的選育工作提供依據(jù),我們對(duì)5個(gè)品系的球等鞭金藻(I.galbana FACHB-861,OA3011-QD,OA3011-LK,OA3011-AY和OA3011-LZ)的種屬關(guān)系、生長(zhǎng)和脂肪酸組成進(jìn)行了比較分析。實(shí)驗(yàn)在相同條件下對(duì)5個(gè)品系球等鞭金藻培養(yǎng)6 d,提取基因組DNA并PCR擴(kuò)增測(cè)定18S rDNA基因序列,每天定時(shí)記錄各品系的OD660,繪制生長(zhǎng)曲線,在進(jìn)入穩(wěn)定期后收獲并進(jìn)行生物量的測(cè)定及脂肪酸的氣相色譜分析。18S rDNA基因序列的進(jìn)化分析表明,OA3011-LK、OA3011-AY和OA3011-LZ具有一致的序列,而OA3011-QD因1個(gè)堿基的差異與其它OA3011出現(xiàn)了分離,FACHB-861與OA3011品系親緣關(guān)系較遠(yuǎn);對(duì)生長(zhǎng)和脂肪酸進(jìn)行對(duì)比分析后,結(jié)果顯示,5個(gè)品系的球等鞭金藻在不同的時(shí)間達(dá)到穩(wěn)定期,其中FACHB-861表現(xiàn)出了與其他品系明顯不同的生長(zhǎng)模式。FACHB-861和OA-3011品系的生物量具有顯著差異,OA3011-LK、OA3011-AY和OA3011-LZ之間也有顯著差異(P0.05),但特異生長(zhǎng)率各品系間無(wú)顯著差異(P0.05)。FACHB-861和OA3011-LZ的總脂肪酸(TFA)含量顯著低于OA3011-LK、OA3011-QD和OA3011-AY(P0.05)。FACHB-861的脂肪酸各成分與OA-3011各品系明顯不同。然而,在OA-3011各品系間脂肪酸組成也有明顯差異,這表明球等鞭金藻存在廣泛和頻繁的數(shù)量性狀表型差異。總之,地理隔離造成的這些差異為球等鞭金藻的選育提供了可行性。選擇來(lái)自山東煙臺(tái)(OA3011-YT)以及海南省(OA3011-HN)的球等鞭金藻進(jìn)行膠群相細(xì)胞模擬雜交,OA3011-YT品系進(jìn)行膠群相模擬自交,利用優(yōu)化后的瓊脂平板涂布技術(shù)獲得單克隆并在F/2液體基中培養(yǎng)出F1代個(gè)體,作為模擬自交系F1及模擬雜交系F1,通過(guò)測(cè)定生長(zhǎng)指標(biāo)、脂肪含量和脂肪酸組分篩選出優(yōu)勢(shì)藻株,對(duì)優(yōu)勢(shì)藻株進(jìn)一步進(jìn)行膠群相細(xì)胞雜交、自交,再重復(fù)上述步驟獲得模擬自交系和模擬雜交系的F2代優(yōu)勢(shì)藻株,最后對(duì)F2代藻株進(jìn)行脂肪酸組分的分析。比較F1及F2代藻株脂肪酸組分后發(fā)現(xiàn),通過(guò)定向選育,F1代中DHA和EPA含量較高的藻株在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;模擬雜交系在生長(zhǎng)、脂肪酸含量以及提高DHA、PUFA含量的能力上均優(yōu)于模擬自交系,最終篩選獲得4株DHA含量高并且生長(zhǎng)速度快的藻株,以及2株EPA含量高并且生長(zhǎng)速度快的藻株進(jìn)行保種,未來(lái)將對(duì)其進(jìn)行進(jìn)一步的開(kāi)發(fā)、研究和利用。綜上所述,球等鞭金藻作為一種重要的餌料微藻,具有進(jìn)行高DHA和EPA含量品系的選育的必要性以及可行性,而選擇育種作為一項(xiàng)基礎(chǔ)性科學(xué)研究是很有必要的,不僅可以初步獲得球等鞭金藻的數(shù)量性狀遺傳規(guī)律,增加對(duì)球等鞭金藻遺傳多樣性的了解,而且可以為進(jìn)一步的誘變育種和轉(zhuǎn)基因育種提供優(yōu)良的藻種,優(yōu)化球等鞭金藻的種質(zhì)資源。
[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.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S917.3

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