【作者】 李铁松；

【导师】 王长海；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 对海带(Laminaria japonica Aresch.)中的吲哚乙酸和脱落酸进行分离纯化,优化条件,利用反相高效液相色谱(RP-HPLC)方法对它们进行定性和定量研究,利用质谱(APESI-MS)方法对海带吲哚乙酸和脱落酸进行了鉴定。在此基础上,利用RP-HPLC方法,测定吲哚乙酸和脱落酸在成熟海带的不同藻体部位中及不同生长时期的海带中的含量,详细研究了它们在成熟海带中的分布及其含量与海带不同生长时期的关系,并从生长调节作用、代谢物质含量及氧化损伤的保护作用等方面,研究了海带吲哚乙酸和脱落酸对海洋微藻的生物学效应。获得海带吲哚乙酸的最适分离纯化方法为:避光组织匀浆,匀浆液用超声波进行细胞破碎,90%甲醇浸提,乙醚萃取,硅胶脱色,通过Sephadex LH 20柱层析和高效液相色谱方法进行纯化。海带脱落酸的最适分离纯化方法为:组织匀浆,超声波破碎细胞,80%甲醇浸提,乙酸乙酯萃取,硅胶脱色纯化,通过Sephadex LH 20柱层析和高效液相色谱方法进一步纯化。在此基础上,通过RP-HPLC方法的定性分析和APESI-MS方法的分析结果确定分离目标产物为吲哚乙酸和脱落酸。RP-HPLC方法的定量分析结果表明海带吲哚乙酸的产量为65～95μg/kg·FW,海带脱落酸的产量为65～70μg/kg·FW,该方法操作简便,准确,重现性好。研究表明,成熟海带的不同部位对于吲哚乙酸含量的影响显著(0.01＜p＜0.05),成熟海带下部的吲哚乙酸含量最高,其次为中部和上部;而成熟海带的不同部位对于脱落酸含量的影响并不显著(p＞0.05)。海带的不同生长时期对全海带中吲哚乙酸和脱落酸的含量影响均极显著(p＜0.01),其中吲哚乙酸在海带夹苗初期的幼嫩海带中含量最高,随着海带成熟,其含量逐渐减少,而脱落酸含量却逐渐增加,在成熟期的海带中脱落酸的含量最高。研究表明,在0～100μmol/L浓度范围内,海带吲哚乙酸对小球藻、盐藻和紫球藻的生长均有促进作用;而角毛藻在外加海带吲哚乙酸的浓度≥75μmol/L时,细胞生长被抑制。在0～200μmol/L浓度范围内,海带脱落酸对小球藻、盐藻、球等鞭金藻和紫球藻四种海洋微藻的生长均表现出抑制作用,脱落酸浓度对四种海洋微藻的生长影响均显著(0.01＜p＜0.05),浓度越大,脱落酸对四种海洋微藻生长的抑制作用越明显。四种海洋微藻中,球等鞭金藻对海带脱落酸最为敏感,其次依次是紫球藻、小球藻和盐藻。在实验浓度范围内,海带吲哚乙酸对于海洋微藻胞内可溶性糖含量的影响均不显著,对于盐藻和角毛藻的胞内可溶性蛋白和叶绿素含量影响均不显著;对于小球藻胞内可溶性蛋白和叶绿素的含量影响显著(0.01＜p＜0.05),对紫球藻胞内可溶性蛋白含量的影响显著(0.01＜p＜0.05),对叶绿素合成影响不显著(p＞0.05)。海带脱落酸对紫球藻胞内可溶性蛋白及胞内可溶性糖含量影响均极显著(0.001＜p＜0.005),对叶绿素含量影响不显著(p＞0.05);对小球藻胞内可溶性蛋白含量影响显著(0.01＜p＜0.05),对胞内可溶性糖和叶绿素含量影响不显著(p＞0.05);对盐藻和球等边金藻胞内可溶性蛋白及胞内可溶性糖含量影响均不显著(p＞0.05),而对叶绿素含量影响均极显著(p＜0.005)。研究表明,海带脱落酸对小球藻、盐藻、球等鞭金藻和紫球藻四种海洋微藻因除草剂百草枯所引起的细胞氧化损伤均具有保护作用,其中海带脱落酸对盐藻细胞氧化损伤的保护作用最为明显。更多还原

【Abstract】 A study is planned to optimize the isolution and purification conditions on auxin indole-3-acetic acid(IAA)and abscisic acid(ABA),to establish their identification and quantification of tAA and ABA in the kelp(Laminariajaponica Aresch.)extracts,on the base of that,to investigate their distribution in the different parts of the mature kelp and the changes of their content in the kelp with different growth,and to research on their bioactivities on the marine microalgae.Conditions on isolation and purification of auxin IAA in the kelp extracts were optimized and the optimal process was established including cell burst by ultrasonic, extraction with 90%methanol and ether,decoloration with silica gel,and purification by Sephadex LH 20 and Reverse-phase high performance liquid chromatography(RP-HPLC). The optimized conditions on isolation and purification of ABA in the kelp extracts were investigated and the optimal process was also established including cell burst by ultrasonic, extraction with 80%methanol and ethyl acetate,decoloration with silica gel,and purification by Sephadex LH 20 and RP-HPLC.The identification of IAA and ABA was based on co-chromatography and comparative chromatography with the standard,analysis of UV spectra,and atmospheric pressure electrospray mass spectrometry(APESI-MS).Evidence is provided for IAA of 65～95/μg/Kg·fresh weight and ABA of 65～70μg/Kg·fresh weight in the extracted samples by the method of RP-HPLC.IAA and ABA are isolated by silica gel, Sephadex LH 20 and HPLC.The processes lay the foundation for further study on their bioactivities with convenience,quickness,accurateness and better fidelity.The results show that the distribution of auxin IAA is not equal in the different parts of the mature kelp and the effect of the different parts in the mature kelp on the content of auxin IAA is statistically marked(0.01＜p＜0.05).The content of auxin IAA in the bottom of the mature kelp is highest,secondly in the middle and fewest in the top.However,the distribution of ABA is basically equal in the different parts of the mature kelp.And the effect of the mature kelp with different parts on the content of ABA is not marked(p＞0.05).The results show that the effect of the kelp with different growth on the content of auxin IAA andABA is statistically marked(p＜0.01).The content of auxin IAA is highest in the childish kelp with the initial stage when clamped in rafts,and along with growing up,the content of auxin IAA gradually decreases.But the content of ABA gradually increases in the kelp along with growing up,and the content of ABA is highest in the mature kelp.The research shows that exogenously added IAA in the kelp enhanced the growth of Chlorella sp.,Dunaliella salina and Porphyridium Cruentum except Chaetoceros muelleri.. IAA in the kelp significantly inhibited the accumulation of cellular dissolvable proteins in Chlorella sp.(0.01＜p＜0.05)and Porphyridium Cruentum(0.01＜p＜0.05).And it had a very significant effect on the chlorophyll biosynthesis of Chlorella sp.(0.0001＜p＜0.0005).But it was not obvious to regulate biosynthesis of cellular polysaccharide in the four marine microalgae(p＞0.05).The research shows that the growth of Chlorella sp.,Dunaliella salina,Isochrysis galbana and Porphyridium Cruentum is all inhibited in the treatments with ABA in the extracted samples.And the results are significantly different that the effects of ABA in the extracted samples with different concentrations on their growth(0.01＜p＜0.05)and the inhibition becomes distincter and distincter in the treatments with the increasement of ABA in the extracted samples.In the four marine,microalgae,Isochrysis galbana is best impressible for ABA in the extracted samples,and secondly Porphyridium Cruentum,Chlorella sp.and Dunaliella salina.The result shows that,for Porphyridium Cruentum,it is significantly different that the effects of ABA in the extracted samples with different concentrations on the content of cellular solvable proteins and Cellular dissolvable saccharide(0.001＜p＜0.005), however,there is not significantly different that the effects of ABA in the extracted samples with different concentrations on the content of chlorophyll(p＞0.05);for Chlorella sp.,there is significantly different that the effects of ABA in the extracted samples with different concentrations on cellular solvable proteins(0.01＜p＜0.05),but it is not significantly different that the effects of ABA in the extracted samples with different concentrations on the content of cellular dissolvable saccharide and chlorophyll(p＞0.05);for both Dunaliella salina and lsochrysis galbana,there is not significantly different that the effects of ABA in the extracted samples with different concentrations on cellular solvable proteins and cellular dissolvable saccharide(p＞0.05),but it is chlorophyll that the effects of ABA in the extracted samples with different concentrations on the content in both the two algae are significantly different, respectively Dunaliella salina(0.0001＜p＜0.0005)and Isochrysis galbana(0.001＜p＜0.005). In addition,ABA in the extracted samples can protect the four marine microalgae cells from oxidantion stress by the paraquat.And the protection of ABA in the extracted samples is most marked for Dunalieila salina in the four marine algae.更多还原