原壳小球藻中玉米黄质环氧化酶基因的克隆和分析
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摘要
类胡萝卜素(carotenoids)是存在于生物体(包括植物、光合及非光合细菌、藻类和真菌)内的一类天然色素的总称,具有重要的生理功能,如抗氧化、抵御癌症、黄斑眼病、心血管疾病等。利用微藻生产类胡萝卜素是近几年来研究的热点,原壳小球藻中叶黄素含量较高,作为极具潜力的叶黄素源而备受关注。通过对原壳小球藻类胡萝卜素代谢途径的改造来提高它的合成量具有一定的工业化应用前景。
本研究以原壳小球藻为实验材料,围绕其生物量的快速测定方法、植物激素对其生长的影响、以及玉米黄质环氧化酶(ZEP)基因cDNA的克隆以及生物信息学分析展开研究和讨论,为进一步揭示小球藻的生长条件、测定方法及其类胡萝卜素合成的分子代谢机制奠定基础。主要的研究内容及结果如下:
1、为了满足后期工业化生产中藻细胞干重测量的精准要求,对小球藻的细胞密度与吸光值、干重与吸光值之间的线性关系进行了分析,得到一条根据光密度值获得原壳小球藻干重的便捷方法。
2、通过不同植物激素处理原壳小球藻,找到了加快小球藻生长速率的方法。研究发现,0.5mg/L吲哚丁酸(IBA)的处理效果较佳,而脱落酸(ABA)对小球藻的生长促进作用不明显。
3、根据NCBI数据库中zep基因的保守序列设计简并引物,利用PCR扩增获得了zep基因的核心片段,其长度为972 bp,将此片段的核苷酸序列与衣藻(Chlamydomonas reinhardtii)的zep基因序列相比,其同源性可达77%。这表明,所扩增的片段为zep基因的保守序列。采用cDNA末端快速扩增技术(RACE)获得了zep基因的cDNA全长序列,其长度为2616 bp。在NCBI上进行Blast比对,发现该序列与衣藻有80%的相似性。该序列包含一个完整的开放阅读框(ORF),长度为1680 bp,编码559个氨基酸。利用各种生物信息软件对ZEP蛋白的相关信息进行了分析。zep基因的分子进化树显示,在藻类中小球藻与衣藻的亲缘关系较近。
4、为研究zep基因在逆境下以及在脱落酸(ABA)合成中的作用,本实验用不同浓度的盐及ABA处理原壳小球藻,通过荧光定量PCR方法检测zep基因表达量的变化,结果发现,在盐处理下,zep基因的表达量明显上调,而在ABA处理下该基因下调,由此分析发现了zep基因在逆境响应中具有抵御作用以及zep基因在ABA合成途径中具有关键作用。
Carotenoids are a category of natural pigment, which exist in many organisms, such as plants, photosynthetic or non-photosynthetic bacteria, alga and fungi. They have important physiological function of anti-oxidation, prevention of cancer, macular eye disease and cardiovascular disease. Chlorella has aroused great attention due to its high cellular carotenoids content, especially Chlorella protothecoides. It can produce a large amount of lutein, which is a potential resource for lutein production. It will be a commercial application prospect to enhance carotenoids biosynthesis and production with C. protothecoides by transforming the carotenoids biosynthesis pathway.
Using the green alga, C. protothecoides CS-41, this research was focused on the development methods of biomass determination, stimulation effect on algal growth with plant phytohormone; cloning and analysis of zeaxanthin epoxidase gene. The main research contents and results are as follows:
1. For the sake of satisfying the requirements for the precise determination of cell dry weight for industrial application purpose, a good linear relationship between the absorbance and microscopic counting/ dry weight was established, providing a convenient method of biomass determination by optical density measurement.
2. The growth rate of C. protothecoides CS-41 was increased through the treatments with phytohormones of appropriate concentration. 0.5mg/L IBA was the most effective. On the contrary, ABA treatment had little effect on the growth.
3. The degenerate primer sets were designed with the conservative sequence of zep gene found in NCBI database and a core fragments of 972 bp was obtained. RACE (rapid-amplification of cDNA ends) essay and RT-PCR were used to isolate the full-length cDNA of zep from C. protothecoides CS-41. A cDNA of 2616 bp was cloned with an open reading frame of 1680 bp, which encoded a putative ZEP from C. protothecoides CS-41. The phylogenetic tree established with MEGE 4.1 software revealed that the genetic relationship between C. protothecoides CS-41 and Chlamydomonas reinhardtii is close.
4. With the aim of exploring the functions of zep gene under the stress-environments and the function in ABA biosynthesis, fluorescence quantitative PCR was performed to detect the expression of zep gene under NaCl and ABA treatments. The results revealed that zep gene expression was up-regulated under salt stress treatment,which clarified that zep gene played an important role in stress response. Under ABA treatment, zep gene was down-regulated, which revealed that zep gene took part in ABA biosynthesis and had an important function.
本研究以原壳小球藻为实验材料,围绕其生物量的快速测定方法、植物激素对其生长的影响、以及玉米黄质环氧化酶(ZEP)基因cDNA的克隆以及生物信息学分析展开研究和讨论,为进一步揭示小球藻的生长条件、测定方法及其类胡萝卜素合成的分子代谢机制奠定基础。主要的研究内容及结果如下:
1、为了满足后期工业化生产中藻细胞干重测量的精准要求,对小球藻的细胞密度与吸光值、干重与吸光值之间的线性关系进行了分析,得到一条根据光密度值获得原壳小球藻干重的便捷方法。
2、通过不同植物激素处理原壳小球藻,找到了加快小球藻生长速率的方法。研究发现,0.5mg/L吲哚丁酸(IBA)的处理效果较佳,而脱落酸(ABA)对小球藻的生长促进作用不明显。
3、根据NCBI数据库中zep基因的保守序列设计简并引物,利用PCR扩增获得了zep基因的核心片段,其长度为972 bp,将此片段的核苷酸序列与衣藻(Chlamydomonas reinhardtii)的zep基因序列相比,其同源性可达77%。这表明,所扩增的片段为zep基因的保守序列。采用cDNA末端快速扩增技术(RACE)获得了zep基因的cDNA全长序列,其长度为2616 bp。在NCBI上进行Blast比对,发现该序列与衣藻有80%的相似性。该序列包含一个完整的开放阅读框(ORF),长度为1680 bp,编码559个氨基酸。利用各种生物信息软件对ZEP蛋白的相关信息进行了分析。zep基因的分子进化树显示,在藻类中小球藻与衣藻的亲缘关系较近。
4、为研究zep基因在逆境下以及在脱落酸(ABA)合成中的作用,本实验用不同浓度的盐及ABA处理原壳小球藻,通过荧光定量PCR方法检测zep基因表达量的变化,结果发现,在盐处理下,zep基因的表达量明显上调,而在ABA处理下该基因下调,由此分析发现了zep基因在逆境响应中具有抵御作用以及zep基因在ABA合成途径中具有关键作用。
Carotenoids are a category of natural pigment, which exist in many organisms, such as plants, photosynthetic or non-photosynthetic bacteria, alga and fungi. They have important physiological function of anti-oxidation, prevention of cancer, macular eye disease and cardiovascular disease. Chlorella has aroused great attention due to its high cellular carotenoids content, especially Chlorella protothecoides. It can produce a large amount of lutein, which is a potential resource for lutein production. It will be a commercial application prospect to enhance carotenoids biosynthesis and production with C. protothecoides by transforming the carotenoids biosynthesis pathway.
Using the green alga, C. protothecoides CS-41, this research was focused on the development methods of biomass determination, stimulation effect on algal growth with plant phytohormone; cloning and analysis of zeaxanthin epoxidase gene. The main research contents and results are as follows:
1. For the sake of satisfying the requirements for the precise determination of cell dry weight for industrial application purpose, a good linear relationship between the absorbance and microscopic counting/ dry weight was established, providing a convenient method of biomass determination by optical density measurement.
2. The growth rate of C. protothecoides CS-41 was increased through the treatments with phytohormones of appropriate concentration. 0.5mg/L IBA was the most effective. On the contrary, ABA treatment had little effect on the growth.
3. The degenerate primer sets were designed with the conservative sequence of zep gene found in NCBI database and a core fragments of 972 bp was obtained. RACE (rapid-amplification of cDNA ends) essay and RT-PCR were used to isolate the full-length cDNA of zep from C. protothecoides CS-41. A cDNA of 2616 bp was cloned with an open reading frame of 1680 bp, which encoded a putative ZEP from C. protothecoides CS-41. The phylogenetic tree established with MEGE 4.1 software revealed that the genetic relationship between C. protothecoides CS-41 and Chlamydomonas reinhardtii is close.
4. With the aim of exploring the functions of zep gene under the stress-environments and the function in ABA biosynthesis, fluorescence quantitative PCR was performed to detect the expression of zep gene under NaCl and ABA treatments. The results revealed that zep gene expression was up-regulated under salt stress treatment,which clarified that zep gene played an important role in stress response. Under ABA treatment, zep gene was down-regulated, which revealed that zep gene took part in ABA biosynthesis and had an important function.
引文
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