【作者】 李雪；

【导师】 包振民； 胡晓丽；

【作者基本信息】 中国海洋大学 ， 生物工程（专业学位）， 2013， 硕士

【摘要】 类胡萝卜素是生物体重要的维生素A原和抗氧化剂，在动物中不能从头合成，只能从食物中获得。虾夷扇贝(Patinopecten yessoensis, Jay,1857)是我国重要的海水养殖生物，其新品种“海大金贝”，富含类胡萝卜素，为研究海洋动物类胡萝卜素吸收、转运及代谢机制提供了模型材料。本研究在虾夷扇贝中克隆了硬脂酰辅酶A去饱和酶基因（Stearoyl-CoA desaturase，SCD），分析并发现了其在“海大金贝”和普通虾夷扇贝中的时空表达差异；进一步利简并基因组高通量测序技术，对虾夷扇贝类胡萝卜素积累进行了全基因组关联分析（Genome-WideAssociation Studies, GWAS），筛查到了与类胡萝卜素积累相关的单核苷酸多态性（single nucleotide polymorphism, SNP）位点，并对相关基因进行了功能注释分析。论文研究为深入解析虾夷扇贝类胡萝卜素积累的分子机制打下了基础，并为富含类胡萝卜素扇贝的选育提供了大量候选遗传位点。1虾夷扇贝PySCD基因克隆及其在“海大金贝”和普通扇贝中的表达差异分析克隆了PySCD基因的cDNA全长序列并获得及基因组序列。PySCD含有4个外显子，3个内含子；cDNA全长2559bp，包含990bp开放阅读框（open readingform，ORF）；编码的氨基酸序列具有SCD蛋白特征结构域：4个跨膜结构域和3个组氨酸结构域。在PySCD基因5’侧翼序列发现了固醇类调节因子（Sterol-regulatory element binding protein, SREBP）结合位点、CCAAT识别位点、视黄醛X受体（retinoic acid X receptor, RXR）结合位点等代谢调控元件；在转录起始位点上游5kb附近发现重复单元37bp、重复次数25左右的小卫星序列。利用real-time qRT-PCR（quantitative reverse transcription PCR）技术检测了PySCD基因在虾夷扇贝胚胎幼虫时期（受精卵、囊胚、原肠、D型幼虫、面盘幼虫）和成体组织（外套膜、鳃丝、性腺、肾、闭壳肌、肝胰腺）的表达水平。该基因在受精卵和面盘幼虫的表达量显著高于其他胚胎幼虫时期，受精卵中的高表达提示PySCD mRNA可能来源于卵细胞，之后表达量下降，至面盘幼虫期显著提高；成体组织中，PySCD基因在肝胰腺、性腺和闭壳肌的表达量较高，其中肝胰腺的表达量显著高于其他组织。比较“海大金贝”和普通虾夷扇贝的PySCD基因时空表达显示：在面盘幼虫期，“海大金贝”PySCD基因表达量显著高于普通虾夷扇贝，可能在该时期“海大金贝”的脂类代谢水平提高，进而促进其类胡萝卜素的积累；在成体组织，“海大金贝”的消化腺、性腺、闭壳肌中PySCD基因表达量显著高于普通虾夷扇贝，前期研究显示，在这些组织中，“海大金贝”的类胡萝卜素含量显著高于普通虾夷扇贝。上述结果暗示，PySCD基因的高表达可能促进了类胡萝卜素在“海大金贝”的积累，且“海大金贝”的类胡萝卜素积累与脂类代谢可能存在一定的相关性。2虾夷扇贝类胡萝卜素积累的全基因组关联分析（GWAS）采用基因组简化技术2b-RAD，对12只“海大金贝”和12只普通虾夷扇贝进行了类胡萝卜素积累的GWAS分析。测序数据总量为6G，共获得172,875,615条reads序列，聚类得到287,909种IIB型内切酶酶切标签，平均测序深度40×以上，包含89221个可分型SNP位点。从以上标签中筛查到60个在“海大金贝”和普通虾夷扇贝间基因型频率差异显著的SNP位点，其中8个位点在另外两个独立的群体中得到验证。利用虾夷扇贝基因组序列，对8个在“海大金贝”和普通虾夷扇贝中基因型频率有差异SNP所在的标签进行了基因注释。获得99条上述SNP附近的基因序列，对这些基因进行Kyoto Encyclopedia of Genes and Genomes (KEGG)代谢通路定位，定位到脂类代谢、信号传导、免疫相关通路。对这些基因进行进一步的功能分析将有助于解析“海大金贝”积累类胡萝卜素的分子机制。更多还原

【Abstract】 Carotenoids which function as retinoid (vitamin A) precursors andantioxidants, are obtained from the diet and cannot be synthesized de novo inanimals. Yesso scallop (Patinopecten yessoensis, Jay,1857) is importantaquaculture species in China. The new variety “Haida Golden Scallop”developed from this species is enriched in carotenoids compared with thenormal ones, providing a good studying model for the mechanism underlyingcarotenoid absorption, transformation and metabolism in animals. In this study,we cloned the Stearoyl-CoA desaturase (SCD) gene from P. yessoensis, and foundthe expression differences of this gene between “Haida Golden Scallop” andnormal P. yessoensis. Further genome-wide association study (GWAS) identifiedSNPs (single nucleotide polymorphisms) which showed significant gentotypedifferences between “Haida Golden Scallop” and normal P. yessoensis. This studywill provide important information to our understanding of the molecularmechanism of carotenoids accumulation in P. yessoensis and candidate geneticloci for scallop breeding aiming at improving carotenoid content.1Cloning of Yesso scallop SCD gene (PySCD) and analysis of the expressiondifference of this gene between normal scallop and “Haida golden scallop”The PySCD gene had4exons and3introns and a full-length cDNA of2559bpcontains a990bp open reading frame (ORF) encoding329amino acids. Thepredicted protein of PySCD had4transmembrane domain and3histidine-boxmotifs which was the characteristic feature of PySCD gene family. Several putativebinding sites for transcription factors, including sterol-regulatory element binding protein (SREBP) binding sites, CCAAT recognition sites and retinoic acidX receptor (RXR) binding sites were present in the5’ flanking region. Meanwhile,a minisatellite DNA with unit length of37bp and repeat number of25was foundat5kp upstream of the transcription initiation site.Expression levels of PySCD during the developmental stages (fertilized eggs,blastulae, gastrulae, D-shaped larvae, and veliger larvae) and in adult tissues(mantle, gill, gonad, kidney, striated muscle, and digestive gland) were analyzedusing real-time qRT-PCR (quantitative reverse transcription PCR). PySCDtranscripts were detected in all of the developmental stages sampled withsignificantly higher expression levels in the newly fertilized eggs and veligerlarvae than in those of other stages. In adult scallops, PySCD were detected in allthe sampled tissues, and digestive gland, gonad and striated muscle exhibitedsignificantly higher expression levels of PySCD than other tissues. A comparisonof PySCD expression showed that significantly more PySCD transcripts weredetected in “Haida golden scallop” than normal scallop in veliger larvae. In adulttissues, significantly higher expression level of PySCD was found in the digestivegland, striated muscle, and gonad of “Haida golden scallop” than that in thecorresponding tissues of normal Yesso scallops. The higher expression level ofPySCD in “Haida golden scallop” than that in ordinary yesso scallop implied thathigh expression of PySCD maight promote the carotenoids accumulation and thecarotenoids accumulation in “Haida golden scallop” maight be related with lipidmetabolism.2Identification of SNPs associated with carotenoids accumulation in Yessoscallop using GWASGWAS was proformed in12“Haida golden scallops” and12normal scallopsusing2b-rad protocol to identify SNPs associated with carotenoids accumulation.A total of6G base pairs and172,875,615reads (40-fold sequencing coverage)were produced by sequencing the2b-rad libraries, and then287,909tags which contained89,221SNPs were obtained. Sixty of these SNPs exhibited significantgenotype frequencies between “Haida golden scallop” and normal yesso scallop,and8of them were further verified in other two different population groups.The8SNPs which showed significantly difference genotype frequenciesbetween “Haida Golden scallop” and normal yesso scallop were furtherannotated based on the genome sequences of Yesso scallop. Nighty nine geneswere found around these SNPs, which were mapped to Kyoto Encyclopedia ofGenes and Genomes (KEGG) pathways including lipid metabolism, signaltransduction and immune system. Further functional analysis of these genescould deepen our understanding of the molecular mechanism underlyingcarotenoid accumulation in “Haida golden scallop”.更多还原