甘薯淀粉合成相关基因的克隆与分子标记开发

发布时间：2018-01-01 17:11

本文关键词：甘薯淀粉合成相关基因的克隆与分子标记开发　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：甘薯(Ipomoea batatas(L.)Lam)是重要的块根类粮食作物,生物产量和淀粉产量高,淀粉是衡量甘薯品质的一项重要指标。高淀粉型甘薯育种一直是国内外甘薯育种的主要目标。甘薯是六倍体作物,自交不亲和、遗传背景复杂,甘薯EST信息和核酸序列相对较少,其分子生物学研究落后于玉米、小麦等作物。目前在甘薯淀粉含量方面的遗传研究多停留在QTL定位水平,鉴定到的分子标记与主效QTL距离较远并且标记检测程序复杂,不利于其在育种工作中的应用。本研究以淀粉含量不同的甘薯为材料,通过同源克隆方法从甘薯中克隆淀粉合成酶基因,开发与淀粉含量相关的分子标记,并在我国的甘薯常规品种中进行验证,有利于应用分子标记对甘薯淀粉含量的改良,主要结果如下:(1)利用同源克隆技术从甘薯中分离了淀粉合成相关酶基因IbSSS cDNA序列,又从NCBI数据库中获得IbGBSSI、IbISA、IbAGPase、IbSBEⅠ、IbSBEⅡcDNA序列,通过生物信息学方法分析甘薯淀粉合成相关酶基因的核苷酸序列、一级结构、二级结构、三级结构、相对分子质量、等电点、功能结构域、等信息。通过同源克隆,我们得到IbSSS编码区全长1974bp,编码657个氨基酸。IbSSS蛋白属于Glycosyltransferase_GTB_type超家族,转运肽预测结果显示IbSSS蛋白定位于叶绿体中,蛋白同源比对结果显示IbSSS氨基酸序列与马铃薯、番茄、木薯同源性分别为72%、71%和70%。(2)根据已有的cDNA序列,在高淀粉漯徐薯8号和低淀粉的郑薯20中克隆IbGBSSI、IbAGPase四个大亚基,两个小亚基的基因组序列以及IbSSS的基因组序列,基因结构分析表明IbAGPaseLS1,IbAGPaseLS2,lbAGPaseLS3,IbAGPaseLS4都含含有13个内含子,IbAGPaseSS1,IbAGPaseSS2都含含有8个内含子。IbAGPase四个大亚基基因的内含子数目比较一致,两个小亚基基因的内含子数目一致。IbGBSSI含有12个内含子,IbSSS含有14个内含子。(3)利用qRT-PCR技术分析这些基因在块根膨大不同时期的表达水平,结果表明,在块根膨大的初期,IbSBEⅠ和IbSBEⅡ在两个品种中的转录本水平持续增高。IbSBEⅠ在高淀粉的漯徐薯8号中表达量高,而IbSBEⅡ在低淀粉的郑薯20中表达量高。在郑薯20中,IbIS4的表达量在块根膨大的初期逐步升高,在前中期达到最高,而在漯徐薯8号中,IbISA在前期,中期的表达量保持平稳,在后期表达量却明显提升,而且发现IbIS4在郑薯20中的表达量明显高于漯徐薯8号。IbGBSSI的表达量变化与IbSBEⅠ,IbSBEⅡ,IbISA类似,推测它们可能在块根膨大的初期协同作用促进淀粉的快速合成。总之,这四个基因在漯徐薯8号和郑薯20两个品种中表达模式有显著的差异。(4)根据从淀粉含量有明显差异的漯徐薯8号和郑薯20中克隆获得的8个基因组序列,通过分析每一个基因在高淀粉和低淀粉品种的序列差异,直接开发与功能相关的SNP和Indel标记,但是这部分结果不理想,并没有验证出与功能相关的SNP位点。(5)根据高低淀粉池转录组数据开发SNP分子标记,选取了26个SNP位点和Indel位点,并在两个亲本和F1代的高、低淀粉池中进行验证,初步分析得到两个SNP位点和1个Indel位点可能与淀粉含量有关。
[Abstract]:Sweet potato (Ipomoea batatas (L.) Lam) is an important food crop of the tuber yield and starch, high yield, starch is an important indicator to measure the quality of sweet potato. The high starch Sweetpotato Breeding have been one of the main aims of sweet potato breeding. Sweet potato is six times body crops, self incompatibility, complex genetic background sweet potato, EST and nucleic acid sequence information is relatively small, the molecular biology research behind corn, wheat and other crops. The genetic research on sweet potato starch content stays in the QTL level, and the identification of molecular markers to the major QTL distance and mark detection procedure is complex and not conducive to its application in plant breeding the sweet potato starch content. In this study, with different materials, through the method of homologous cloning cloning starch synthase gene from sweet potato, the development of molecular markers associated with starch content in sweet potato, and often in China Verify compliance varieties, is conducive to the application of molecular marker on the improvement of starch content of sweet potato, the main results are as follows: (1) by homology cloning from starch synthesis related enzyme gene IbSSS sequence of cDNA from sweet potato, and obtained from the NCBI database IbGBSSI, IbISA, IbAGPase, IbSBE I and IbSBE II cDNA sequence analysis. The nucleotide sequence of potato starch synthesis related enzyme gene by bioinformatics methods, primary structure, secondary structure of two, three level structure, relative molecular mass, isoelectric point, functional domains, and other information. By homologous cloning, we obtained the full-length 1974bp IbSSS encoding region, 657 amino acids encoding.IbSSS protein belongs to Glycosyltransferase_GTB_type superfamily the prediction results show that, the transit peptide of IbSSS protein localized in chloroplast protein homology, the results showed that IbSSS amino acid sequence with potato, tomato, cassava homology were 72%, 71% And 70%. (2) according to the sequence of cDNA in the Luohe Xushu No. 8 high starch and low starch Zhengshu 20 clone IbGBSSI, IbAGPase four subunit, two genomic sequences of the small subunit and IbSSS genomic sequence analysis showed that IbAGPaseLS1, IbAGPaseLS2, lbAGPaseLS3 gene structure, containing IbAGPaseLS4 13 introns, IbAGPaseSS1, IbAGPaseSS2 with the number of.IbAGPase containing 8 introns and four subunit gene intron consensus, intron number.IbGBSSI two small subunit gene contains 12 introns, IbSSS contains 14 introns. (3) analysis showed that the gene expression level in the root enlargement in different periods, the use of qRT-PCR technology, in the early stage of tuber enlargement, the transcription of IbSBE I and IbSBE II in two varieties in the level of.IbSBE increased continuously in high starch Luohe Xushu No. 8 high expression, and IbSBE II In the low starch Zhengshu 20 high expression. In Zhengshu 20, the early expression of IbIS4 in root enlargement increased gradually, reached the highest in the middle, and in Luohe Xushu No. 8, IbISA in the early, mid expression remained stable, but the expression in the later improved significantly. And found that the expression changes and the expression of IbIS4 in IbSBE I, Zhengshu 20 was significantly higher than that in Luohe Xushu No. 8.IbGBSSI IbSBE II, similar to IbISA, that they may be rapid synthesis of synergistic effect in the early stage of tuber enlargement, promote starch. In short, these four genes have significant differences in expression patterns in Luohe Xushu No. 8 and Zhengshu 20 two varieties. (4) according to the 8 genome sequences from the starch content showed significant differences in Luohe Xushu 8 and Zhengshu 20 cloned, by analyzing the sequence difference of each gene in high and low starch varieties, direct development and function SN P and Indel markers, but the results are not satisfactory, and no validation of SNP loci and related functions. (5) according to the level of starch pool transcriptome data developed SNP markers, selected 26 SNP sites and Indel sites, and in two of parents and F1 generation, verify the low starch pool in a preliminary analysis of two SNP loci and 1 Indel loci may be associated with starch content.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S531;Q943.2

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