【作者】 胡焕春；

【导师】 张方东；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 在玉米不同自交系基因组间存在着丰富的SNPs（single nucleotidepolymorphisms）和InDels（insertion/deletion polymorphisms）,这为开发SNP分子标记、鉴别不同等位基因型提供了便利。目前已有多种检测SNP的方法,其中等位基因特异PCR（Allele-specific PCR,AS-PCR）是近年来开发的一种基于PCR技术的快速、简便、费用低廉的检测等位基因型的技术,但是因其特异引物3’端的第一个碱基有可能与模板错配从而影响了检测结果的可靠性。为了准确地检测玉米的等位基因型,可以根据玉米基因组丰富的SNPs和InDels设计同时包含多个SNP和InDel位点的等位基因特异PCR引物,这种引物可以克服单个SNP或InDel位点可能存在着错配的缺点来准确地检测玉米等位基因型,并进一步检测等位基因在杂交种中的表达情况。为了验证利用该方法是否可以方便、准确地检测玉米等位基因型,并检测等位基因在杂交种中的表达情况。本课题以一个淹水胁迫后在自交系Mo17和Hz32之间存在着表达差异的基因ZmZF为例来开展研究,旨在建立一种快捷准确的鉴别等位基因型以及检测等位基因在杂交种中表达的方法。研究结果如下:（1）根据已经公布的ZmZF基因cDNA序列设计的引物在两个亲本材料受水淹处理后的cDNA中扩增出了大部分ZmZF基因cDNA中的3’UTR和一小部分的编码区序列,在这两个部分序列间共包含了3个SNP位点和3个InDel位点。（2）根据等位基因特异PCR方法的原理,把SNP和InDel位点分别设计在正反分型引物中,然后用这些引物进行PCR,通过琼脂糖凝胶电泳检测PCR产物来鉴别ZmZF-Mo17和ZmZF-Hz32基因型的cDNA模板。通过比较,初步筛选到了包含SNP位点的正向引物Pa、Pg和包含InDel位点的反向引物Ph、Pm能够准确地区分出这两种等位基因型。进一步地对这两对分型引物在不同Mg²⁺和dNTP浓度下的稳定性作了检测,发现这两对分型引物在一定浓度范围内都能稳定地区分这两种等位基因型。另外用实时荧光定量PCR方法检测也能够准确地鉴别出这两种等位基因型。这些结果说明结合了SNP和InDel位点的分型引物比只包含一个SNP位点的分型引物检测的准确性强,利用该引物可以快捷准确地进行玉米基因分型和等位基因在杂交种中的表达检测。（3）用分型引物做半定量RT-PCR检测ZmZF等位基因在杂交种以及两个亲本根系中的表达发现:在杂交种中与在亲本中一样,与0h的对照相比,在淹水胁迫后,ZmZF-Mo17表达量明显升高,而ZmZF-Hz32表达量比较稳定,并且比ZmZF-Mo17低。水淹8h后的不同材料的叶片用半定量RT-PCR检测发现这两个等位基因在叶片中的表达和根系中一样也存在着差异。用分型引物做实时荧光定量PCR检测后发现:两个等位基因在杂交种中的表达与在两亲本中一样存在着差异,但淹水胁迫24h后,在杂交种中ZmZF-Mo17的表达量较16h少。这些结果表明:在淹水胁迫下,玉米杂交种中也存在着等位基因表达的差异,这种差异性可能是由这两个基因Cis结构之间的差异引起的。更多还原

【Abstract】 The maize genome have much abundance of single nucleotide polymorphisms （SNPs） and insertion/deletion polymorphisms（InDels）, so it’s very useful for developing molecular markers and discriminating alleles. At present, there are many procedures to assay SNPs and InDels. Allele-specific PCR is a simple, low-cost technique to discriminate the alleles quickly, but because of the mismatch between the specific primer and template made by the SNP site and 3’-end of primer, the result maybe not so accurate. For mazie allele discrimination, we can use the abundance of these polymorphisms when its be transformed to allele-specific PCR primers containing both of them, so this type of allele specific primer may reslove the problem of mismatched amplification made by single SNP or InDel site.In order to test whether this is a simple method to discriminate the alleles reliably and detect the alleles expression variation, the gene of ZmZF（ Zea mays zinc finger-like protein） which is presumed to be a transcription factor gene induced by flooding stress and differently expressed in roots between two inbreds of Mo17 and Hz32 was chosen. In this study, we are want to establish a simple and accurate technique to discriminate alleles and detect the alleles expression variation in maize hybrid. The main results were:（1）Using a pair of primers designed according to the cDNA sequence published in the NCBI, we have amplified most of the gene’s 3’UTR and small parts of the encoding sequence of the two alleles of ZmZF-Mo17 and ZmZF-Hz32. After sequencing,we have found three SNP sites and three InDel sites between the two alleles.（2）According to the AS-PCR primer design principle, we have designed several paris of genotyping primers which containing a SNP sites in the forward primer and InDel sites in the reverse primer. After tested, the allele specific primer of Pa、Ph（matched to the ZmZF-Hz32 for A type） and Pg、Pm（matched to the ZmZF-Mo17 for G type） were found. Further more, we have tested the stability of the genotyping primers in different Mg²⁺ and dNTP concentration. The results showed that the allele specific primers discriminated the alleles from parental inbred Mo17 and Hz32 specificly and perfectly. The allele specific primers also can be used in Real-Time fluorescence PCR method. （3）Using the semi-quantitative RT-PCR by the genotyping primers, the expression level of the alleles in the roots of the hybrid of F₁ and the parents were tested.The results showed that in the hybrid, the expression lever of the ZmZF-Hz32 allele has no obvious difference during the time of waterlogging ,but the allele of ZmZF-Mo17 was increased from the time on, and the expression lever of the parental allele of Hz32 was much more lower than the allele from the parent of Mo17. Using semi-quantitative RT-PCR to test the expression level of the leves also got the same results as the roots at Oh and 8h. Using the Real-Time fluorescence quantitative PCR method, we still found the expression variation in the hybrid and the two parental inbreds, but in the hybrid, at the 24h, the expression lever of the allele from the parent of Mo17 was lower than the 16h. These results suggested that the differential expression lever between alleles was common in the hybrid by flooding stress, and the allelic expression variation maybe caused by the difference of the alleles Cis factors.更多还原