【作者】 张忠琴；

【导师】 孙颖； 孙大业；

【作者基本信息】 河北师范大学 ， 细胞生物学， 2002， 硕士

【摘要】 我室前期工作表明，百合花粉管质膜上存在类整合素α、β3，并可能通过介导花粉管与雌蕊基质间的相互作用调节花粉管的生长。而要进一步从分子水平上证实类整合素的存在以及研究它的功能，必须克隆其基因。根据动物整合素α、β亚基的胞质域保守序列设计简并引物，利用3′RACE的方法扩增α、β亚基的3′末端序列。实验过程中经过各种尝试与改进之后，一共扩得9个片断，测序分析表明它们均不是目的片段。 在进行以上实验的过程中，有一个克隆含有引物序列，但没有按推定的阅读框阅读，BLAST比对表明它与各物种中的酪蛋白激酶(CK2)α亚基有很高的同源性。酪蛋白激酶CK2是目前发现的普遍存在于真核生物中的丝氨酸／苏氨酸型蛋白激酶，进化上具有高度的保守性并参与了多种生理功能的调控，因而克隆它的基因具有重要意义。通过5′RACE获得了CK2α的全长cDNA。接下来用两对不同的引物进行PCR反应，进一步确定了CK2α编码区序列。百合花粉管CK2αcDNA全长1480bp，含有一个999bp的开放阅读框，编码了333个氨基酸，推测的分子量为39.2KDa。GeneBank注册号为AF517838。它与其它植物中的CK2。基因同源性很高，如与拟南芥的同源性为90％，与玉米的同源性为94％；与动物中的CK2α基因同源性也较高如与人的同源性为76％。 通过使用两对不同引物进行PCR反应还发现在萌发的百合花粉中存在两个CK2α的转录本。它们不同之处主要是在5′非翻译区，一个转录本在5′非翻译区比另一个多62bp，推测此区域可能与CK2α翻译水平上的调控有关。 至今，还没有人在花粉中检测CK2α的存在以及研究它的功能。百合花粉CK2α全长cDNA的克隆，将有助于研究它在花粉中的生理生化功能。更多还原

【Abstract】 Our previous data suggested that the a , p 3 integrin-like proteins be present in lily pollen tube and mediate the interaction between pollen and extracellular matrix of pistil to regulate pollen tube growth. To elucidate further the presense and function of integrin-like protein ,we try to clone 3’ terminus seqence of integrin-like gene from germinated pollen of lily by 3’ RACE using degenerate primers corresponding to the conserved cytoplasmic regions of a and 3 subunits. However, it proved fruitless athough great efforts have been done. We isolated nine amplified DNA fragments.but none of them is related to the integrin gene.Among these amplified DNA fragments,one clone contains primer sequence but does not translate according to the putitive integrin amino acid sequence. It possess high homology with those of CK2(Casein Kinase 2) a subunits from various species. It is now well established that protein Kinase CK2 is a pleiotropic and abiquitous serine or threonine kinase,which is highly conserved during evolution.We obtained the complete cDNA sequence of CK2 a via 5’ RACE. Followed by PCR using two different pairs of primers, we further ascertain the sequence of the coding regions of CK2 a . The complete cDNA sequence of CK2 a from lily pollen contain 999 bp open reading frames, encoding a polypeptide of 333 amino acid and has a predicted molecular weight of 39.2KDa. Sequence analysis of the cDNA showed a high level of homology to the CK2 a from other plant species, for example 90 % homology with Arabidopsis and 94 % homology with maize. It also 76 % homology with human. Moreover, it contains all of the conserved amino acids of CK2 a in various species. The sequence of CK2 a from lily pollen have been deposited in the GeneBank datebase(accession number , AF517838)By PCR, we also proved the persence of two transcripts in germinated lily pollen . The main difference between the two transcripts is lie in the 5’-untranslated region. One is 62bp larger than the other. This region may be jp related to the regulation of protein translation..Anyway, CK2 a cDXA cloning is the first work ever done in plant pollen. It would contribute to understanding the regulation mechanism of pollen tube growth only when more detailed studies of the biochemistry and physiological functions of CK2 had been done in pollen.更多还原