【作者】 黄胜和；

【导师】 黄占景； 沈银柱；

【作者基本信息】 河北师范大学 ， 遗传学， 2005， 硕士

【摘要】 植物耐盐性研究不仅对作物遗传改良有重要意义,而且还是植物基础生物学研究的一个重要组成部分。因此,植物耐盐相关基因的克隆受到人们广泛的关注。近年来,随着分子生物学的迅速发展,科学家们发现并克隆了一些植物耐盐相关基因,但离人们的期望值还相差很远。本研究在本室用cDNA-AFLP得到G09-94号差异cDNA序列的基础上,通过EST比对进行电子克隆,得到一含完整ORF区的cDNA序列,再以此cDNA序列进行Blastn,发现与水稻一未知功能的基因同源性达90%。设计引物,进行PCR,测序结果与电子克隆结果完全一致,证明克隆成功。Northern杂交显示,0%NaCl处理的H8706-34和RH8706-49幼苗、1%NaCl处理的H8706-34和RH8706-49幼苗都表达此基因,但1%NaCl处理的RH8706-49表达量明显增高,因此,将此基因命名为TaSC(Triticum asetivum L. Salt-tolerant Correlative),并作为一新基因被GenBank接受(AY956330)。表达谱基因芯片数据库显示,用1%NaCl处理RH8706-49小麦幼苗,此基因在叶片中为短时间表达下调,长时间表达上调。半定量RT-PCR结果证实,用1%NaCl处理,H8706-34根部TaSC基因表达量先下降,然后缓慢上升到正常量,而RH8706-49中短时间表达下调,长时间表达上调。比对EST库结果表明,TaSC基因在根、叶、种子、幼穗、子房等组织器官中都有表达,而比对Mapped wheat ESTs库结果可以将TaSC基因定位于染色体5BL、5DL、7DS上。将DNA序列翻译成蛋白,用多个生物软件或专业网站预测其性质、结构、定位和功能,结果显示,此蛋白pI值为3.96,分子量为15315.76Da,有4个跨膜区和1个信号肽区,定位在质膜上,与一未知功能的假定膜蛋白同源性为82.2%,与另一未知功能的蛋白家族(UFP220)同源性达79.8%,并且具有豆蔻酰化、酪氨酸硫酸化、酪蛋白激酶Ⅱ磷酸化和蛋白激酶C磷酸化等功能位点。因此推测TaSC基因是组成型表达,而且没有组织器官特异性,同时,可能是植物逆境胁迫反应网络中的一个中下游基因,其表达产物参与了多条信号转导途径,将上游反应信号转导到下游反应元件。以克隆载体为基础,构建了双元表达载体p1300-TS和p1300-TS-GP,并与对照质粒p1300、p1300-GP一起转化农杆菌GV3101,采用浸花法转化Columbia型拟南芥,经潮霉素筛选得到Ap1300、Ap1300-GP、Ap1300-TS和Ap1300-TS-GP转基因拟南芥。更多还原

【Abstract】 It is not only very important to improve crops to research the salt-tolerance in plants, but also is a part of basic researchs. So much attention is paid to the cloning of the salt-tolerant correlative gene in plants. At present, some salt-tolerant correlative gene were cloned along with development of molecule biology, but this is not achieve people’ s desires. A cDNA sequence with whole ORF was obtained according as the G09-94 cDNA fragment of cDNA-AFLP by e-clone and is 90% homology to a rice gene whose fuction is unknowned by blastn. The sequencing result confirms the success of e-clone. Northern blot shows the expression in H 8706-34 treated by 0% NaCl, H 8706-34 treated by 1% NaCl, RH 8706-49 treated by 0% NaCl and RH 8706-49 treated by 1% NaCl, but the quantity of expression in RH 8706-49 treated by 1% NaCl is beyond the others. So the gene is named as TaSC ( Triticum asetivum L Salt-tolerant Correlative) and was registered at GenBank as a new gene with the accession number of AY956330. DNA microarrays for gene expression profiles show that the quantity of expression in RH8706-49 leaves in short time is decreased and the one in long time is increased under 1%NaCl stress. Half-quantity RT-PCR was done. The quantity of expression in H8706-34 roots decrease earlier and are the natural quantity later. The result in RH8706-49 is the same of the DNA microarrays for gene expression profiles. TaSC gene can be expressed in roots, leaves, grains, spike, endosperm and so on according to blast ESTs;and it is assigned in 5BL 、 5DL 、 7DS of wheat chromosomes according to blast Mapped wheat ESTs. The character, structures, location and functions of the TaSC protein were analysed by using many softwares for molecular biology and many professional networks . The protein has four membrane-spanning segments and one signal peptide segment and its pI is 3.96 ; its molecular weight is 15315.76 Da. Furthermore, TaSC protein is localized in the plasma membrane and is 82.2% homology to a predicted membrane protein whose fuction is unknowned and 79.8 % homology to an uncharacterized protein family(UPF0220). It has the site of N-myristoylation, Tyrosine sulfation, Casein kinase II phosphorylation and protein kinase C phosphorylation. So it can be presumed that TaSC gene is a constitute-expressed gene on middle or lower position of the responsed networks under adversity stress in plant and is not tissue-specific. And TaSC protein may play a role in many signal transduction更多还原