【作者】 单曙光；

【导师】 程宪国；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 锌指蛋白(zinc finger protein)是一类具有“指状”结构域的转录因子,主要功能是调控基因的表达,锌指蛋白种类较多,但以C2H2型最多。C2H2型锌指蛋白基因最早在非洲爪蟾中发现,植物中发现的第一个C2H2型锌指蛋白基因是在矮牵牛中分离得到的ZPT2-1,之后陆续在矮牵牛、拟南芥、棉花、大豆、水稻等植物中相继分离到了该类型的锌指蛋白基因,并发现在植物中都含有QALGGH高度保守序列,而动物锌指蛋白中不存在。已有的研究发现C2H2型锌指蛋白主要参与植物的生长发育以及调控植物的抗逆性。本研究的GmC2H2转录因子基因是依据GenBank中大豆EST数据库的保守序列,设计特异性的引物,利用PCR、RT-PCR等技术获得的一个cDNA开放阅读框序列,它是大豆中一个编码经典C2H2型锌指蛋白的基因,并在Genbank中注册,其登陆号为:DQ055134。该基因全长765 bp,开放阅读框长度为516 bp,其编码172个氨基酸的锌指蛋白,分子量约为19KD。在本实验室前期研究的基础上,将GmC2H2基因的全长编码序列分别构建到16318hGFP亚细胞定位载体上,将16318hGFP -GmC2H2转化原生质体进行亚细胞定位分析得知,GmC2H2锌指蛋白在细胞核中表达。将GmC2H2基因的全长编码序列构建到pCAMBIA1304植物表达载体上,借助优化的floral-dip法转化模式植物拟南芥,利用潮霉素Hygromycine(45-50mg·L-1)抗性筛选,经PCR扩增检测,获得19株转基因阳性苗。通过孟德尔定律筛选分离比接近3:1的株系,外源基因可能为单拷贝插入的株系,已得到3个纯合株系,分别为株系1、株系4、株系7。经GUS组织染色分析,在携带目的基因的pCAMBIA1304空载体的拟南芥幼苗中,GUS基因在整个植株体内表达,而在携带目的基因GmC2H2的转基因拟南芥中,表达部位集中在根部。将GmC2H2基因的突变体MZF1、MZF2、MZF4、MZF8、MZF10序列分别构建到pCAMBIA1304植物表达载体上,利用同样方法转化拟南芥获得纯合系转基因拟南芥,用作后续该GmC2H2转录因子各元件的功能研究。为进一步验证GmC2H2的功能,对野生型和转基因型拟南芥植株进行脱落酸(ABA,200μmol·L-1)处理和低温(1°C)、高盐(NaCl,250mmol·L-1)胁迫处理。研究表明,在ABA处理和低温、盐胁迫处理后,转基因植株的长势明显好于野生型植株;ABA处理和低温、盐胁迫导致野生型和转基因型拟南芥植株丙二醛含量都升高,但野生型植株中丙二醛含量增加量更为显著;转基因拟南芥中可溶性糖和脯氨酸的含量显著高于野生型;转基因植株的细胞损伤率要低于野生型拟南芥。推测可能是目的基因GmC2H2的插入导致了转基因拟南芥植株的抗逆性明显高于野生型拟南芥。经ABA处理和低温、盐胁迫后,对转基因拟南芥与野生型植株进行real-time qPCR分析,结果表明,胁迫处理后的转基因拟南芥中的目的基因GmC2H2的表达量,无论在根还是叶中,都明显高于未处理的转基因拟南芥。在低温和ABA处理条件下,携带有目的基因GmC2H2的拟南芥叶和根中,抗冻基因cor6.6的表达量要高于野生型拟南芥植株。在低温处理条件下,转基因拟南芥叶中EPSPS基因的表达量要高于野生型拟南芥,而根中则表现不明显;而在ABA处理条件下,转基因拟南芥无论是根还是叶中,EPSPS基因的表达量都要高于野生型拟南芥。更多还原

【Abstract】 Zinc finger protein is a kind of transcription factor that has a "finger" structural domain, and it can regulate the gene expression. There are various types of zinc finger proteins, but most of them belong to the C2H2-type. C2H2 type zinc finger protein was first discovered in Xenopus tropicalis, and a C2H2 zinc finger protein gene named ZPT2-1 which was isolated from the petunia was first found in plants, and the gene of this type zinc finger protein have gradually been isolated from the petunia, Arabidopsis thaliana, cotton, soybeans, rice and other plants. And a highly conserved sequence of QALGGH was found in plantkingdom, while it did not exist in zinc finger proteins of animal. Previous studies have found that C2H2 zinc finger protein mainly involved in plant growth and development and regulation of plant stress resistance.In this study, based on the conserved sequence of soybean EST database in GenBank, the GmC2H2 transcription factor gene is isolated from soybean by PCR and RT-PCR techniques using specific primers, and it contains a cDNA open reading frame encoding classic C2H2 type zinc finger protein, and the accession number in Genbank is DQ055134. The full length of this gene is 765 bp with an open reading frame of 516 bp length which encodes 172 amino acids with 19 kD of molecular weight.On the basis of previous studies in this laboratory, the full-length GmC2H2 gene coding sequences were constructed to 16318hGFP vector for subcellular localization, then 16318hGFP-GmC2H2 was transformed to protoplast, finally analyzed the subcellular localization. Data showed that the GmC2H2 zinc finger protein expressed in the nucleus. The full-length GmC2H2 cDNA sequence was constructed to the plant expression vector of pCAMBIA 1304, and with the optimization of the floral-dip method into the model plant Arabidopsis, 19 transgenic seedlings were generated by using of Hygromycine (45-50mg?L-1) resistance screening and the PCR amplification test. According to the Mendel’s law, some lines with the ratio of nearly 3:1 were isolated, in which the foreign gene may be inserted as single copy. There were three homozygous lines named line-1, line-4 and line-7. The results of histochemical staining for GUS showed that transgenic Arabidopsis seedlings of 12-day carrying the target vector pCAMBIA1304, the GUS gene were expressed throughout the whole plant, and were mainly concentrated in the roots of the transgenic Arabidopsis carrying the target GmC2H2 gene. In addition, the mutants of GmC2H2 gene which were named MZF1、MZF2、MZF4、MZF8、MZF10 were respectively constructed to the expression vectors to explore the function of elements of GmC2H2 transcription factor in regulating plant growth and development or plant stress resistance.For further identifying the function of GmC2H2 gene, wild-type and transgenic genotypes of Arabidopsis thaliana plants were treated with low temperature (1°C), abscisic acid (ABA, 200μmol?L-1) and high salt (NaCl, 250mmol?L-1) treatment. Studies showed that under the treatment of low temperature, ABA and salt treatment, transgenic plants grew better than the wild-type plants, and the contents of MDA in both of them were increased, but the contents of MDA in the wild-type Arabidopsis were more significant. The contents of soluble sugar and proline in the transgenic Arabidopsis were significantly higher than that in the wild type, and cell injury rates of transgenic plants was lower compared to the wild type Arabidopsis, indicating that the insertion of GmC2H2 gene may lead to a significant higher resistance of stress in transgenic Arabidopsis plants than wild-type Arabidopsis.The results of real-time qPCR showed that expression of GmC2H2 gene in the transgenic Arabidopsis was significantly higher after the treatment of low temperature and ABA treatment than untreated transgenic Arabidopsis both in roots and leaves. Under the treatment of low temperature and ABA, the accumulation of antifreeze gene cor6.6 in leaves and roots of those Arabidopsis plants was higher than that in the wild-type plants. Under low temperature conditions, the accumulation of EPSPS gene in leaves of the transgenic Arabidopsis was higher than that of the wild-type, while it was not obvious in the roots. For ABA treatment, the accumulation of EPSPS gene was higher in transgenic Arabidopsis than the wild-type both in leaves and roots.更多还原