【作者】 刘娜；

【导师】 李冰；

【作者基本信息】 河北师范大学 ， 植物学， 2008， 硕士

【摘要】 J-蛋白是一类包含J-结构域的蛋白质,其中一些成员具有分子伴侣或热激蛋白的功能。拟南芥中存在94种J-蛋白,如此多的J-蛋白的存在预示它们可能在植物的生命活动中发挥重要的功能。此前关于J-蛋白的研究主要集中在分子伴侣功能方面,它们在生长发育及抗逆方面的功能目前报道较少。AtJ50是拟南芥J-蛋白家族中的一员,基因组序列分析显示AtJ50可以被分泌到胞外。关于AtJ50的组织定位、亚细胞定位以及在生长发育和环境胁迫中功能的研究尚未见报道。本项目以拟南芥野生型和T-DNA插入突变体为材料,对拟南芥AtJ50基因的表达特性及在生长发育和环境胁迫中的功能做了一个初步的探讨。通过半定量PCR的方法检测了AtJ50基因在植株不同器官中的表达,发现AtJ50基因在叶和花中表达最高,根和茎中次之,果中表达最弱。通过半定量PCR的方法检测了AtJ50基因在不同发育时期叶中的表达,结果表明AtJ50基因在不同发育时期都有表达,但是随着植株的衰老表达明显减弱。筛选了2个不同T-DNA插入位点的AtJ50基因的纯合突变体,鉴定结果显示,在2个突变体中T-DNA都是单拷贝插入的,且都未能检测到AtJ50基因的完全转录本,表明2个突变体中AtJ50基因的确已被敲除,可用于后面功能的研究。对AtJ50突变体进行了生长发育过程中表型的观察,结果显示突变体植株与野生型植株在莲座生长、茎高及生长发育进程方面都没有明显的差异。在热、冷、盐、干旱胁迫条件下,在野生型植株中检测了AtJ50基因的诱导表达。结果表明AtJ50基因表达受热激和盐胁迫诱导,干旱也能微弱增加AtJ50基因的表达量,而冷胁迫对AtJ50基因表达没有影响。检测了AtJ50突变体在热、冷、冻、盐、干旱胁迫条件下抗性的改变。结果显示突变体与野生型在对热、冷、冻、干旱胁迫的抗性方面没有明显区别;而在盐胁迫下AtJ50突变体的生活力明显弱于野生型,且突变体在盐胁迫时种子的生存率也明显低于野生型,说明AtJ50基因可能在植物抗盐性方面发挥功能。通过RT-PCR或PCR方法克隆了AtJ50基因的表达区序列及AtJ50基因的启动子序列,为进一步的组织、亚细胞定位及遗传转化做准备工作。更多还原

【Abstract】 J-protein is a kind of protein which contains J-domain. Some of them act as molecular chaperones or heat shock proteins. A total of 94 J-proteins were identified in the genome of the model flowering plant Arabidopsis thaliana, implying that J-proteins is likely to play important role in growth and development of plant. At present, the researches about J-proteins are mainly focus on the molecular chaperone. There is less report for function of J-proteins on growth, deveploment and adaptability to environmental stress. AtJ50 is one of the J-protein family members in Arabidopsis thaliana. Based on the result of sequence analysis, AtJ50 could be secreted to the apoplast. It has not been reported yet to the tissue and subcelluar localization of AtJ50, as well as its function on growth, development and tolerance to environmental stress. In this project, with the wile type Arabidopsis and T-DNA insertion mutant, we conducted some primary researches on the expression characteristic, the functions on growth, development and adaptability to the environmental stress.The expression of AtJ50 gene in different organs was detected by semi-quantitative PCR. The result showed that the expression level of AtJ50 gene was most high in leaves and flowers, less in roots and stems, least in siliques. The expression of AtJ50 gene on different development stages was also checked by semi-quantitative PCR. The result indicated that AtJ50 gene expressed on every developmental stage, but the expression level decreased with the plant’s caducity.We screened two homozygous T-DNA insertion mutants with two different insertion sites. There was only single T-DNA copy in both mutants. The result with RT-PCR showed that there was not full transcript of AtJ50 gene in mutants,. The phenotypes of AtJ50 mutants were observed and analyzed. The results indicated that there was no obvious difference on the rosette size, stem height and the development stage between wild type and AtJ50 mutants.Under the heat, cool, salt, drought condition, we detected the induced expression of AtJ50 gene in the wild type. The results showed that the expression of AtJ50 gene could be induced obviously during heat and salt stresses, induced slightly during drought stress. However, the expression of AtJ50 gene was not affected by cool stress. In addition, the tolerance of AtJ50 mutants was detected under various stresse conditions. The results showed that the viability of seedlings had no obvious differenece between wild type and AtJ50 mutants under heat, cool, freeze and drought stress conditions. However the viability of AtJ50 mutants was weaker and ratio of germination was also lower than wild type under salt stress. The results suggest that AtJ50 gene might be involved in the adaptation of plant to salt stress.In addition, we cloned the cDNA sequences and promoter sequences of AtJ50 gene, which is preliminary work for the further researchs including the tissue and subcellular localization and genetic transformation.更多还原