锌转运蛋白AsZNT1基因上游调控序列的功能鉴定

发布时间：2018-01-22 14:12

本文关键词： 重金属 AM真菌 AsZNT1启动子顺式元件　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着人类自然活动的不断加强,各种重金属污染物通过多种途径进入到土壤中,由于难降解和不溶性使得重金属通过食物链传递,最终危害人类健康,目前重金属污染问题已成为全球亟待解决的问题之一。从研究角度和技术措施上来讲,生物修复的应用前景是很广阔的。生物修复包括很多方面,其中利用植物和微生物的共生作用来修复重金属污染的土壤成为当今的研究热点。丛枝菌根真菌在自然界中分布广泛,能与大多数的高等植物形成共生体系,不仅可以提高植物对多种营养元素的吸收,还能够增强宿主植物对非生物胁迫的抗逆性。而且菌根真菌是直接联系土壤和植物根系的一类,因此,研究和利用菌根真菌缓解重金属污染土壤对农业发展具有重要的指导意义。目前,从分子水平方面解释重金属的吸收转运调控机制还不清楚。本研究中,分析和鉴定了紫云英中ZIP家族的锌转运蛋白基因的5’端上游序列的功能,主要的研究成果如下:1.通过两轮反向PCR设计特异性引物,扩增到了As ZNT1基因上游1.8kb的启动子序列。通过PLACE和plant CARE软件预测了顺式元件及它们的位置,显示有几个重要的顺式元件存在于As ZNT1启动子的序列中,包括与磷调控相关的P1BS、与菌根和磷调控相关的34bp元件、可能参与菌根和锌调控的MYCS-like、ZRE-like元件。2.组织特异性表达实验表明,As ZNT1基因主要在分生组织区、生长区、幼嫩侧根发生处和成熟组织的中柱中表达,PAs ZNT1::GUS融合表达没有在根的表皮和皮层中发现,揭示了As ZNT1可能参与锌或其他金属离子的吸收转运。3.通过一系列的截短实验,检测到了与磷饥饿应答相关的元件P1BS,及一个新的串联重复序列34bp元件参与了As ZNT1基因的表达调控,而MYCS-like、ZRE-like不应答基因的调控。4.通过over-lap PCR构建特异性的缺失载体,进一步验证了已知的磷酸盐转运因子的顺式元件P1BS和新发现的34-bp元件是菌根真菌和外界锌浓度应答的调控元件,它们参与了As ZNT1基因的表达调控。
[Abstract]:With the continuous strengthening of human natural activities, a variety of heavy metal pollutants into the soil through a variety of channels, because of the difficult degradation and insolubility of heavy metals through the food chain transfer, and ultimately endanger human health. At present, heavy metal pollution has become one of the most urgent problems in the world. From the point of view of research and technical measures, the application prospect of bioremediation is very broad. Bioremediation includes many aspects. The symbiotic action of plant and microorganism to remediate the soil contaminated by heavy metals has become a research hotspot. Arbuscular mycorrhizal fungi are widely distributed in nature and can form symbiotic system with most higher plants. It can not only enhance the absorption of many nutrient elements in plants, but also enhance the resistance of host plants to abiotic stress. Mycorrhizal fungi are a kind of plant roots directly linked to soil and plant roots. It is important to study and use mycorrhizal fungi to alleviate heavy metal contaminated soil. At present, it is not clear to explain the regulation mechanism of heavy metal absorption and transport from the molecular level. The function of 5 '-terminal upstream sequence of zinc transporter gene of ZIP family was analyzed and identified. The main research results were as follows: 1. The specific primers were designed by two rounds reverse PCR. The promoter sequence of 1.8kb upstream of as ZNT1 gene was amplified. The cis elements and their location were predicted by PLACE and plant CARE software. Several important cis-elements were found in the sequence of as ZNT1 promoter, including P1BSs associated with phosphorus regulation and 34bp elements associated with mycorrhizal and phosphorus regulation. MyCS-like ZRE-like element. 2. The results of tissue specific expression showed that the gene of as ZNT1 was mainly in the meristem region and growth region. The expression of pas ZNT1::GUS fusion was not found in the epidermis and cortex of the root. It was revealed that as ZNT1 may be involved in the absorption and transport of zinc or other metal ions. Through a series of truncation experiments, P1BS related to phosphorus starvation response was detected. And a new tandem repeat 34bp element involved in the regulation of as ZNT1 gene expression while MYCS-like. ZRE-like does not respond to gene regulation. 4. Construction of specific deletion vector through over-lap PCR. It was further verified that the cis element P1BS of the known phosphate transporter and the newly discovered 34-bp element were the regulatory elements for the response of mycorrhizal fungi and external zinc concentration. They are involved in the regulation of as ZNT1 gene expression.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;X53

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