拟南芥新基因At234的克隆及其在非生物与生物胁迫中的功能分析

发布时间：2018-04-13 05:25

  本文选题：拟南芥 + RNAi文库　； 参考：《郑州大学》2016年硕士论文

【摘要】：本研究对拟南芥RNAi突变体文库进行高温胁迫筛选,获得拟南芥突变体阳性单株,PCR鉴定测序获得目的基因片段,长度为206bp,并命名该基因为At234。通过拟南芥TAIR网站数据库序列比对结果发现,At234位于拟南芥第2号染色体上,无内含子结构,基因全长380bp,5’-UTR长度为44bp,3’-UTR长度为102bp,其ORF为234bp,编码77个氨基酸残基,所翻译蛋白质的分子量为8.944kDa,等电点为9.98。为了进一步验证该基因的功能,我们分别设定了RNAi植株与植物病原菌互作、重金属胁迫、干旱胁迫和高盐胁迫等生物与非生物胁迫验证实验。具体研究结果如下:确定野生型拟南芥的极限耐受高温温度阈值,最终采用65℃高温分别处理拟南芥野生型与RNAi文库(250库与350库)突变体各30min,在两个RNAi文库中都筛选出了若干株拟南芥耐极限高温的RNAi突变体,通过PCR克隆获得目的基因,均为At234基因的突变株。继而通过潮霉素筛选培养,单株收种,获得拟南芥RNAi突变体的纯合子。采用本实验室自建的植物病原菌互作瓶内检测体系分析at234植株与病原菌的互作反应。研究发现,在组培条件下at234植株较野生型拟南芥对棉花大丽轮枝菌和大豆核盘菌均表现为明显感病;对玉米灰葡萄孢菌和茄病镰刀菌均表现为明显抗病;而在和小麦赤霉孢菌的互作中at234植株和野生型拟南芥均为正常感病。在组培条件下,at234植株较野生型拟南芥表现出高抗CdCl2(100mg/L)和Pb(NO3)2(1g/L)等重金属盐;在添加甘露醇(5%)的固体培养基中,at234植株较野生型拟南芥表现出明显的抗旱性;在含有12g/L NaCl的高盐胁迫培养基中,at234植株根系发育正常,表现出较强的高盐耐受性。综上所述,At234是拟南芥参与生物胁迫与非生物胁迫反应中的一个关键基因,其具体作用机制还有待进一步的研究。
[Abstract]:In this study, the RNAi mutant library of Arabidopsis thaliana was screened under high temperature stress.The results of TAIR database alignment of Arabidopsis thaliana showed that At234 was located on chromosome 2 of Arabidopsis thaliana and had no intron structure.The molecular weight of the translated protein is 8.944 kDa and the isoelectric point is 9.98.In order to further verify the function of the gene, we set up biological and abiotic stress verification experiments such as RNAi plant and plant pathogen interaction, heavy metal stress, drought stress and high salt stress.The specific results are as follows: determine the threshold of extreme tolerance to high temperature in wild type Arabidopsis thaliana.The wild-type Arabidopsis wild-type mutant and the RNAi library (U250 library and 350 library) were treated at 65 鈩，

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