摘要：脂磷酸磷酸酶（LPPs）是一组磷酸转移酶家族的酶，它有四种亚型[1]LPP1、LPP2、LPP3、LPP4。高等植物中LPPs的研究主要集中在拟南芥中，它们的作用是催化水解焦磷酸甘油二脂 (DGPP)去磷酸化生成磷脂酸（PA），并进一步催化磷脂酸（PA）去磷酸化生成二酰基甘油 (DAG)[2, 3]。其中, LPP1能广泛的应答生物或非生物胁迫[4]。为了研究LPP1应答盐害的分子机理，我们利用GUS进行组织特异性染色，并加以盐处理生理分析野生型和lpp1的耐盐表型。结果表明，AtLPP1主要表达在根和叶等组织部位，lpp1纯合缺失突变体对盐胁迫更加敏感，种子的萌发率、生物量降低及细胞的Na+/K+离子含量发生紊乱，而外源加入PA能有效的缓解盐胁迫效应。通过上述工作我们可以初步揭示LPP1蛋白抗盐的分子机制，为抗逆农作物新品种的培育提供理论基础和基因资源。41919

毕业论文关键词：拟南芥；脂磷酸磷酸酶(LPP1)；β-葡萄糖苷酸酶（GUS）；盐胁迫

The molecular mechanism of Lipid Phosphate Phosphatase(LPP1) responses to salt stress in Arabidopsis thaliana

Abstract： Lipid phosphate phosphatases(LPPs) are a group of enzymes that belong to a phosphatase/pho sphotransferase family. LPPs consist of four isoforms:LPP1、LPP2、LPP3、LPP4[1]. The research of lipid ph osphate phosphatase(LPPs) in higher plants mainly concentrate on Arabidopsis thaliana, They responsible for the dephosphorylation of DGPP to PA and of PA to diacylgylcerol are encoded by four genes in Arabidopsis[2-3]. Whereas, LPP1 responses to a wide range of the biological and abiotic stress[4].In order to discuss the molecular mechanisms of LPP1 responses to salt stress, histochemical staining of GUS activity was performed with transgenic plants ,and the phenotypes analysis with salt stress was further studied by using wild type and lpp1 mutants. It reveals that AtLPP1 was mainly expressed in root and leaves, moreover, the decrement of seed germination, biomass and the imbalance of Na+ with K+ in lpp1 mutants with salt, while those effects can be relieved by adding exogenous PA. Through the above works, LPP1 protein molecular mechanism of resistance to salt stress will be revealed preliminarily, which provide theoretical basis for breeding of new varieties of crops aspect and genetic resources.

Key words: Arabidopsis thaliana; lipid phosphate phosphatase(LPP1);GUS; salt stress 

目  录

摘要1

关键词1

Abstract1

Key words1

引言1

1 材料与方法2

1.1 材料2

1.2方法3

1.2.1 拟南芥基因组总DNA、RNA的提取3

1.2.2 实验引物设计3

1.2.3 基因克隆及酶切酶连转化反应4

1.2.4菌落PCR-阳性克隆筛选5

1.2.5 拟南芥植株的转染与GUS报告基因的检测5

1.2.6 拟南芥发芽率的测定方法5

1.2.7拟南芥离子含量的测定方法5

1.2.8 拟南芥生物量的测定方法6

2 结果与分析6

2.1 AtLPP1基因组织特异性表达部位的定位6

2.1.1 AtLPP1:GUS转基因植株的构建6

2.1.2 AtLPP1:GUS组织表达定位6

2.2 拟南芥中LPP1耐盐性机制分析7

2.2.1 lpp1纯和突变体植株的鉴定筛选7

2.2.2 利用CRISPR Cas9技术构建lpp1纯和突变体株系8

2.2.3 构建AtLPP1基因的回补型和过表达型转基因株系9

2.2.4 拟南芥发芽率的测定9

2.2.5 拟南芥生物量的测定10

2.2.6 拟南芥离子含量的测定10

3 讨论 11

3.1 AtLPP1基因表达的定位分析11

3.2 LPP1的耐盐机理探究11

致谢11

参考文献12

附录13

拟南芥脂磷酸磷酸酶（LPP1）应答盐害的分子机理

引言：随着全球环境的恶化，全球气候变暖，海平面不断上升等自然因素以及工业生产发展，环境污染加剧，淡水资源匮乏，海水入侵，农田灌溉方法不当等人为因素使土盐渍化、沙漠化日益严重。而干旱高盐、低温、土地盐碱化等逆境因素严重影响农作物的生长和发育，并致其产量和质量显著下降[5]。因此，如何提高农作物的抗逆能力是农业生产中所面临的重大科学问题。