【作者】 王丽玲；

【导师】 章文华；

【作者基本信息】 南京农业大学 ， 植物学， 2008， 硕士

【摘要】 采用电子克隆的方法从拟南芥中分离表达脂磷酸磷酸酶的其中一个基因AtLPP1(At2g01180)。AtLPP1编码328个氨基酸残基,它与拟南芥中其他三个LPPs基因有着高度的同源性,均可能包括六个跨膜区,有着高度保守的acidPPc结构域。定量RT-PCR分析的结果表明,AtLPP1在拟南芥叶片、茎、根、花等各个组织中均有表达;同时通过花浸泡法将构建的AtLPP1启动子区域驱动的GUS表达载体转入拟南芥中,获得AtLPP1启动子区域驱动的GUS转基因拟南芥植株。取生长期的拟南芥各组织进行GUS染色分析表明AtLPP1在多个组织中特异表达。构建了AtLPP1::YFP瞬时表达载体,采用PEG4000转化拟南芥原生质体,结果表明AtLPP1::YFP可能定位于原生质体细胞质膜和细胞内膜上。以往的研究表明拟南芥中AtLPP1基因受Gy,mastoparan,UV-B和harpin等的诱导,AtLPP2基本不受各种胁迫影响,相当于管家基因(Pierrugues et al.2001)。同时Katagiri等发现AtLPP2在种子萌发过程中参与PA调控途径。我们采用AtLPP1的缺失突变体lpp1进行LPP1的耐盐性生理分析,发现lpp1对盐胁迫敏感,将生长一个月的拟南芥植株用含有100mM NaCl的Hoagland营养液浇灌,一周后测定植株叶片的离子含量,发现AtLPP1的缺失突变体lpp1ma~+离子含量明显升高,同时K~+含量也较野生型明显降低,这说明AtLPP1的缺失影响了植株体内离子的平衡,这也是影响植物耐盐性水平下降的一个重要因素。Pi元素含量分析,我们发现lpp1植株较野生型水平略低,这可能是因为AtLPP1的缺失导致了植物体内PA含量下降。据此,我们在含NaCl的MS培养基中加入外源PA发现lpp1植株所受的盐胁迫明显降低,这说明AtLPP1缺失所导致的植株耐盐性下降与PA的合成有关,内源PA含量测定发现不管是在正常生长条件下还是盐胁迫下,lpp1植株体内PA含量比野生型明显降低。这进一步说明LPP1在植物体内的作用是生成PA。比较分析盐胁迫下lpp1和野生型萌发情况,我们发现100 mM NaCl下lpp1萌发率大幅下降,这进一步说明AtLPP1的缺失从种子萌发阶段开始影响植株的生长,降低了植株的耐盐性。更多还原

【Abstract】 In order to isolate lipid phosphate phosphatase gene from Arabidopsis,the amino acid sequence was get from Arabidopsis database.The cDNA fragment containing complete ORFs of 984bp was cloned from total RNA prepared from Arabidopsis leaves by the RT-PCR approach,and designated as AtLPP1.The predicted proteins of AtLPPl display high similarity to the other three LPPs,containing of 6 TM domains and a highly conserved acidPPc domain.The quantitative RT-PCR approach showed that the expression profiles of AtLPP1 was expressed in roots,leaves,siliques,flowers,stems.We also constructed GUS expression vector drived by AtLPP1 promoter and transformed to Arabidopsis by floral dip. The histochemical staining for GUS displayed the same results as the quantitative RT-PCR approach.In order to study the subcelluar localization of LPP1,we constructed the AtLPP1::YFP transiently expression vector and transformed to Arabidopsis protoplasts by PEG4000.The YFP fluorescence suggested that localized in plasma membrane and intracellular membrane.The previous research showed AtLPP1 was regulated by Gy,mastoparan,UV-B and harpin,while AtLPP2 did not affected by any stress,like a house-keeping gene(Pierrugues et al.2001).Katagiri et al(2005) found AtLPP2 is a negative regulator that participates early in the ABA signaling network during seed germination.We used AtLPP1-deficit mutant lppl to analyse the physiology of salt tolerance.We found lppl was sensitive to salt. One-month-old lppl and wide-type were watered with one-fourth strength Hoagland nutrient solution plus 100 mM NaCl.The ion analys indicated that lppl plants accumulate more Na~+ and retain less K~+ compared with wild-type plants.These suggest that AtLPP1 gene deficit affect ion homeostasis in plant and reduce the plant salt tolerance.We also found the content of Pi in lppl mutant was lower than wide-type after NaCl treatment. That’s maybe because the AtLPP1 gene deficit result the lower of PA in plant.Based on this, we added PA to MS medium contained 100mM NaCl,and we found the lppl mutant can recovered from the salt stress more.These results suggest that PA contributes to the increase of salt tolerance in plant and the AtLPP1 gene deficient results the PA’s lack.The contant of endogenesis PA assay confirm this conclusion more.Under salt stress,PA in lppl mutant was more decreasing than wide-type plant.These suggest that LPP1 in plant mainly reduced PA increase.Regarding germination,lppl mutant seeds were more sensitive than the wild-type seeds,also ABA.The germination rate was less than the wild type on MS plus 100mM NaCl.This may suggest that AtLPP1 is also involved in seed germination.更多还原