【作者】 张学杰；

【导师】 李法曾； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2004， 博士

【摘要】 盐胁迫包括渗透胁迫和离子胁迫以及由此引起的次级胁迫如氧化胁迫等，造成植物生长延滞，甚至死亡。土壤中高浓度的盐导致了世界上许多作物大幅减产。植物耐盐基因工程被认为是培养耐盐作物、开发利用盐荒地最有效、最经济的手段，但缺乏有效的目的基因是限制植物耐盐基因工程进一步发展的瓶颈。本论文的主要目的是在寻找植物耐盐基因工程有效目的基因方面做一些有益的探索。 1．过量表达盐地碱蓬cDNA文库对拟南芥耐盐性的影响 我们建立了一个过量表达或共抑制盐地碱蓬cDNA文库的系统，该系统被称为SARE系统。双元载体由pCAMBIA1200和pRT105组装而成，由CaMV 35S启动子驱动盐地碱蓬cDNA文库表达。该载体具有潮霉素抗性，并可以利用侧翼引物进行PCR反应确定cDNA插入。我们将双元载体通过农杆菌介导用花序浸泡法转入拟南芥中，共筛选到2000多个转化子，其中大约15.7％为单位点插入。PCR扩增结果表明插入片断集中在800-2000bp之间。经初步筛选，19、27和185三个株系在萌发和幼苗时期表现出较强的耐盐性。在200mmol／LNaCl条件下，转基因株系的生长明显优于野生型株系。PCR扩增得到三个长度分别约为800bp、1000bp和1200bp的片断，其序列测定与分析正在进行中。 2．过量表达SsNHX1-AVP1对拟南芥耐盐性的影响 我们构建了同时过量表达盐地碱蓬液泡膜Na~+／H~+ antiporter基因SsNHX1与拟南芥液泡膜H~+-PPase基因AVP1的双价载体并导入拟南芥中进行表达，筛选得到了343个纯系。PCR、RT-PCR、Southern、Northern以及Western结果证明目的基因已经整合到转基因植株中并进行了表达。在正常生长情况下，转基因植株叶片液泡膜H~+-PPase活性明显高于野生型植株，说明转入的基因确实发挥了作用。在不含NaCl的MS培养基上，转基因植株的生长与野生型植株没有明显差异，说明外源基因的插入和过量表达并没有影响植物的正常生长。 在200mmol／L NaCl处理下，尽管转基因植株和野生型植株的生长都受到抑制，但转基因植株表现出更强的耐盐性。外源基因的表达可以提高种子萌发率，增加鲜重和干重，积累较多的K~+，维持较高的细胞质K~+／Na~+比和较大的净光合速率，增加脯氨酸含量，降低渗透势。结果表明同时过量表达盐地碱蓬液泡膜Na~+／H~+逆向转运蛋白基因SsNHX1及拟南芥焦磷酸酶基因AVP1明显提高了拟南芥植株的耐盐性，进一步证实离子区隔化在植物抗盐方面的重要作用，为利用分子生物学手段进行作物耐盐新品种的培育奠定了基础。过纽裹达越地喊蔽cDNA文库及&刀月万户训王，Pl对拟南芥耐趁性的形晌和盆地翻斑曰D7荟因。ONA的克隆与裹达分析3.盐地碱蓬质体。一3脂肪酸去饱和酶基因的克隆与表达分析 利用EST随机挑取克隆测序的策略从盐生植物盐地碱蓬叶片cDNA文库中分离得到编码质体。一3脂肪酸去饱和酶基因的全长cDNA序列。该基因开放阅读框长1272bP，编码423个氨基酸残基。推定的氨基酸序列与烟草、芝麻和紫苏。一3脂肪酸去饱和酶基因分别有80%、79%和78%的相似性。推定该蛋白分子量为48485.7D，等电点为9.04。该蛋白具有四个跨膜区和三个组氨酸簇(HDCGH，HGWRISHRTHH，HHDIGTHVIHH)，完全与植物。一3脂肪酸去饱和酶基因的结构相一致。该序列氨基端前51bp富含经基氨基酸(丝氨酸和苏氨酸)，表现出质体蛋白转运肤的特征。 我们也研究了盐地碱蓬质体。一3脂肪酸去饱和酶基因在不同胁迫下的表达变化，N。件hem分析表明该基因在盐地碱蓬地上部分的表达明显受盐、低温胁迫及伤害所诱导。400 mmol几NaCI处理或伤害处理3h时，盐地碱蓬质体。一3脂肪酸去饱和酶基因转录水平达到第一个高峰，12h时达到第二个高峰。SsFAD夕的mRNA水平在Zoommol/L和400班unol几NaCI处理24h时出现两次高峰。15℃低温处理时，SsFAD7的mRNA水平在处理12h时达到最高，24h时表达下降，48h时又出现高峰，以后逐渐下降。同时，我们构建了植物表达载体pCAMBIA3301一SsFAD夕并转入拟南芥中，筛选得到了21个转化子。本论文主要创新点: l)我们建立了一个新的过量表达或共抑制盐地碱蓬cDNA文库的系统，并将其转入拟南芥中。我们得到了三个在萌发和幼苗时期表现出较强耐盐性的株系。我们认为该系统对于分离盐地碱蓬耐盐相关基因会有价值的。 2)构建了同时含有液泡膜Na+舰+antiporter与H十一PPase基因的双元植物表达载体并转入拟南芥，筛选到在100和ZoomMol/L NaCI胁迫环境下生长发育均明显好于野生型的转基因拟南芥植株 101和127，进一步验证了液泡Na+区隔化在植物耐敖方面的重要作用。 3)从盐生植物盐地碱蓬cDNA文库中克隆得到编码质体。一3脂肪酸去饱和酶基因的cDNA序列，这是第一个从盐生植物中克隆得到的质体。一3脂肪酸去饱和酶基因序列;并较为详细的研究了盐地碱蓬质体。一3脂肪酸去饱和酶基因在不同胁迫下的表达变化，发现该基因在软地碱蓬地上部分的表达明显受盐、低温胁迫及伤害所诱导。更多还原

【Abstract】 Salt stress consisting of osmotic and ionic stress, and then the secondary stress such as oxidative stress arising by them, can bring on .growth inhibition and even death of plants. High concentrations of salt in soils account for large decreases in the yield of a wild variety of crops all over the world. Plant salt-tolerant genetic engineering was considered as the most effective and economical mean to bring up salt-tolerant crops and to exploit salt wasteland. The lack of efficient genes has been the bottleneck to limit its development. The main objective of the dissertation was to do some work to find new efficient genes for plant salt-tolerant genetic engineering.1. Effects of overexpression ofSuaeda salsa cDNA library on the salt tolerance in Arabidopsis thalianaWe have developed a system named sense/antisense RNA expression (SARE) to over-express or co-suppress random Suaeda salsa cDNAs. The binary vector containing a novel Suaeda cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter consisted of the part of pCAMBIA1200 and pRT105. The vector offers selection with hygromycin-B and the ability to identify the cDNA insert using PCR with flanking primers. We introduced this overexpression library into Arabidopsis by floral-dip upon Agrobacterium /wme/ac/’ens-mediated transformation and selected over 2000 transformants. Only about 15.7% of Tl plants have one insert. The insert size is ca 800-2000bp. There were three lines, 19, 27 and 185, which exhibited higher salt tolerance during the period of seeds germination and seedling. The transgenic Arabidopsis plants grew much better than wild type plants under 200 mmol/L NaCl condition. Three insert fragments, which sizes are ca SOObp, lOOObp and 1200bp respectively, have been obtained from the three transgenic lines by PCR amplification. The DNA sequence and analysis is under goning.2. Effects of co-overexpression ofSsNHXJ and AVPl on the salt tolerance in ArabidopsisThe bivalence vector carrying both SsNHXI and AVPl have been constructed and thenintroduced into Arabidopsis. We have got 343 pure lines. PCRs RT-PCR, Southern Northernand Western blotting analysis testified that both extraneous genes had been integrated into all of the transgenic plant genomes. Under normal condition, the PPase activities of transgenic plants were obviously higher than that of wild type plants, which indicated that the extraneous genes had played its role on transgenic plants. No difference was observed in growth between transgenic plants and wild type plants in the absence of NaCl stress conditions, which indicated that the insertion and overexpression ofSsNHXI and AVPl had no effect on the normal growth of Arabidopsis plants.The growth of both transgenic and wild type Arabidopsis plants were suppressed after a 200mmol/L NaCl treatment, but the transgenic plants had higher salt tolerance than wild type plants. The expression of extraneous genes can improve seeds germination, increase fresh and and dry weight, accumulate more K"1", maintain a high cytosolic K/Na ratio and photosynthetic rate (Pn), increase the content of proline and then reduce the osmotic potential. These results indicated that transgenic Arabidopsis plants co-overexpressed ofSsNHXI and AVPl had greater salt tolerance than wild type plants, which further indicated the importance of Na compartmentation in plant salt tolerance. This can be used in genetic engineering for improving salt tolerance of crop plants.3. cDNA cloning and expression of Suaeda salsa FAD7 gene (plastid w -3 fatty acid desaturase)Full cDN A sequence encoding plastid w -3 fatty acid desaturase (FAD7) were isolated from the cDNA library of halophyte Suaeda salsa through EST analysis. The cDNA contained a 1470-bp putative open reading frame encoding for 489 amino acids with a predicted molecular mass of 54.29 kDa and a predicted isoelectric point of 9.04. The deduced amino acid sequence shows 80%, 79% and 78% identity with Nicotiana tabacum, Sesamum indicum and Perilla frutescens w -3 fatty acid desaturases respect更多还原