【作者】 张桦；

【导师】 张富春；

【作者基本信息】 新疆农业大学 ， 草业科学， 2011， 博士

【摘要】 新牧1号杂花苜蓿(Medicago varua Xinmu No.1)是中等程度耐盐,在轻微盐碱地区有大面积种植,已取得良好的经济效益和社会效益。但其耐盐能力仍然有限,要在盐碱地广泛种植,必须进一步提高新牧1号杂花苜蓿的耐盐能力。本研究从不同类型的抗逆相关基因入手,通过分析基因的功能和表达调控,为研究苜蓿耐盐分子机制和进一步分子育种提供依据。(1)通过同源克隆策略和RT-PCR技术,克隆了新牧1号杂花苜蓿的MvNHX1、MvP5CS和MvDREB1基因,并以新牧1号苜蓿actin基因为内参基因,通过半定量RT-PCR和荧光定量PCR分析在盐胁迫下0-24 h下MvNHX1、MvP5CS和MvDREB1基因表达情况。MvNHX1和MvP5CS基因在盐胁迫后表达均明显上调,MvDREB1基因在盐胁迫前后表达量变化不大,说明,MvNHX1和MvP5CS与苜蓿的耐盐性相关。(2)通过染色体步移法克隆了MvNHX1和MvP5CS基因的启动子,利用生物信息学方法,对MvNHX1和MvP5CS基因的启动子和MvNHX1和MvP5CS蛋白的结构和功能以及细胞中的定位进行了分析。启动子分析表明两个基因的启动子序列中均含有通用启动元件和上游调控元件,MvP5CS基因启动子还具有高水平转录应答元件。序列分析发现MvNHX1属于Na+/H+反向运输蛋白家族。MvP5CS氨基酸序列比对与已知的其它植物的P5CS氨基酸序列同源性都在70%以上,且具有P5CS酶的六个保守区。进一步预测分析表明MvNHX1是一种跨膜的蛋白质,具有信号肽,位于质膜上；MvP5CS是一种亲水蛋白,位于细胞质中。(3)利用农杆菌介导的叶盘转化法,将新牧1号苜蓿的MvNHX1和MvP5CS基因转入烟草,经分子检测证实MvNHX1和MvP5CS基因已成功转入烟草基因组中。对T1代转基因烟草在盐胁迫下RT-PCR分析表明,盐胁迫前后MvNHX1和MvP5CS基因在转基因烟草中稳定表达。盐胁迫和干旱胁迫下转MvNHX1和MvP5CS基因烟草萌发率均高于非转基因烟草。转MvNHX1基因烟草在耐盐性上表现较好,转MvP5CS基因在抗旱性上较好。盐胁迫下脯氨酸含量分析表明转MvNHX1和MvP5CS基因烟草比非转基因烟草在胁迫后的脯氨酸含量增幅大,而转MvP5CS基因烟草增幅比转MvNHX1基因烟草还要明显。转MvNHX1和MvP5CS基因烟草的MDA含量增加缓慢,而对照非转基因烟草MDA含量急剧增加,转MvP5CS基因烟草比转MvNHX1基因烟草MDA积累更慢。综合分析表明,转MvNHX1和MvP5CS基因可以提高烟草的耐盐性的抗旱性。MvP5CS基因在渗透调节和保护细胞膜方面作用更大。更多还原

【Abstract】 Medicago varia Xinmu No.1 is of moderate salt tolerance and planted widely in the slightly-alkaline land with excellent economic and social benefits. The salt tolerance of Medicago varia Xinmu-1 is still to be improved further for its wide application in alkaline land due to its limited salt tolerance. This study offers references for researching salt-tolerance molecular mechanism and future molecular breeding of Medicago varia Xinmu No.l through analysis of gene functions and regulation and control of genetic expression with different types of genes related to adversity tolerance.(1) MvNHX1, MvP5CS and MvDREB1 of Medicago varia Xinmu No.l were cloned with RT-PCR and homology cloning. The RT-PCR and a Real-time PCR assay showed that the level of MvNHX1 and MvP5CS transcriptions both were up-regulated obviously in the seedlings while MvDREB1 transcription showed no remarkable variation after high salinity treatment, which indicated that MvNHX1 and MvP5CS are related to salt tolerance of the plant.(2) The promoters of MvNHX1 and MvP5CS were cloned respectively with Genome walking technology. Promoter analysis showed that both promoters have a common cis-acting element and core promoter element, and MvP5CS promoter also has cis-acting element conferring high-level transcription. With Bioinformatics method, the structure and function as well as location of MvNHX1 and MvP5CS were analyzed. Homology analysis showed that MvNHX1 belongs to Na+/H+ antiportor family, and MvP5CS shared an identity more than 70% with the known P5CS in other plants, in addition to six conservative domains of P5CS enzyme. Further analysis showed that MvNHX1 was a transmembrane protein in plasmalemma with signal peptide. MvP5CS is a hydrophilic protein in cytoplasm, which has the same domain as P5CS in other plants.(3) The MvNHX1 and MvP5CS genes of Medicago varia Xinmu No.l were transferred successfully into tobacco via agrobacterium mediation. RT-PCR analysis showed that the expression level of MvNHX1 and MvP5CS transcription in T1-generation genetic-modified tobacco was steady during salinity treatment. The germination percentage of transgenic tobacco were higher than non-transgenic tobacco under drought and salt stress. MvNHXl transgenic tobacco showed better effect on salt tolerance and MvP5CS on drought resistance. The increment of proline content of transgenic tobacco exceeded non-transgenic tobacco under salt stress, and MvP5CS transgenic tobacco had an increment of proline content higher than MvNHXl transgenic tobacco. While MDA content in non-transgenic tobacco increased sharply under salt stress, MvP5CS genetic-modified tobacco had an accumulation rate of MDA slower than MvNHX1 genetic-modified tobacco. Comprehensive analysis indicated that MvNHX1 and MvP5CS genes both could improve salt tolerance and drought resistance of transgenic tobacco, and MvP5CS had a better effect on osmotic adjustment and protection of cell membrane.更多还原