【作者】 王景艳；

【导师】 刘兆普； 安利佳；

【作者基本信息】 南京农业大学 ， 植物营养学， 2006， 硕士

【摘要】 盐分对植物的伤害主要是Na~+引起的，而Na~+／H~+逆向转运蛋白催化Na~+和H~+的逆向跨膜运输，是植物抵御盐胁迫的主要方式之一，在植物耐盐性方面起着重要的作用。对于植物Na~+／H~+逆向转运蛋白基因的克隆、表达及功能的分析将对植物耐盐基因工程产生重要意义。 本研究通过GenBank上登陆的其它植物液泡膜Na~+／H~+逆向转运蛋白基因的同源序列设计一对简并引物，利用RT-PCR和RLM-RACE技术获得了獐茅液泡膜Na~+／H~+逆向转运蛋白基因的cDNA全序列，命名为AlNHX1。AlNHX1cDNA全长2706bp，5′端非编码区387bp，3′端非编码区696bp，含有1个可能的加polyA信号：AATAA，开放阅读框1623个核苷酸，编码一个由540个氨基酸构成的多肽，含有Na~+／H~+逆向转运蛋白的高度保守序列LFFIYLLPPI，这是Na~+／H~+逆向转运蛋白活力的竞争性抑制剂氨氯吡嗪嘧的结合位点。该基因氨基酸序列同源性与芦苇(Phragmites australis)、水稻(Oryza sativa)、小麦(Triticum aestivum)、大麦(Hordeum vulgare)、玉米(Zea mays)、滨藜(Atriplex gmelini)、盐角草(Salicornia europaea)、拟南芥(Arabidopsis thaliana)的Na~+／H~+逆向转运蛋白基因分别达到了94％、90％、88％、89％、75％、75％、76％、73％。同时根据RLM-RACE的原理和5′端非编码区序列的分析，推测出目的基因上游的转录起始位点可能为a，为该基因表达调控方面的研究奠定了基础。 根据AlNHX1基因全长序列设计引物扩增其编码区，并将其克隆到pUC19载体中，经测序确认，保留未发生突变的克隆体，用于构建其植物表达载体pBI121-NHX。冻融法将pBI121-NHX重组质粒导入根癌农杆菌(Agrobacterium tumefaciens)GV3101中，获得工程菌GV3101-pBI121-NHX。 农杆菌介导法将AlNHX1基因编码区导入烟草(Nictiana tabacum L.cv.89)，获得卡那霉素抗性植株。PCR、RT-PCR及Southern杂交均证明外源AlNHX1基因已整合到烟草基因组中。盐胁迫下转基因烟草的相对电导率显著低于对照，说明转基因烟草的膜结构所受损伤小于对照。转基因烟草具有一定的耐盐性，能在含250mmol／L NaCl的培养基中正常生长3周，而对照则在100mmol／L NaCl处理时便不生根。更多还原

【Abstract】 Salt stress is mainly caused by Na~+. The Na~+/H~+ antiporters catalyze the exchange of Na~+ for H~+ across the membrane, and they play an important role in the mechanisms of plant salt tolerance.The cloning, expression and studying the function of the Na~+/H~+ antiporter will bring momentous sense to the genetic engineering of plant salt tolerance.In order to isolate the full length cDNA of Na~+/H~+ antiporter gene from the tonoplast membrane of Aeluropus littoralis, RT-PCR and RLM-RACE were carried out, and a pair of degenerate primers were designed according the totonoplast Na~+/H~+ antiporter homologous gene region of other plants. The gene was named AlNHX1. AlNHX1 cDNA (2706bp) included a 387bp 5’ UTR, a 696bp 3’ UTR, a putative polyadenylylation signal and a 1623bp open reading frame encoding a 540-amino-acid polypeptide which was 94 %, 90 %, 88 %, 89 %, 75 %, 76 %, 75 %, 73 % to sequences of Phragmites australis, Oryza sativa, Triticum aestivum, Hordeum vulgare, Zea mays , Atriplex gmelini, Salicornia europaea Arabidopsis thaliana, in amino acid homology respectively. The deduced amino acid sequence included the conserved amiloride binding sites of LFFIYLLPPI. A was considered as the possible transcription initiation site based on the principle of RLM-RACE and the analysis of AlNHX1 5’ UTR sequences, and it would establish foundation for the studying of its relating and expression.To amplify the ORF sequence of AlNHX1, Primers were designed according to the full length cDNA sequence of AlNHX1. And the AlNHX1 ORF was insert into the plasnid of pUC19. The right clone which was ensured by sequencing was used for the constructing of the plant expression vector pBI121-NHX. The resultant plasmid pBI121-NHX was transferred into Agrobacterium tumefaciens (GV3101) by the liquid nitrogen freezing thaw method.更多还原