【作者】 安吉翠；

【导师】 彭抒昂；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 柑橘是世界范围内普遍种植的一类非常重要的经济作物,而它在中国的主要种植区之一赣南的土壤中严重缺乏硼素,导致柑橘花而不实或者“猴头果”,降低了柑橘产量,从而影响果农的经济收入。硼是植物生长必需的微量元素,它在土壤中存在的主要形式是B(OH)3,并以中性分子的形式为植物所吸收和运输。植物对硼素的浓度适应范围比较窄,硼素浓度过低或过高对植物生长都会造成不利的影响。在雨水过多地方如江西赣南地区,硼素很容易被雨水淋失而使作物出现缺硼症状；而在干旱地区如澳大利亚,因灌溉水含硼量过高或者施用过量硼肥而硼在土壤中累积造成植物硼毒害。虽然硼素对作物的经济价值影响非常大,但是目前人们对硼素的代谢路径尚不清楚。近期的研究表明,植物中特有的一类水通道蛋白—NIP家族蛋白参了硼素的吸收和运输过程,NIP家族基因运输硼、甘油等大分子的功能成为研究热点。目前在拟南芥、水稻、葡萄等作物中已经克隆得到与硼吸收和运输有关的基因,但是在柑橘中还未见报道。本研究利用电子克隆、染色体步移等技术,从柑橘砧木枳[Poncirus trifoliata (L.) Raf.]中克隆了两个基因CiNIP5和CiNIP6及他们的启动子,分析了基因编码的氨基酸序列的保守性和多样性,研究了他们在枳壳中组织特异表达模式和缺硼、硼毒害胁迫条件下的时空表达情况以及启动子区域存在的关键元件。为了进一步的证实CiNIP5和CiNIP6与硼吸收和运输的关系,还对这两个基因进行功能验证工作。本研究最终证实了柑橘中CiNIP5和CiNIP6分别参与了植株吸收和运输硼的过程,为通过转基因技术提高柑橘耐硼胁迫环境提供了理论基础。主要结果如下：1.采用同源克隆技术从柑橘砧木枳中得到硼运输基因CiNIP5,基因序列包含903bp的开放阅读框,编码300个氨基酸。预测CiNIP5编码蛋白质分子的分子量为31.0kDa,等电点为8.73。多序列比对和系统进化树分析表明,CiNIP5属于水通道蛋白家族中的NIP亚家族,与拟南芥中的AtNIP5;1有高度的相似性。生物信息学分析表明CiNIP5蛋白具有典型的NPA和Ar/R结构域,可以运输比水分子大的中性小分子物质。基因表达分析表明CiNIP5mRNA主要在枳根部表达,在茎和叶中的表达量非常低。低硼胁迫下,CiNIP5mRNA积累量显著升高；在高浓度硼胁迫下,CiNIP5mRNA积累量显著降低。2.采用同源克隆技术从柑橘砧木枳中得到硼运输相关基因CiNIP6,基因序列包含915bp的开放阅读框,此开放阅读框包含五个外显子和四个内含子,编码304个氨基酸。预测CiNIP6编码蛋白质分子的分子量为31.2kDa,等电点为8.61。多序列比对和系统进化树分析表明CiNIP6属于水通道蛋白家族中的NIP亚家族,与拟南芥中的AtNIP6;1有高度的相似性。生物信息学分析表明CiNIP6蛋白具有典型的NPA和Ar/R结构域,其中Ar/R组成为T,I,G和R不同于AtNIP6;1的结构,推测CiNIP6具有不同于AtNIP6;1的功能。基因表达分析表明CiNIP6mRNA主要在枳地上部位表达,在根中的表达量非常低。在低硼和高硼浓度胁迫下,CiNIP6mRNA积累量都显著升高,说明CiNIP6参与调节植物体内硼的平衡分布。3.应用染色体步移技术克隆得到CiNIP5和CiNIP6基因启动子。生物信息学分析表明这两个基因的启动子都具有典型的TATA box和CAAT box,并含有很多逆境响应元件；如干旱、伤害等,还包含乙烯、赤霉素、生长素、光等激素和环境信号响应元件。4.通过生物信息学分析,比对发现CiNIP5的5端UTR和AtNIP5;1的5端UTR具有很高的相似性,都具有两个保守序列：序列1(CAUGUAA)和序列2(UCAAAUCAUGUAA)。这两个序列对于CiNIP5响应环境中硼浓度而上调或下调变化起到决定性作用。5.通过转基因技术,将CiNIP5和CiNIP6基因转入烟草中超表达进行功能分析。结果表明转CiNIP5基因植株的细胞耐逆境能力增强,叶片的光合作用增强,根部累积的硼含量显著增加；转CiNIP6基因植株叶片中硼含量显著增加,但是其他生理指标和对照没有显著差异。说明CiNIP5对于植物适应硼浓度的变化有更重要的意义。更多还原

【Abstract】 Citrus is one of the most important economic crops in the world. The filed where citrus planted in the southern China is serious lack of Boron (B). This leads to reduced product of citrus and economic losses to the farmers. Boron (B) is an essential element for plants. In soil solution, B exists primarily as boric acid [B(OH)3]. It is accepted that plants take up B from soil in the form of boric acid. In agriculture, the range of B concentration between deficiency and toxicity is thought to be narrow. B deficiency is a major problem that impedes crop growth and generally leads to the rapid cessation of root elongation, reduced leaf expansion and reduced fertility, mainly due to reduced cell expansion. On the other hand, B toxity is another problem in food production. Thus, the understanding to the mechanisms that regulate B homeostasis in plants is important with regards to both knowledge of physiology and agricultural practice. Decades of researches showed that, the nodulin26-like intrinsic proteins (NIPs) family, which is a group of highly conserved multifunctional major intrinsic proteins that are unique to plants, trasnsports a variet of uncharged solutes ranging from water to ammonia to glycerol and boric acid. Reacently, some genes involved in boron absorption in Arapbidopsis, rice and grape were cloned, but there was no report in citrus.In our research, we tried to make clear how B was absorbed and transported in the citrus and further to solve the B deficient problem in the citrus product. Two genes were cloned from trifoliate orange [(Poncirus trifoliata (L.) Raf.] using in silico cloning and named as CiNIP5and CiNIP6respectively. Their mRNA levels in different tissues were detected. Both genes were compared with their similar genes from other plants. In addition their ectopic expressions in tobacco were conducted. We found that CiNIP5and CiNIP6could increase the capacity of resistance to boron deficiency, while CiNIP5played more important part than CiNIP6did. The main results were in the following:1. Based on the sequence of AtNIP5;1, we cloned CiNIP5gene from trifoliate orange, containing an open reading frame (ORF) of903bp which encodes a300amino acids polypeptide with a predicted molecular mass of31.0kDa and an isoelectric point of8.73. Analysis of the putative amino acid sequence suggested that CiNIP5belonged to the NIPII subfamily, and showed highly homology with those proteins from other plants with function of transporting B. Bioinformatics analysis revealed that CiNIP5contained two conserved regions-NPA motif and Ar/R filter, which indicated that CiNIP5could transport small uncharged solutes. Analysis of the expression of CiNIP5showed that it was mainly expressed in roots, and it’s expression was upregulted by B deficiency, however, was downregulted by B toxicity. 2. CiNIP6was cloned from trifoliate orange, containing an open reading frame (ORF) of915bp which encodes a304amino acid polypeptide with a predicted molecular mass of31.2kDa and an isoelectric point of8.61. Analysis of the putative amino acid sequence suggested that CiNIP6belonged to the NIPII subfamily, and showed highly homology with other B transporter proteins. Bioinformatics analysis revealed that CiNIP6contained two conserved regions-NPA motif and Ar/R filter. The ar/R filter of CiNIP6consists of Thr (T), Ile (I), Gly (G) and Arg (R), which is different from that of AtNIP6;1. Those changes indicated that CiNIP6might have different functions from AtNIP6;1. Analysis of gene expression revealed that CiNIP6mainly existed in leaves and stems. Both in B deficient and toxic condition, the transcripts of CiNIP6were up-regulated, especially in the toxic situation. This result suggested that CiNIP6participated in the regulation of B homeostasis in citrus.3. The promoter regions of CiNIP5and CiNIP6were cloned by genomic walking PCR. Bioinformatic analysis showed that they both contained typical TATA box and CAAT box, and many stress responsing elements, such as dehydration, wounding, hormone and stress:ethylene, gibberellin, auxin, light and so on.4. By bioinformatics analysis, we found that the5’UTR between CiNIP5and AtNIP5;1were highly similar, they both contained two conserved sequences:1(CAUGUAA) and2(UCAAAUCAUGUAA), which are important for NIP5B-dependent degradation under B toxic condition.5. The transgenic tobacco overexpressing CiNIP5contained more B and had enhanced photosynthetic intensity than in the wild type plants, implicating the overexression of CiNIP5has the ability to enhance the resistance to boron deficiency. The transgenic tobacco overexpression CiNIP6contained more B in the leaves but less in the roots than the wild type plants, implicating that it had function of homeostasis of B in the plants.更多还原