Molecular Cloning And Functional Characterization Of Two Genes (CINIP5and CINIP6)Associated With Boron Transport From Trifoliate Orange [Poncirus Trifoliata (L.)RAF.]

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.