CAJ | 학술논문

利用cDNA快速末端扩增(RACE )技术获得红树植物秋茄液泡膜内在蛋白(TIP)的全长cDNA (GenBank 登录号:AF521135).该cDNA全长为1.1kb,含有一个756个核苷酸的完整开放阅读框,编码252个氨基酸,等电点为5.77,分子量为26.3 kD.该氨基酸序列含有6个跨膜区和两个高度保守的Asparagine-proline-alanine (NPA)基序.与冬葡萄、花椰菜、拟南芥的TIP相比,氨基酸的一致性达到77%～79%.Northern分析表明盐分抑制该基因在红树3个种中的表达,这种下调表达可能降低液泡膜水分渗透,有利于盐胁迫下细胞的保水.
이용cDNA쾌속말단확증(RACE )기술획득홍수식물추가액포막내재단백(TIP)적전장cDNA (GenBank 등록호:AF521135).해cDNA전장위1.1kb,함유일개756개핵감산적완정개방열독광,편마252개안기산,등전점위5.77,분자량위26.3 kD.해안기산서렬함유6개과막구화량개고도보수적Asparagine-proline-alanine (NPA)기서.여동포도、화야채、의남개적TIP상비,안기산적일치성체도77%～79%.Northern분석표명염분억제해기인재홍수3개충중적표체,저충하조표체가능강저액포막수분삼투,유리우염협박하세포적보수.
Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. Candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3′ and 5′end of the KCTIP1 gene were obtained using the SMARTTM RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3′end of this gene was obtained using the GSP1 primer, and a 690 bp fragment corresponding to the 5′end of this gene was obtained using the GSP2 primer. Two primers that flank the putative open reading frame (ORF) were designed to obtain the cDNA containing the complete ORF by RACE PCR reaction. The full-length cDNA of KCTIP1, containing a 756 bp open reading frame (ORF), was approximately 1.1 kb; the start codon was located at the nucleotides of 99-101 and stop codon at the nucleotides of 855-857 followed by a poly (A) tail. The KCTIP1 cDNA sequence in this research was released in GenBank with accession number AF521135. Using ExPASy Proteomics tools provided by EMBL, the isoelectric point and MWt of KCTIP1 are estimated as 5.77 and 26.3 kD respectively. Transmembrane prediction analysis revealed the deduced KCTIP1 protein sequence contains six transmembrane regions at amino acid residues of 20-42, 57-79, 86-108, 113-135, 142-164 and 217-239. Two highly conserved asparagine-proline-alanine (NPA) motifs were located at 85-87 and 199-201 amino acid residues respectively. KCTIP1 is also predicted to contain the Cys residue (Cys 118) that are shown to confer Hg-sensitivity in Arabidopsis γ-TIP and δ-TIP. Similarity analysis showed that KCTIP1 shared 77%～79% amino acid sequence identity with the TIPs from Vitis berlandieri, Brassica oleracea and Arabidopsis thaliana. Expression analyses indicated that KCTIP1 had different expression among species of Mangroves. Expressions of KCTIP1 in Kandelia candel, Rhizophora apoculata and Ceriops tagal were suppressed by salt, and were insensitive to salt stress in unknown species of Mangroves. Previous studied showed that salt conditions might result in large and rapid changes in extracellular water potential and serious disturbance to the cytoplasm. In order to compensate for this imbalance, the relative contribution of water channels to flow across the root could thus vary. K. Candel is a species that is native to intertial zone of tropical and subtropical coast and is well-adapted to salt conditions. The coordinated down-regulation of aquaporins in this plant may decrease membrane wate permeability and thus increase the cellular water co nservation during periods of salt stress. The results reported here are consiste nt with the postulated roles for tonoplast water channels in regulating the hydr aulic permeability of the vacuolar membranes and in adjusting the water homeosta sis of the protoplasm under various physiological conditions. The identification of KCTIP1 as one of saltresponsive genes implies that intracellular osmotic e quilibration is a part of salttolerant mechanisms in Mangroves.