香蕉WAT1基因的鉴定及表达分析

Identification and expression analysis of WAT1 genes in banana

【目的】基于香蕉基因组数据筛选Walls are thin 1(WAT1)基因,分析它们的序列及表达特性。【方法】以拟南芥WAT1为参考序列,通过本地Blast筛选获得香蕉WAT1基因,分析其核苷酸、启动子及编码蛋白特性,并利用实时定量PCR技术研究其在不同组织部位、不同激素和逆境胁迫处理下的表达情况。【结果】筛选获得5个香蕉WAT1基因(命名为MaWAT1-1~5)。蛋白亚细胞定位预测结果显示,MaWAT1-1、MaWAT1-2、MaWAT1-4主要定位在液泡和细胞膜上,MaWAT1-3主要定位在细胞质和细胞膜,MaWAT1-5定位在细胞膜和叶绿体。基因结构分析和系统进化树分析均将MaWAT1s分为两组,MaWAT1-1、MaWAT1-2、MaWAT1-4聚为一组(含6个外显子和5个内含子),MaWAT1-3和MaWAT1-5归为一组(含7个外显子和6个内含子)。启动子顺式作用元件分析结果显示:MaWAT1s启动子含有大量激素和胁迫响应相关元件。实时定量PCR结果显示,MaWAT1-4在叶片中表达量最高,MaWAT1-1在根和假茎中表达量最高,其余均在根中表达量最高;大多数MaWAT1s的表达受GA3、SA、盐胁迫和干旱等显著诱导,受高温显著抑制,同时部分成员的表达受IAA、ABA、JA、低温、机械损伤和枯萎病影响显著。【结论】MaWAT1s的表达受多种激素和逆境影响显著,可能在香蕉生长发育和抗逆防御反应中发挥着重要作用。

【Objective】WAT1 gene, a member of the Medicago truncatula NODULIN 21(MtN21) gene family, encodes a plant-specific protein that plays an important role in the secondary cell wall formation, and the transportation of the nutrients, amino acids, hormones and other substances in plant cells.Recently, its roles in the process of plant resistances to stresses were also widely identified. Up to now,however, no research has been reported on the WAT1 gene in banana. This study aims to reveal the sequence characteristics and expression pattern of the WAT1 genes in banana(Musa nana Lour.) under different phytohormones and stress treatments.【Methods】By using the Arabidopsis thaliana WAT1 gene as reference sequence, the WAT1 genes of banana were screened and selected from the banana genome data by local Blast. Then, their gDNA, CDS nucleotide sequences and encoded protein sequences were subjected to series of bioinformatics analysis. Moreover, their expressions under different phytohormones(including GA3, MeJA, ABA, SA and IAA) and stresses(including salt, low temperature,drought, high temperature, wounding and Fusarium wilt) were studied using quantitative real time PCR(qRT-PCR).【Results】Totally, the five WAT1 genes(respectively located in chromosomes 1, 3, 6, 7, and10, and named as the MaWAT1-1 to MaWAT1-5) were identified from banana. Their gDNA and CDS length respectively ranged from 1 954 bp to 2 877 bp and from 1 086 bp to 1 164 bp. Their encoded proteins respectively contained 361-387 aa with relative molecular weight of 39-42 kD, and an isoelectric point of 8.5-9.2. All these MaWAT1 s were found to be hydrophobic proteins with 10 transmembrane structures but without signal peptide. The subcellular localization prediction result showed that the MaWAT1-1, the MaWAT1-2 and the MaWAT1-4 were mainly located in the vacuole and cell membrane,the MaWAT1-3 was mainly located in cytoplasm and cell membrane, and the MaWAT1-5 was mainly located in cell membrane and chloroplast. Gene structure analysis showed that the banana MaWAT1-1,MaWAT1-2 and MaWAT1-4 owned 6 exons and 5 introns, while the MaWAT1-3 and the MaWAT1-5 had7 exons and 6 introns. Conserved domain prediction revealed that all the MaWAT1 s had two conserved EamA domains covering more than 70% of the whole proteins. The MaWAT1 s were divided into two subgroups by the phylogenetic tree analysis. One subgroup was consisted of the MaWAT1-1, the MaWAT1-2 and the MaWAT1-4(with higher similarity to the Arabidopsis WAT1), and the other subgroup was consisted of the MaWAT1-3 and MaWAT1-5. Promoter cis-acting element analysis showed that all the promoters of the MaWAT1 s contained many phytohormone and stress responsive elements,indicating that the expression of the MaWAT1 s might be regulated by these factors. Furthermore, we also found that the number and types of phytohormone responsive cis-acting elements varied a lot among these promoters. Quantitative real-time PCR(qRT-PCR) was conducted to look at the expression patterns of the MaWAT1 s in different tissues and organs and in response to the different phytohormones.Results showed that the expression of MaWAT1 s varied in different tissues and organs, the MaWAT1-4 showed the highest expression in the leaves, the MaWAT1-1 showed the highest expression in the roots and pseudostemes, while the expression of other the MaWAT1 s was the highest in the roots. The ABA responsive elements were found in the promoters of the MaWAT1-1, the MaWAT1-2 and the MaWAT1-4. Consistently, their expression was found to be induced by the ABA treatment. Notably, under the ABA treatment, the expression of the MaWAT1-2 at all the treatment time points was found to be higher than that in the control. The GA responsive element was found in the MaWAT1-1, the MaWAT1-2 and the MaWAT1-3 promoters. The expression of the MaWAT1-1 was about 6.7 times higher than that of the control at 48 h post the GA3 treatment, and the expression of the MaWAT1-2 at 12 h post the GA3 treatment was about 54.0 times higher than that of the control. The MaWAT1-1 to 4 promoters contained the MeJA responsive elements, and their expression after the MeJA treatment was found to be significantly induced at some time points after the MeJA treatment. The WAT1 was recognized as an auxin transporter. Under the IAA treatment, the expression of the MaWAT1-1, whose promoter contained an auxin responsive element, was 18.2 and 11.8 times higher than that of the control at 3 h and 6 h, respectively.The SA treatment showed a certain induction effect on the expression of the MaWAT1 s. The expression of the MaWAT1-3 to 5, whose promoters contained SA responsive elements, was significantly induced by the SA treatment at some time points. However, the expression of the MaWAT1-1 and the MaWAT1-2, whose promoters contained no such elements but several other stress responsive elements, was more significantly induced by the SA treatment and the highest expression was respectively 7.7-fold and 10.0-fold of the control. Besides, many stress responsive elements, such as drought-, low temperature-, high temperature-, anaerobic-, fungal induction-, defense stress-responsive elements, were found in the MaWAT1 s promoter. The qRT-PCR results also showed that the expression of the MaWAT1 s was significantly affected by the stress treatments and their expression patterns differed. The expression of the MaWAT1 s was significantly induced by the salt stress. For example, the expression level of the MaWAT1-1 was about 4.8 times higher than that of the control at 48 h post salt treatment, and the expression of the MaWAT1-2 at 4 h and 48 h post salt treatment was respectively 15.9 and 15.4 times of the control. Notably, the expression of the MaWAT1-3 at 48 h post salt treatment was about 103.6 times of the control. Under the drought stress, the expression of the MaWAT1-1 to 4 was mostly induced. The expression of the MaWAT1-2 was significantly higher(9.7 times) than that of the control after 4 days of drought treatment. The expression of all the MaWAT1 s was significantly suppressed by the 38 ℃ high temperature stress treatment. Under the low temperature treatment, the expression levels of the MaWAT1-1 and the MaWAT1-2 were significantly increased at 24 h and 12 h, respectively, while the expression of the MaWAT1-3 to 5 was down-regulated by the low temperature treatment. Under the wounding treatment, the expression patterns of the MaWAT1 s also varied. The expression of the MaWAT1-1 to 3 was induced to different extents after the wounding treatment, while the expression of the MaWAT1-4 was significantly down-regulated in the early stages(0.5 d and 1 d) but significantly increased 3 d after the treatment, and the expression of the MaWAT1-5 after the wounding treatment was lower than that of the control. Under the Fusarium wilt treatment, the expression levels of the MaWAT1-2 and the MaWAT1-3 were significantly higher than that of the control at some time points, while the others were lower or significantly lower than that of the control.【Conclusion】Results obtained in this study revealed that the expression of the MaWAT1 s was greatly influenced by several phytohormones and stresses, indicating that they might function in the development and stress responses in banana.