5羟色胺-N-乙酰转移酶(SNAT)是褪黑素合成过程中的关键酶之一.为揭示香蕉SNAT基因的功能,该研究对香蕉SNAT进行了全基因组鉴定,获得其中2个成员(MaSNAT1和MaSNAT2)并对它们进行克隆验证,结果发现仅MaSNAT2表达.对MaSNAT2进行了系列生物信息学分析,并通过实时荧光定量PCR技术研究其在MeJA、ABA、GA3、褪黑素及低温处理下的表达模式.结果显示:(l)MaSNATT2基因CDS长度为741 bp,编码246个氨基酸;MaSNAT2蛋白定位于叶绿体,具有GNAT超家族典型保守Acetyltransf_7结构域;MaSNAT2二级结构主要由α螺旋、β折叠和无规则卷曲组成,三级结构与水稻OsSNAT相似度为81.60％.(2)MaSNAT2蛋白与小果野蕉SNAT相似度最高,亲缘关系最近;MaSNAT2启动子具有MeJA、ABA和GA3等激素响应元件.(3)qRT-PCR结果显示,MaSNAT2基因的表达受ABA抑制;GA3处理8 h和48 h后MaSNAT2表达量分别升高15.1倍和16.2倍;MeJA处理4 h后MaSNAT2表达量提高至5.2倍;褪黑素及低温处理能显著诱导MaS-NAT2的表达.研究表明,MaSNAT2属于GNAT超家族,其表达受GA3和MeJA诱导,受ABA抑制;该基因在香蕉响应低温胁迫过程中也发挥重要作用.

Ever since their discovery, introns within the coding sequence (CDS) of transcripts have been paid great attention. However, the introns located in the untranslated regions (UTRs) are often ignored. Here, we identified, characterized and compared the UTR introns (UIs) from six citrus species. Results showed that the average intron number of UTRs is greatly lower than that of CDSs. Among all six citrus species, the number and density of 5′UTR introns (5UIs) are higher than those of 3′UTR introns (3UIs). The UI densities varied greatly among different citrus species. There are 11 and 9 types of splice site (SS) pairs for the UIs of C. sinensis and C. medica, respectively. However, the UIs of the other four citrus species all own only three kinds of SS pairs. The ‘GT-AG’, accounting for more than 95% of both 5UIs and 3UIs SS pairs for all the six species, is the most popular type. Moreover, 81 5UIs and 26 3UIs were identified as common UIs among the six citrus species, and the transcripts containing these common UIs were mostly involved in gene expression or gene expression regulation. Our study revealed that the UIs’ length, abundance, density and SS pair types varied among different citrus species and that many UI-containing genes play important roles in gene expression regulation. Our findings have great implications for future citrus UI function research.

为研究印度梨形孢和尖孢镰刀菌古巴专化型热带4号小种(FocTR4)对香蕉根系微生物群落结构的影响,利用Illu-mina HiSeqXten测序平台分别对接种印度梨形孢(S+)、FocTR4(F+)和2种菌(SF)的香蕉根系进行了宏基因组测序分析.结果表明,接种印度梨形孢和FocTR4后,香蕉根系的微生物组成和物种多样性发生了显著变化.在门分类水平上,各组的优势菌门组成相似,但S+组中的厚壁菌门(Firmicutes)、F+组中的放线菌门(Actinobacteria)和SF组中的变形菌门(Pro-teobacteria)相对丰度显著提高,分别为对照组(接种PDB溶液)的1.97、1.84和2.47倍;在属分类水平上,各组优势菌属组成相似,但S+组中葡萄球菌属(Staphylococcus)的占比(26.24％)高于其他组,F+组显著富集链霉菌属(Streptomyces),占比达到44.38％,而SF组的根瘤菌属(Rhizobium)占比(58.91％)显著高于其他组.

克隆香蕉含VQ基序蛋白基因MaVQ1,研究其序列特征及其在不同激素处理和逆境胁迫下的表达模式.采用RT-PCR技术从'天宝蕉'中克隆了该基因,对其进行生物信息学分析,并利用实时定量PCR技术(qRT-PCR)研究它在不同组织部位和不同激素、不同逆境胁迫处理下的表达情况.结果显示:MaVQ1编码序列(CDS)长为459 bp,可编码一个分子式为C732H1164N210O207S3、分子量为16 314.76、等电点为10.19的不稳定亲水性蛋白.MaVQ1含有保守的VQ结构域,不含跨膜结构和信号肽,与小果野蕉VQ亲缘关系最近.亚细胞定位预测结果显示MaVQ1主要定位在细胞核.蛋白互作预测结果显示MaVQ1与其他香蕉VQ蛋白以及WRKY互作系数最高.启动子顺式作用元件预测结果显示MaVQ1启动子包含多种光响应元件、激素响应元件和逆境胁迫相关作用元件.转录因子结合位点分析结果显示其启动子上存在大量的ERF结合位点.qRT-PCR结果显示:MaVQ1在不同组织部位中的表达无显著差异,其表达受茉莉酸、脱落酸和低温显著诱导,受高温和干旱抑制.本研究表明,与其他植物VQ类似,MaVQ1的表达受多种激素和逆境影响,暗示其可能在香蕉抗逆防御反应过程中发挥着重要调节作用.

Accumulated evidence has shown that CDS introns (CIs) play important roles in regulating gene expression. However, research on UTR introns (UIs) is limited. In this study, UIs (including 5′UTR and 3′UTR introns (5UIs and 3UIs)) were identified from the Atalantia buxifolia genome. The length and nucleotide distribution characteristics of both 5UIs and 3UIs and the distributions of cis-acting elements and transcription factor binding sites (TFBSs) in 5UIs were investigated. Moreover, PageMan enrichment analysis was applied to show the possible roles of transcripts containing UIs (UI-Ts). In total, 1077 5UIs and 866 3UIs were identified from 897 5UI-Ts and 670 3UI-Ts, respectively. Among them, 765 (85.28%) 5UI-Ts and 527 (78.66%) 3UI-Ts contained only one UI, and 94 (6.38%) UI-Ts contained both 5UI and 3UI. The UI density was lower than that of CDS introns, but their mean and median intron sizes were ~2 times those of the CDS introns. The A. buxifolia 5UIs were rich in gene-expression-enhancement-related elements and contained many TFBSs for BBR-BPC, MIKC_MADS, AP2 and Dof TFs, indicating that 5UIs play a role in regulating or enhancing the expression of downstream genes. Enrichment analysis revealed that UI-Ts involved in ‘not assigned’ and ‘RNA’ pathways were significantly enriched. Noteworthily, 119 (85.61%) of the 3UI-Ts were genes encoding pentatricopeptide (PPR) repeat-containing proteins. These results will be helpful for the future study of the regulatory roles of UIs in A. buxifolia.

Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis-elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.

The LOX genes have been identified and characterized in many plant species, but studies on the banana LOX genes are very limited. In this study, we respectively identified 18 MaLOX, 11 MbLOX, and 12 MiLOX genes from the Musa acuminata, M. balbisiana and M. itinerans genome data, investigated their gene structures and characterized the physicochemical properties of their encoded proteins. Banana LOXs showed a preference for using and ending with G/C and their encoded proteins can be classified into 9-LOX, Type I 13-LOX and Type II 13-LOX subfamilies. The expansion of the MaLOXs might result from the combined actions of genome-wide, tandem, and segmental duplications. However, tandem and segmental duplications contribute to the expansion of MbLOXs. Transcriptome data based gene expression analysis showed that MaLOX1, 4, and 7 were highly expressed in fruit and their expression levels were significantly regulated by ethylene. And 11, 12 and 7 MaLOXs were found to be low temperature-, high temperature-, and Fusarium oxysporum f. sp. Cubense tropical race 4 (FocTR4)-responsive, respectively. MaLOX8, 9 and 13 are responsive to all the three stresses, MaLOX4 and MaLOX12 are high temperature- and FocTR4-responsive; MaLOX6 and MaLOX17 are significantly induced by low temperature and FocTR4; and the expression of MaLOX7 and MaLOX16 are only affected by high temperature. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression levels of several MaLOXs are regulated by MeJA and FocTR4, indicating that they can increase the resistance of banana by regulating the JA pathway. Additionally, the weighted gene co-expression network analysis (WGCNA) of MaLOXs revealed 3 models respectively for 5 (MaLOX7-11), 3 (MaLOX6, 13, and 17), and 1 (MaLOX12) MaLOX genes. Our findings can provide valuable information for the characterization, evolution, diversity and functionality of MaLOX, MbLOX and MiLOX genes and are helpful for understanding the roles of LOXs in banana growth and development and adaptations to different stresses.

为探究一个受低温诱导的香蕉MaTIFY9基因的序列特征及在不同激素和逆境胁迫处理下的表达模式,分别使用PCR和反转录PCR (RT-PCR)技术克隆该基因的gDNA和cDNA序列,分析其核苷酸和编码蛋白序列特性,利用实时荧光定量PCR (qRT-PCR)技术研究其在不同器官和不同激素、逆境胁迫处理下的表达情况.结果 显示:MaTIFY9编码了一个分子式为C841H1346N236O249S7,氨基酸大小为172 aa,分子量为18989.93,等电点为9.55的不稳定亲水性蛋白.MaTIFY9无信号肽和跨膜结构,具有TIFY和Jas (CCT-2)两个保守结构域,属于JAZ亚家族,与小果野蕉TIFY9亲缘关系最近.亚细胞定位预测结果显示MaTIFY9主要定位于细胞核.MaTIFY9启动子含有大量的光响应相关元件,此外还含有许多防御及应激、SA和ABA响应元件.qRT-PCR结果显示MaTIFY9在根中表达量最高,其次是假茎和球茎,叶最低;其表达受脱落酸ABA、水杨酸SA和枯萎病显著诱导,受高温抑制.本研究表明,与其他植物TIFy类似,MaTIFY表达受多种激素和逆境调控,可能在香蕉响应非生物和生物逆境胁迫过程中发挥重要作用.

Fusarium wilt disease, caused by Fusarium oxysporum f.sp. cubense (Foc), has been recognized as the most devastating disease to banana. The regulatory role of long non-coding RNAs (lncRNAs) in plant defense has been verified in many plant species. However, the understanding of their role during early FocTR4 (Foc tropical race 4) infection stage is very limited. In this study, lncRNA sequencing was used to reveal banana root transcriptome profile changes during early FocTR4 infection stages. Quantitative real time PCR (qRT-PCR) was performed to confirm the expression of eight differentially expressed (DE) lncRNAs (DELs) and their predicted target genes (DETs), and three DE genes (DEGs). Totally, 12,109 lncRNAs, 36,519 mRNAs and 2642 novel genes were obtained, of which 1398 (including 78 DELs, 1220 DE known genes and 100 DE novel genes) were identified as FocTR4 responsive DE transcripts. Gene function analysis revealed that most DEGs were involved in biosynthesis of secondary metabolites, plant–pathogen interaction, plant hormone signal transduction, phenylalanine metabolism, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism and so on. Coincidently, many DETs have been identified as DEGs in previous transcriptome studies. Moreover, many DETs were found to be involved in ribosome, oxidative phosphorylation, lipoic acid metabolism, ubiquitin mediated proteolysis, N-glycan biosynthesis, protein processing in endoplasmic reticulum and DNA damage response pathways. QRT-PCR result showed the expression patterns of the selected transcripts were mostly consistent with our lncRNA sequencing data. Our present study showed the regulatory role of lncRNAs on known biotic and abiotic stress responsive genes and some new-found FocTR4 responsive genes, which can provide new insights into FocTR4-induced changes in the banana root transcriptome during the early pathogen infection stage.

[目的]研究印度梨形孢对金柑种子萌发及幼苗生长的影响.[方法]分别试验了印度梨形孢发酵液不同浸种时长(0、1、2、4、8、12和16 h)对'尤溪金柑'种子萌发和幼苗生长的影响.[结果]印度梨形孢发酵液浸种对金柑种子萌发率和幼苗叶片数无显著影响,但浸种1 h组金柑种子萌发率最高;不同时长浸种对金柑幼苗根系长度和地上部高度的促进效果呈波动变化,1 h浸种组最佳;播种后前2个月浸种1 h和4 h可以提高的金柑幼苗根系重量,但后期仅长时间浸种(≥8 h)表现出促进作用;播种后1个月浸种2 h和4 h对地上部重量具有促进效果,但后期仅浸种12 h表现为促进作用.[结论]该研究可为印度梨形孢在金柑实生育苗中的应用奠定基础.

Introns exist not only in coding sequences (CDSs) but also in untranslated regions (UTRs) of a gene. Recent studies in animals and model plants such as Arabidopsis have revealed that the UTR-introns (UIs) are widely presented in most genomes and involved in regulation of gene expression or RNA stability. In the present study, we identified introns at both 5′UTRs (5UIs) and 3′UTRs (3UIs) of sweet orange genes, investigated their size and nucleotide distribution characteristics, and explored the distribution of cis-elements in the UI sequences. Functional category of genes with predicted UIs were further analyzed using GO, KEGG, and PageMan enrichment. In addition, the organ-dependent splicing and abundance of selected UI-containing genes in root, leaf, and stem were experimentally determined. Totally, we identified 825 UI- and 570 3UI-containing transcripts, corresponding to 617 and 469 genes, respectively. Among them, 74 genes contain both 5UI and 3UI. Nucleotide distribution analysis showed that 5UI distribution is biased at both ends of 5′UTR whiles 3UI distribution is biased close to the start site of 3′UTR. Cis- elements analysis revealed that 5UI and 3UI sequences were rich of promoter-enhancing related elements, indicating that they might function in regulating the expression through them. Function enrichment analysis revealed that genes containing 5UI are significantly enriched in the RNA transport pathway. While, genes containing 3UI are significantly enriched in splicesome. Notably, many pentatricopeptide repeat-containing protein genes and the disease resistance genes were identified to be 3UI-containing. RT-PCR result confirmed the existence of UIs in the eight selected gene transcripts whereas alternative splicing events were found in some of them. Meanwhile, qRT-PCR result showed that UIs were differentially expressed among organs, and significant correlation was found between some genes and their UIs, for example: The expression of VPS28 and its 3UI was significantly negative correlated. This is the first report about the UIs in sweet orange from genome-wide level, which could provide evidence for further understanding of the role of UIs in gene expression regulation.

[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MaWATs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.

为揭示甜橙组蛋白乙酰转移酶1基因(CsHAC1)在柑橘黄龙病(HLB)侵染过程中的响应机制,利用PCR和RT-PCR分别克隆该基因gDNA和cDNA序列,并进行系列生物信息学分析.同时,还对CsHAC1互作蛋白进行预测并研究它们在感染HLB的柑橘中的表达情况.结果显示,该基因编码序列(CDS)全长为5 307 bp,预测可编码含有1 768个氨基酸、无信号肽和跨膜结构的蛋白质.亚细胞定位预测的结果显示CsHAC1主要定位在细胞核.CsHAC1含有ZnFTAZ、PHD、HAT KAT11、ZnF ZZ等多个保守结构域,蛋白互作预测结果显示CsHAC1与ZC3H19L、SUMOs和HAM1L等蛋白存在互作关系.启动子顺式作用元件预测结果显示CsHAC1启动子除含有大量光响应元件外还含有一些逆境(如低温、防御和应激、厌氧等)和激素(如脱落酸、生长素、水杨酸等)相关元件.通过分析CsHAC1及其互作蛋白编码基因在感染黄龙病的柑橘根和叶片中的表达情况发现,CsHAC1在叶片中的表达受HLB诱导,且与HAM1L的表达呈显著负相关.本研究结果表明CsHAC1可能和它的互作蛋白基因一起通过表观遗传调控参与柑橘对HLB侵染的响应.

In this study, we reported the complete chloroplast genome of Fortunella crassifolia Swingle using the HiSeq-4000 sequencing. The chloroplast genome size is 160,229?bp, which consists of a large single-copy region (87,774?bp), a small single-copy region (18,721?bp), and a pair of IR regions (26,867?bp). The chloroplast genome contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs, and 4 rRNAs. Phylogenetic maximum likelihood analysis showed that F. crassifolia was closest to Hongkong kumquat (F. hindsii). The complete chloroplast genome would be subsequently used for citrus species researches.

通过盆栽试验,研究不同剂量的枯草芽孢杆菌Bacillus subtilis对嘉宝果Myrciaria cauli flora Berg实生苗地上部生长量和叶绿素含量的影响.结果表明:枯草芽孢杆菌在一定程度上对嘉宝果的株高、茎粗、冠幅、各主枝新梢数、各主枝最长新梢以及叶绿素含量均有促进作用.其中,每盆3 g的枯草芽孢杆菌对嘉宝果的促生作用最为显著.栽培生产中将枯草芽孢杆菌剂量范围控制在每667m2900～1 800 g对促进嘉宝果生长发育最为适宜.

为改善3年生嘉宝果(Myrciaria cauliflora Berg)的栽培基质并促进其生长和发育,通过田间盆栽试验研究了有机碳对其实生苗地上部生长量和叶绿素含量的影响.结果表明:有机碳对嘉宝果实生苗地上部生长量和叶绿素含量均有一定的促进作用,以每盆150 mL有机碳效果最为显著.生产中将有机碳剂量控制在每盆100～200 mL对改良嘉宝果生长的土壤基质最为适宜,能有效促进其生长发育.