植物表皮蜡质构成了植物地上器官的疏水层,是保护植物免受机械损伤和病原体入侵的第一道屏障。对环境胁迫十分敏感,能够通过调节自身结构组成和含量来抵御外界环境的胁迫,对植物生长、发育及适应环境具有重要意义。此外,表皮蜡质还能使植物表面光泽度发生变化。黄瓜果实光泽性状是其重要的外观品质性状之一,对黄瓜的商品价值意义重大。本研究从黄瓜中同源克隆了WIN1/SHINE1基因,命名为CsWIN1。基因结构和氨基酸序列分析发现,该基因序列全长1456 bp,包含2个外显子,1个内含子,编码区序列（CDS）全长741 bp,编码247个氨基酸,与拟南芥WIN1/SHINE1基因序列相似性达59%,含有一个AP2保守结构域。荧光定量PCR结果表明:CsWIN1在黄瓜植株的叶片和幼果中高表达。在叶片中的特异高表达说明CsWIN1的功能很可能与叶表皮细胞发育相关;在发育初期随着果实的成熟CsWIN1的表达量逐渐上升,但在开花前的各花器官相对低表达,说明CsWIN1可能在果实发育初期发挥功能。烟草亚细胞定位结果显示CsWIN1蛋白定位于细胞核。通过在拟南芥中过表达CsWIN1,发现其与已报道的WIN1/SHINE1过表达株系表型相似。通过对CsWIN1转基因株系的基因表达分析发现,如CER1,CER2和KCS1等蜡质合成相关基因的表达受到了调控。根据以上研究结果,推测黄瓜CsWIN1基因与拟南芥WIN1/SHINE1基因在表皮蜡质合成调控上的功能是保守的,通过调控下游蜡质合成相关基因的表达从而影响蜡质的合成与代谢。此外,通过双荧光素酶报告基因转录激活实验发现,控制毛状体发育并决定表皮细胞命运早期发育阶段的关键基因Tril能够通过激活CsWIN1pro:LUC的表达来激活CsWIN1的表达水平,说明黄瓜表皮毛的发育可能与表皮蜡质的形成相关。目前,对黄瓜表皮蜡质的研究报道较少,关于黄瓜表皮蜡质的合成途径和相关分子机制仍不甚清楚。本研究通过对黄瓜CsWIN1的克隆与功能的初步验证,为进一步研究黄瓜表皮蜡质这一重要品质性状以及其与表皮毛发育的关系奠定了基础。

黄瓜（Cucumis sativus L.）是全球范围内广泛被食用的蔬菜,在众多蔬菜作物中,其首个完成基因组测序,推动了分子育种、功能基因组学及比较基因组学研究的进程。Tnt1为一类来自烟草（Nicotiana tabacum）的反转录转座子,在组织培养过程中能被再诱导转座,在植物有性繁殖时能稳定遗传和分离,因此在很多植物构建突变体库的工作中都发挥了重要的作用,但是利用Tnt1构建黄瓜突变体库的研究还未见报道。为了探索Tnt1在构建黄瓜突变体库上的应用价值,本研究将成功插入Tnt1的转基因黄瓜后代（T2代）进行自交获得T3代,并对T3代的子叶节进行离体培养获得再生植株,随后通过PCR检测鉴定再生植株及其后代是否含有Tnt1插入,通过Southern blot鉴定Tnt1在组织后代中拷贝数的变化,然后采用染色体步移技术分析后代植株中Tnt1的插入位点,初步证明了Tnt1在黄瓜中具有“复制”和再转座的特性,也获得部分突变材料。主要结果如下:1.将含有Tnt1的转基因黄瓜NCT10植株（T2）与黄瓜自交系’SA0422’进行杂交,再将F1与’SA0422’回交,获得97株BC1F1代植株,经特异性引物PCR鉴定,其中87株含有Tnt1插入,根据后代分离比推算NCT10至少含有3个Tnt1拷贝。2.将含有Tnt1的黄瓜NCT10、NCT05家系（T3）种子的子叶节进行组织培养,共获得145株再生植株。其中23粒NCT10-2种子最终获得再生植株为56株,每片子叶获得的再生芽平均为1～2个。3.采用Southern blot检测T2代NCT10和NCT05以及再生植株T10-1～10-x所包含的Tnt1拷贝数,再次确认了Tnt1插入原始黄瓜材料的基因组中,并可随着组织培养激活其转座活性,在黄瓜基因组中实现转座。4.采用染色体步移技术对NCT10、NCT05株系及其再生苗T10-1～10-x随机抽取10株进行Tnt1插入位点的侧翼序列分离,最终获得部分材料的Tnt1插入位置。结果显示,Tnt1具有位点插入随机性,可插入基因外显子区,也可插入基因间隔区。

BackgroundTrichomes are excellent model systems for the analysis of cell differentiation and play essential roles in plant protection. From cucumber inbred line 'WD1', we identified an EMS-induced trichome abnormally developing mutant, nps, which exhibited smaller, denser and no pyramid-shaped head trichomes.ResultsUsing F-2 and BC1 populations constructed from a cross between nps and '9930', the genetic analysis showed that the nps trait is controlled by a single recessive nuclear gene. We identified CsNps by map-based cloning with 576 individuals of the F-2 population generated from the cross of nps and inbred line '9930'. The CsNps was located at a 13.4-kb genomic region on chromosome 3, which region contains three predicted genes. Sequence analysis showed that only one single nucleotide mutation (C -> T) between 9930 and nps was found in the second exon of Csa3G748220, a plant-specific class I HD-Zip gene. The result of allelism test also indicated that nps is a novel allelic mutant of Mict (Micro-trichome). Thus, nps was renamed mict-L130F. By comparing the transcriptome of mict-L130F vs WD1 and 06-2 (mict) vs 06-1 (wildtype, near-isogenic line of 06-2), several potential target genes that may be related to trichome development were identified.ConclusionsOur results demonstrate that Mict-L130F is involved in the morphogenesis of trichomes. Map-based cloning of the Mict-L130F gene could promote the study of trichome development in cucumber.

Trichomes that cover the epidermis of aerial plant organs play multiple roles in plant protection. Compared with a unicellular trichome in model plants, the development mechanism of the multicellular trichome is largely unclear. Notably, variations in trichome development are often accompanied by defects in the biosynthesis of cuticle and secondary metabolites; however, major questions about the interactions between developmental differences in trichomes and defects in metabolic pathways remain unanswered. Here, we characterized the glabrous mutant mict/csgl1/cstbh via combined metabolomic and transcriptomic analyses to extend our limited knowledge regarding multicellular trichome development and metabolism in cucumber. Mict was found to be explicitly expressed within trichome cells. Transcriptomic analysis indicated that genes involved in flavonoid and cuticle metabolism are significantly downregulated in mict mutants. Further metabolomic analysis confirmed that flavonoids, lipids, and cuticle compositions are dramatically altered in mict mutants. Additional studies revealed that Mict regulates flavonoid, lipid, and cuticle biosynthesis by likely directly binding to downstream functional genes, such as CsTT4, CsFLS1, CsCER26, and CsMYB36. These findings suggest that specific metabolic pathways (e.g., flavonoids and cuticle components) are co-regulated by Mict and provide insights into transcriptional regulation mechanisms of multicellular trichome development and its specific metabolism in cucumber.

Trichomes and fruit spines are important traits that directly affect the appearance quality and commercial value of cucumber (Cucumis sativus). Tril (Trichome-less), encodes a HD-Zip IV transcription factor that plays a crucial role in the initiation of trichomes and fruit spines, but little is known about the details of the regulatory mechanisms involved. In this study, analysis of tissue expression patterns indicated that Tril is expressed and functions in the early stages of organ initiation and development. Expression of Tril under the control of its own promoter (the TrilPro::Tril-3*flag fragment) could partly rescue the mutant phenotypes of tril, csgl3 (cucumber glabrous 3, an allelic mutant of tril), and fs1 (few spines 1, a fragment substitution in the Tril promoter region), providing further evidence that Tril is responsible for the initiation of trichomes and fruit spines. In lines with dense spine, fs1-type lines, and transgenic lines of different backgrounds containing the TrilPro::Tril-3*flag foreign fragment, spine density increased in conjunction with increases in Tril expression, indicating that Tril has a gene dosage effect on fruit spine density in cucumber. Numerous Spines (NS) is a negative regulatory factor of fruit spine density. Characterization of the molecular and genetic interaction between Tril and NS/ns demonstrated that Tril functions upstream of NS with respect to spine initiation. Overall, our results reveal a novel regulatory mechanism governing the effect of Tril on fruit spine development, and provide a reference for future work on breeding for physical quality in cucumber.

The development of trichomes (spines) on cucumber fruits is an important agronomic trait. It has been reported that two MYB family members, CsMYB6 (Csa3G824850) and CsTRY (Csa5G139610) act as negative regulators of trichome or fruit spine initiation. To further study the functions of these two genes, we overexpressed them in tobacco, and found that the flowers and seed coats of transformants overexpressing CsTRY displayed an unexpected defect in pigmentation that was not observed in plants overexpressing CsMYB6. Moreover, the expression of key genes in the flavonoid synthesis pathway was repressed in CsTRY overexpressing plants, which resulted in the decrease of several important flavonoid secondary metabolites. In addition, CsTRY could interact with the AN1 homologous gene CsAN1 (Csa7G044190) in cucumber, which further confirmed that CsTRY not only regulates the development of fruit spines, but also functions in the synthesis of flavonoids, acting as the repressor of anthocyanin synthesis.