Camellia sinensis CsMYB4a participates in regulation of stamen growth by interaction with auxin signaling transduction repressor CsAUX/IAA4

Subgroup 4(Sg4)members of the R2R3-MYB are generally known as negative regulators of the phenylpropanoid pathway in plants.Our previous research showed that a R2R3-MYB Sg4 member from Camellia sinensis(CsMYB4a)inhibits expression of some genes in the phenylpropanoid pathway,but its physiological function in the tea plant remained unknown.Here,CsMYB4a was found to be highly expressed in anther and filaments,and participated in regulating filament growth.Transcriptome analysis and exogenous auxin treatment showed that the target of CsMYB4a might be the auxin signal pathway.Auxin/indole-3-acetic acid 4(AUX/IAA4),a repressor in auxin signal transduction,was detected from a yeast two-hybrid screen using CsMYB4a as bait.Gene silencing assays showed that both CsIAA4 and CsMYB4a regulate filament growth.Tobacco plants overexpressing CsIAA4 were insensitive to exogenous a-NAA,consistent with overexpression of CsMYB4a.Protein-protein interaction experiments revealed that CsMYB4a interacts with N-terminal of CsIAA4 to prevent CsIAA4 degradation.Knock out of the endogenous NtIAA4 gene,a CsIAA4 homolog,in tobacco alleviated filament growth inhibition and a-NAA insensitivity in plants overexpressing CsMYB4a.All results strongly suggest that CsMYB4a works synergistically with CsIAA4 and participates in regulation of the auxin pathway in stamen.

Chlorophyllase is transcriptionally regulated by CsMYB308/CsDOF3 in young leaves of tea plant

Chlorophyll contributes to tea coloration, which is an important factor in tea quality. Chlorophyll metabolism is induced by light, but the transcriptional regulation responsible for light-induced chlorophyll metabolism is largely unknown in tea leaves. Here, we characterized a chlorophyllase1 gene CsCLH1 from young tea leaves and showed it is essential for chlorophyll metabolism, using transient overexpression and silencing in tea leaves and ectopic overexpression in Arabidopsis. CsCLH1 was significantly induced by high light. The DOF protein CsDOF3, an upstream direct regulator of CsCLH1, was also identified. Acting as a nuclear-localized transcriptional factor, CsDOF3 responded for light and repressed CsCLH1 transcription and increased chlorophyll content by directly binding to the AAAG cis-element in the CsCLH1 promoter. CsDOF3was able to physically interact with the R2R3-MYB transcription factor CsMYB308 and interfere with transcriptional activity of CsCLH1. In addition, CsMYB308 binds to the CsCLH1 promoter to enhance CsCLH1 expression and decrease chlorophyll content. CsMYB308 and CsDOF3 act as an antagonistic complex to regulate CsCLH1 transcription and chlorophyll in young leaves. Collectively, the study adds to the understanding of the transcriptional regulation of chlorophyll in tea leaves in response to light and provides a basis for improving the appearance of tea.

茶树CsMYB基因启动子的克隆与功能分析

以茶树新品系"1005"嫩芽为材料,克隆得到CsMYB基因gDNA全长序列1 828 bp,通过染色体步移技术,分离得到CsMYB基因启动子片段1 038 bp,命名为proMYB。生物信息学分析表明,CsMYBgDNA由2个内含子和3个外显子组成,该启动子含有与提高基因转录水平相关的5′UTR Py-rich stretch顺式作用元件、启动子核心元件TATA-box和CAAT-box,还存在有激素响应元件、光响应元件、逆境应答元件以及大量功能未知或功能特异的顺式作用元件,说明proMYB具有诱导型启动子特性,CsMYB基因在茶树植物体中可能参与多种非生物胁迫应答和激素信号传导;通过烟草的瞬时表达结果表明,该启动子能驱动下游基因表达,具有启动子活性。

黄瓜抗旱相关基因CsMYB94的克隆与表达分析

干旱胁迫是黄瓜质量和产量最严重威胁之一。为了更好地了解黄瓜响应水分胁迫的分子机理,从前期工作中得到一个与干旱有关且显著上调表达的差异表达基因CsaV3_3G039670(log_(2)FC=1.10),其在拟南芥中同源基因AtMYB94通过调节植株表皮蜡质的含量来提高抗旱性,因此命名为CsMYB94。CsMYB94基因CDS序列全长为885 bp,编码294个氨基酸。通过对蛋白结构域分析,CDS序列中含有2个DNA-结合结构域,属于R2R3类型的MYB转录因子。蛋白分析发现,CsMYB94蛋白分子式为C_(1426)H_(2242)N_(404)O_(454)S_(11);预期分子量为3.27 ku;理论等电点为6.53;为疏水性蛋白质,且为不稳定蛋白。通过亚细胞定位软件分析发现,CsMYB94蛋白被定位在细胞核。通过构建系统进化树分析表明,黄瓜CsMYB94与白藻中MYB94亲缘关系最近,与南瓜、苦瓜和甜瓜中的MYB94亲缘关系较近。通过qRT-PCR分析结果表明,CsMYB94在黄瓜叶片中表达量最高,而在雄花、卷须和茎中的表达量相对较低。因此,推测CsMYB94也是通过调节黄瓜蜡质含量来响应干旱胁迫,具体调控路径还需进一步研究。

茶树CsMYB-like基因的克隆及表达分析

MYB是一类重要的转录因子,广泛参与植物生长发育、次生代谢调控和非生物胁迫响应等生物学过程。本研究从茶树中成功克隆了一条CsMYB-like转录因子,CDS序列全长为1113 bp,编码370个氨基酸。生物信息学分析表明其与蓝果树(Nyssa sinensis)、葡萄(Vitis vinifera)等MYB-like氨基酸序列有较高的相似性。亚细胞定位分析表明CsMYB-like基因编码的蛋白主要定位于细胞核。实时荧光定量PCR结果表明,CsMYB-like基因在不同的茶树品种中表达量不同,并且在不同浓度的纳米硒肥处理下表达有差异,在稀释100、200和300倍处理下,该基因的相对表达量分别降低了71.7%、90.3%和51.3%,而400倍处理对该基因的表达无显著影响,表明一定浓度的纳米硒肥能显著抑制CsMYB-like基因的表达。

黄瓜CsMYB108基因的克隆与序列分析

中国黄瓜品种繁多,目前黄瓜杂种优势是选育新品种的关键,杂种优势是一种植物共有的独特生物现象,然而黄瓜杂种优势的分子机理研究还尚未深入。为探究黄瓜杂种优势形成的机理,本研究中父本D0708-2田间表现为雌雄同株,早熟性佳,但是抗病抗逆性差,母本D1104-2-4田间表现为强雌性系,抗病抗逆性佳。以超亲优势的杂交种C15-114为试验材料,筛选出一个与父母本的差异基因Cs MYB108,并运用基因克隆技术克隆了该基因并对该蛋白进行生物信息学分析,挖掘该基因在黄瓜遭遇逆境胁迫过程中所参与的作用机制。结果表明:CsMYB108转录因子全长675 bp,编码225个氨基酸,该转录因子编码的蛋白质属于细胞核中的疏水性蛋白质,并无明显信号肽和无跨膜结构。与甜瓜构成同一进化分支,同源性较高。本研究结果为黄瓜的杂种优势提供理论依据,也为进一步试验研究提供基因材料。