Lin Fucheng

烟草HD-ZIP IV转录因子 NtPDF2 基因的克隆及表达分析

为了明确NtPDF2在烟草中的生物学功能及其调控腺毛分化发育的作用机制，同源克隆了烟草 NtPDF2 基因，并对其序列、基因结构、进化关系以及表达模式进行分析。结果表明： NtPDF2 CDS全长为2 199 bp，编码732个氨基酸残基。烟草PDF2蛋白质序列高度保守，尤其是C端序列，均含有3个保守结构域(Homeobox domain，START domain和SAD domain)和1个保守LZ(Leucine zipper)元件。烟草 PDF2 基因结构高度保守，均由10个外显子和9个内含子组成。进化分析表明，普通烟草 NtPDF2 基因是由林烟草 ML1 基因进化而来。表达分析显示，烟草 PDF2 基因表达具有明显的时空特异性。干旱、黑暗和磷饥饿胁迫可显著下调 NtPDF2 基因的表达，而盐胁迫对其没有明显影响； NtPDF2 基因的表达在赤霉素(GA)、茉莉酸甲酯(MeJA)、脱落酸(ABA)和打顶处理条件下均显著上调，这暗示烟草 NtPDF2 基因参与了调控多种非生物胁迫和激素应答过程。

利用CRISPR/Cas9技术的烟草 NtLS 基因敲除分析

为了培育无腋芽或少腋芽烟草，基于CRISPR/Cas9系统，对烟草 NtLS 基因进行靶向基因组编辑，构建了2个特异靶向 NtLS 基因的CRISPR/Cas9载体cLS1和cLS2，并借助农杆菌介导的烟草转基因技术转化烤烟品种K326，获得20株抗卡那霉素的阳性烟株。10株阳性烟株经过PCR扩增、测序和比对，检测到2株发生蛋白翻译错误的T 0 突变烟株。研究结果不仅为腋芽形成的分子机理研究创制了有价值的突变体，而且为培育无腋芽或少腋芽优质烤烟品系提供了遗传材料。

Cloning, subcellular localization, and expression analysis of NtGGPPS 4 gene in Nicotiana tabacum

Geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP). It is an important branching enzyme for the synthesis of terpenoids, which are the precursors of the main aroma components in tobacco and cigarette. A new cDNA sequence encoding GGPPS (named NtGGPPS4) was cloned from the tobacco (Nicotiana tabacum) by RACE (Rapid amplification of cDNA ends) method. NtGGPPS4 had a 1 119 bp ORF (Open reading frame) that encoded 372 amino acids. The deduced amino acid sequence shared a high similarity with other plant GGPPSs, and contained two Asp-rich motifs characteristic of the isoprenyl synthase family. RT-qPCR analysis showed that NtGGPPS4 was ubiquitously expressed in all organs at all growth stages and its expression increased significantly in stem after topping. Transient expression of GFP-NtGGPPS4 in tobacco protoplast indicated that NtGGPPS4 was localized in mitochondria.Geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP). It is an important branching enzyme for the synthesis of terpenoids, which are the precursors of the main aroma components in tobacco and cigarette. A new cDNA sequence encoding GGPPS (named NtGGPPS4) was cloned from the tobacco (Nicotiana tabacum) by RACE (Rapid amplification of cDNA ends) method. NtGGPPS4 had a 1 119 bp ORF (Open reading frame) that encoded 372 amino acids. The deduced amino acid sequence shared a high similarity with other plant GGPPSs, and contained two Asp-rich motifs characteristic of the isoprenyl synthase family. RT-qPCR analysis showed that NtGGPPS4 was ubiquitously expressed in all organs at all growth stages and its expression increased significantly in stem after topping. Transient expression of GFP-NtGGPPS4 in tobacco protoplast indicated that NtGGPPS4 was localized in mitochondria.

Sequence analysis and expression pattern ofMGTA1 gene in rice blast pathogenMagnaporthe grisea

MGTA1, a putative fungal Zn(II)2Cys6 transcriptional activator-encoding gene, was isolated from rice blast pathogenMagnaporthe grisea, which is homologous toCLTA1 fromColletotrichum lindemuthianum with 51% identity at protein level.MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide.MGTA1 gene exists as a single copy in genomes of 7 strains ofM. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy11.