Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.     

烟夜蛾精氨酸激酶基因的克隆及mRNA表达分析

为了深入了解精氨酸激酶基因的作用和寻求害虫防治新的分子靶标, 本研究采用RT-PCR和RACE技术, 从烟夜蛾Helicoverpa assulta脂肪体中克隆了精氨酸激酶cDNA序列, 命名为HassAK（GenBank登录号: HQ336337）, 并采用荧光定量PCR测定了HassAK基因在不同发育阶段（4龄幼虫第1天到化蛹第1天）、 不同组织（头部、 中肠、 脂肪体、 体壁和腹足）和不同温度条件下的表达情况。测序和序列分析结果表明, HassAK基因阅读框架全长1 068 bp, 编码355个氨基酸残基, 预测蛋白质分子量和等电点分别为40.0 kD和5.76。氨基酸序列分析表明, 该序列具有精氨酸激酶典型的酶活性部位、 酶活性中心位点和能形成离子偶结构的保守区。序列比对结果表明, HassAK与其他昆虫AK的氨基酸序列具有70%以上的一致性。荧光定量分析结果显示, HassAK基因在幼虫头部、 中肠、 脂肪体、 体壁和腹足均可表达, 其中以腹足和中肠内的表达水平较高。时序表达分析表明, 预蛹期HassAK基因的表达量达到高峰。此外, 高温和低温均诱导HassAK基因的表达, 说明该基因可能参与昆虫抵御外界不良环境。     

Genome‐wide identification and expression analysis reveal the potential function of ethylene responsive factor gene family in response to Botrytis cinerea infection and ovule development in grapes (Vitis vinifera L.)

• The prevention of Botrytis cinerea infection and the study of grape seedlessness are very important for grape industries. Finding correlated regulatory genes is an important approach towards understanding their molecular mechanisms.
• Ethylene responsive factor (ERF) gene family play critical roles in defence networks and the growth of plants. To date, no large‐scale study of the ERF proteins associated with pathogen defence and ovule development has been performed in grape (Vitis vinifera L.). In the present study, we identified 113 ERF genes (VvERF) and named them based on their chromosome locations. The ERF genes could be divided into 11 groups based on a multiple sequence alignment and a phylogenetic comparison with homologues from Arabidopsis thaliana. Synteny analysis and Ka/Ks ratio calculation suggested that segmental and tandem duplications contributed to the expansion of the ERF gene family. The evolutionary relationships between the VvERF genes were investigated by exon–intron structure characterisation, and an analysis of the cis‐acting regulatory elements in their promoters suggested potential regulation after stress or hormone treatments.
• Expression profiling after infection with the fungus, B. cinerea, indicated that ERF genes function in responses to pathogen attack. In addition, the expression levels of most ERF genes were much higher during ovule development in seedless grapes, suggesting a role in ovule abortion related to seedlessness.
• Taken together, these results indicate that VvERF proteins are involved in responses to Botrytis cinerea infection and in grape ovule development. This information may help guide strategies to improve grape production.