Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei, Anhui 230036, China.
J Plant Physiol. 2013 Feb 15;170(3):272-82. doi: 10.1016/j.jplph.2012.10.010. Epub 2012 Dec 8.
Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is considered to be a key enzyme in lignin biosynthesis, but little was known about CADs in tea plants (Camellia sinensis). A full-length cDNA sequence (CsCAD2) was isolated by suppressive subtractive hybridization (SSH) in Ectropis oblique feeding-induced tea plants, and another two full-length cDNA sequences (CsCAD1 and CsCAD3) were obtained from a transcriptome obtained by deep sequencing. However, they showed only 20-54% identities. Phylogenetic analysis revealed that they belonged to three different families. DNA gel blotting analysis revealed that two copies of CsCAD1 and CsCAD2 genes existed in tea genome, but CsCAD3 likely had only one copy. Recombinant proteins of these CsCADs were produced in Escherichia coli. The activity of purified recombinant CsCAD2 protein was up to 0.43 μmol min(-1) mg(-1). However, the other two recombinant proteins had lower activities, probably due to incomplete refolding. qRT-PCR analysis indicated that while CsCAD3 was strongly up-regulated in tea plants after E. oblique attack and mechanical damage, CsCAD1 and CsCAD2 showed only moderate or no changes in transcript levels. Treatment of defence-related hormones methyl jasmonate (MeJA) and salicylic acid (SA) elevated the expression of CsCAD1 and CsCAD2, but decreased the transcript abundance of CsCAD3. The transcript levels of CsCAD2 did not change after applying abscisic acid (ABA), whereas CsCAD1 and CsCAD3 were induced. These results suggested that these three CsCAD genes in tea plants may play a role in defense against insects and pathogens and adaptation to abiotic stresses and these genes likely have divergant functions.
肉桂醇脱氢酶（CAD；EC 1.1.1.195）被认为是木质素生物合成中的关键酶，但茶树（Camellia sinensis）中的 CAD 知之甚少。通过抑制性消减杂交（SSH）在斜纹夜蛾诱导的茶树中分离得到全长 cDNA 序列（CsCAD2），并从深度测序获得的转录组中获得另外两个全长 cDNA 序列（CsCAD1 和 CsCAD3）。然而，它们仅显示出 20-54%的同一性。系统发育分析表明，它们属于三个不同的家族。DNA 凝胶印迹分析表明，茶树基因组中存在两个拷贝的 CsCAD1 和 CsCAD2 基因，但 CsCAD3 可能只有一个拷贝。这些 CsCAD 的重组蛋白在大肠杆菌中产生。纯化的重组 CsCAD2 蛋白的活性高达 0.43 μmol min(-1) mg(-1)。然而，其他两种重组蛋白的活性较低，可能是由于不完全重折叠。qRT-PCR 分析表明，斜纹夜蛾攻击和机械损伤后，CsCAD3 在茶树中强烈上调，而 CsCAD1 和 CsCAD2 的转录水平仅表现出中度或无变化。防御相关激素茉莉酸甲酯（MeJA）和水杨酸（SA）处理可提高 CsCAD1 和 CsCAD2 的表达，但降低 CsCAD3 的转录丰度。应用脱落酸（ABA）后 CsCAD2 的转录水平没有变化，而 CsCAD1 和 CsCAD3 则被诱导。这些结果表明，茶树中的这三个 CsCAD 基因可能在抵御昆虫和病原体以及适应非生物胁迫方面发挥作用，这些基因可能具有不同的功能。
