新疆核桃WJ-CAD和ZJ-CAD基因克隆及时空表达表达分析

Cloning and Expression Analysis of WJ-CAD and ZJ-CAD Genes of Walnut in Xinjiang

肉桂醇脱氢酶(CAD)是木质素生物合成中最后一步反应酶。本研究采用RT-PCR技术克隆露仁核桃WJ-CAD基因和硬壳完整核桃ZJ-CAD基因,研究CAD基因在硬壳完整和露仁核桃内果皮中发育过程中的表达特性。WJ-CAD基因cDNA序列含有788 bp ORF,编码258个氨基酸,分子量84.57 kD,理论等电点5.05;ZJ-CAD基因cDNA序列含有666 bp ORF,编码332个氨基酸,分子量81.93 kD,理论等电点5.09;对所编码的蛋白预测分析表明均属于FrmA超基因家族;实时荧光定量PCR结果表明,WJ-CAD基因的相对表达量整体呈现下降-上升-下降的趋势,ZJ-CAD基因的相对表达量整体均呈现上升-下降-上升-下降的趋势,2个基因均在花后65 d表达量达到最高峰,其后期表达量均较低,CAD基因在"温138"核桃内果皮中的表达量远低于同期"纸皮"的表达量。推测"温138"核桃露仁现象由于花后65 d后木质素合成量降低,导致缺乏木质素的积累或影响木质素单体的组成,从而造成内果皮发育不完整(露仁),初步证明CAD基因参与调控木质素合成,露仁现象可能由于硬核期缺乏木质素的积累。

Cinnamyl alcohol dehydrogenase(CAD) is the last step enzyme in lignin biosynthesis. In this study, RT-PCR technology was used to clone WJ-CAD gene in bared-nut walnut and ZJ-CAD gene in hard shell intact walnut,and the expression characteristics of CAD gene in endocarp development of bared-nut walnut and hard shell intact walnut were studied. The c DNA sequence in WJ-CAD gene contained 788 bp ORF, encoding 258 amino acids,with the molecular weight of 84.57 kD and isoelectric point as 5.05. The cDNA sequence in ZJ-CAD gene contained 666 bp ORF, encoding 332 amino acids, and the molecular weight and isoelectric point were 81.93 kD and 5.09 respectively. The predicted expression of the encoded protein indicated that there were belonging to the FrmA superfamily. The results of Real-time fluorescence quantitative PCR showed that the relative expression levels of WJ-CAD gene were relatively low in the late stages, and the overall expression of WJ-CAD gene showed a r ising-decreasing-rising trend. The relative expression level of ZJ-CAD gene was higher in the early stage and ZJ-CAD gene was low in the later stage, with a rising-decreasing-rising-declined trend. The expression levels of both genes peaked at 65 d after flowering, and their expression levels were lower at later stage, the expression of CAD gene in "Wen138" walnut endocarp was much lower than that of "Zhipi" at the same time. It is speculated that the phenomenon of "Wen138" bared-nut walnut is reduced due to the decrease of lignin synthesis after 65 days after flowering, resulting in the lack of lignin accumulation or the composition of lignin monomer, resulting in incomplete development of endocarp(bared-nut), preliminary proof of CAD genes are involved in the regulation of lignin synthesis, and the Lunen phenomenon may be due to the lack of accumulation of lignin in the hard core stage.