核桃内果皮4-香豆酸辅酶A连接酶基因克隆及表达分析

doi:10.7668/hbnxb.201751031

摘要/Abstract

摘要： 为研究新疆核桃露仁机制，以露仁核桃种质温138为研究对象，其突变纸皮核桃为对照，采用RT-PCR技术克隆4CL基因并进行生物信息学分析；采用实时荧光定量PCR方法研究核桃硬化过程中4CL基因相对表达量，找到4CL基因在露仁核桃内果皮中硬核发育过程中的表达特性。结果表明，WJ-4CL基因cDNA序列包括552 bp ORF，编码184个氨基酸，分子质量20.10 ku，理论等电点5.83；ZJ-4CL基因包含453 bp ORF，编码151个氨基酸，分子质量16.52 ku，理论等电点9.35；系统进化树表明，温138与纸皮核桃聚为同一支，其亲缘关系较近。实时荧光定量PCR分析表明，露仁核桃4CL基因在花后100 d的相对表达量为花后51 d相对表达量的117倍，硬壳完整核桃4CL基因在花后86 d相对表达量为花后51 d相对表达量的205倍，初步证明4CL基因在硬壳完整和露仁核桃的不同发育时期表达量存在明显差异。表明4CL基因参与核桃内果皮（硬壳）硬化过程且影响内果皮木质素的合成，从而造成核桃内果皮发育不全，出现露仁现象。

关键词: 核桃, 木质素, 4-香豆酸辅酶A连接酶, 基因克隆, 表达分析

Abstract: In order to study the mechanism of Xinjiang walnut dew, the germplasm Wen 138 of bared-nut walnut was taken as the research object, and the mutant Zhipi walnut was used as the control. The 4CL gene was cloned by RT-PCR and analyzed by bioinformatics method. Fluorescence quantitative PCR was used to study the relative expression of 4CL gene in the process of walnut hardening, and the expression characteristics of 4CL in the development of hard shell walnut and the endocarp of bared-nut walnut were studied. The results showed that the cDNA sequence in WJ-4CL gene contained a 552 bp ORF, encoding 184 amino acids, with the molecular weight of 20.10 ku and isoelectric point of 5.83. The cDNA sequence in ZJ-4CL gene contained a 453 bp ORF, encoding 151 amino acids, and the molecular weight and isoelectric point were 16.52 ku and 9.35, respectively. The phylogenetic tree indicated that Wen 138 and Zhipi were clustered together, and their genetic relationship was relatively close. Real-time fluorescent quantitative PCR analysis indicated that relative expression of WJ-4CL gene in the 100 d after flowering was 117 times that in the 51 d after flowering, while the relative expression of ZJ-4CL gene in the 86 d after flowering was 205 times that in the 51 d after flowering, which preliminarily certified that the expression of 4CL gene was different in different development stages in the two walnut species. The results proved that the 4CL gene participated in the development of walnut endocarp harden processing and affected the synthesis of lignin in the endocarp, resulting in the hypoplasia of walnut endocarp and bared-nut.

Key words: Walnut, Lignin, 4-hydroxycinnamate CoA ligase, Gene cloning, Expression analysis

中图分类号:

Q78
S662.03

郭永翠, 秦江南, 朱玲, 张锐. 核桃内果皮4-香豆酸辅酶A连接酶基因克隆及表达分析[J]. 华北农学报, 2019, 34(2): 79-86. doi: 10.7668/hbnxb.201751031.

GUO Yongcui, QIN Jiangnan, ZHU Ling, ZHANG Rui. Cloning and Expression Analysis of 4-coumarate CoA Ligase Gene in Walnut Endocarp[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(2): 79-86. doi: 10.7668/hbnxb.201751031.

http://www.hbnxb.net/CN/Y2019/V34/I2/79

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