核桃内果皮4-香豆酸辅酶A连接酶基因克隆及表达分析
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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。
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 4 CL gene was cloned by RT-PCR and analyzed by bioinformatics method. Fluorescence quantitative PCR was used to study the relative expression of 4 CL gene in the process of walnut hardening, and the expression characteristics of 4 CL 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-4 CL 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-4 CL 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-4 CL gene in the 100 d after flowering was 117 times that in the 51 d after flowering, while the relative expression of ZJ-4 CL gene in the 86 d after flowering was 205 times that in the 51 d after flowering, which preliminarily certified that the expression of 4 CL gene was different in different development stages in the two walnut species. The results proved that the 4 CL 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.
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 4 CL gene was cloned by RT-PCR and analyzed by bioinformatics method. Fluorescence quantitative PCR was used to study the relative expression of 4 CL gene in the process of walnut hardening, and the expression characteristics of 4 CL 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-4 CL 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-4 CL 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-4 CL gene in the 100 d after flowering was 117 times that in the 51 d after flowering, while the relative expression of ZJ-4 CL gene in the 86 d after flowering was 205 times that in the 51 d after flowering, which preliminarily certified that the expression of 4 CL gene was different in different development stages in the two walnut species. The results proved that the 4 CL 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.
引文
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[2] 徐凯,夏玉芳,李允祥,谢钊俊.贞丰核桃主要经济性状评价[J].种子,2016,35(4):1001-4705.doi:10.16590/j.cnki.1001-4705.2016.04.118.Xu K,Xia Y F,Li Y X,Xie Z J.Evaluation to the main economic characters of Zhenfeng walnut[J].Seed,2016,35(4):1001-4705.
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[4] 武际,郭熙盛,鲁剑巍,万水霞,王允青,许征宇,张晓玲.不同水稻栽培模式下小麦秸秆腐解特征及对土壤生物学特性和养分状况的影响[J].生态学报,2013,33(2):565-575.doi:10.5846/stxb201111201769.Wu J,Guo X C,Lu J W,Wan S X,Wang Y Q,Xu Z Y,Zhang X L.Decomposition characteristics of wheat straw and effects on soil biological properties and nutrient status under different rice cultivation[J].Acta Ecologica Sinica,2013,33(2):565-575.
[5] 文菁,赵书岗,王红霞,张志华,李夕勃.核桃硬壳发育期内果皮木质素与相关酶活性的变化[J].园艺学报,2015,42(11):2144-2152.doi:10.16420/j.issn.0513-353x.2015-0261.Wen J,Zhao S G,Wang H X,Zhang Z H,Li X B.Changes of lignin content and its related enzyme activities in endocarp during walnut shell development period[J].Acta Horticulturae Sinica,2015,42(11):2144-2152.
[6] 邹丽秋,王彩霞,匡雪君,李滢,孙超.黄酮类化合物合成途径及合成生物学研究进展[J].中国中药杂志,2016,41(22):4124-4128.doi:10.4268/cjcmm20162207.Zou L Q,Wang C X,Kuang X J,Li Y,Sun C.Advance in flavonoids biosynthetic pathway and synthetic biology[J].China Journal of Chinese Materia Medica,2016,41(22):4124-4128.
[7] Duthie G,Crozier A.Plant-derived phenolic antioxidants[J].Current Opinion in Lipidology,2000,11(1):43-47.doi:10.1097/00075197-200011000-00006.
[8] 曹运鹏,方志,李姝妹,闫冲冲,丁庆庆,程曦,林毅,郭宁,蔡永萍.砀山酥梨4CL基因家族的全基因组鉴定与分析[J].遗传,2015,37(7):711-719.doi:10.16288/j.yczz.15-069.Cao Y P,Fang Z,Li S M,Yan C C,Ding Q Q,Cheng X,Lin Y,Guo N,Cai Y P.Genome-wide identification and analyses of 4CL gene families in Pyrus bretschneideri Rehd[J].Hereditas,2015,37(7):711-719.
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[16] 黄满芬,王恒,方荣俊,赵卫国,张林,潘刚,刘利.桑树肉桂酸-4-羟化酶基因(MmC4H)的克隆及在不同桑品种间的表达差异[J].蚕业科学,2014,40(4):592-600.doi:10.13441/j.cnki.cykx.2014.04.005.Huang M F,Wang H,Fang R J,Zhao W G,Zhang L,Pan G,Liu L.Cloning and differential expression of cinnamic acid 4-Hydroxylase gene in different mulberry varieties[J].Science of Sericulture,2014,40(4):592-600.
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