桑树硝酸还原酶基因MaNR的克隆及其表达分析

doi:10.3864/j.issn.0578-1752.2014.12.019

摘要/Abstract

摘要： 【目的】以桑树栽培品种桂优62号为材料，克隆桑树硝酸还原酶（nitrate reductase，NR）基因全长cDNA和DNA序列并分析其序列特征，在此基础上研究桑树硝酸还原酶基因在桑树细胞脱分化和分化过程中的表达特征以及影响因素，为进一步阐述硝酸还原酶在桑树生长发育中的作用机制奠定基础。【方法】基于单倍体川桑（Morus notabilis Schneid）基因组数据（http://morus.swu.edu.cn/morusdb）注释的scaffold570序列设计特异引物，硝酸钾处理桑树叶片48 h后使用RNAiso Plus（TaKaRa）和改良CTAB法提取总RNA及基因组DNA，分别以cDNA和DNA为模板，用RT-PCR法克隆获得桑树NR全长cDNA和DNA序列，运用生物信息学手段对推定的氨基酸序列进行分析。以桑胚轴为外植体，在无菌条件下，接种在不同氮源、生长调节物质的培养基中，通过桑树胚轴离体再生过程和real-time PCR方法研究硝酸还原酶基因在离体再生过程中的表达差异以及不同氮源、生长调节物质对NR表达的影响。【结果】克隆获得的桑树NR全长CDS序列，长2 730 bp，编码909个氨基酸，推导的蛋白质分子量为102.84 kD，等电点为6.76。基因组序列长5 142 bp，包含5个外显子和4个内含子，具有完整的5个结构域。通过氨基酸序列比对发现，其序列高度保守，与川桑NR序列同源性达95%，与蔷薇科果树NR序列同源性达78%。聚类分析表明，单子叶植物聚为一组，桑树与蔷薇科果树聚为一组。GenBank登录号分别为KF992020.1和KF992021.1。NR在桑根中表达量最高，叶中表达次之，茎中表达最少；谷氨酸显著提高NR在桑叶中的表达量，GA3显著提高NR在桑茎中的表达量。桑胚轴在单一的NH4+-N培养基中，不能被诱导出愈伤组织及丛生芽，在单一的NO3--N培养基中可以诱导出愈伤组织和丛生芽；但丛生芽继代培养在单一NH4+-N的培养基中和单一的NO3--N培养基中都可以生长。real-time PCR结果显示，NR在子叶和胚轴中的表达量高于胚根。桑胚轴在离体诱导愈伤组织和丛生芽分化过程中，NR的表达量逐渐增加，丛生芽形成后NR的表达量降低并趋于稳定。NH4+-N和NO3-N对NR在继代丛生芽中没有显著影响，谷氨酸对NR在继代丛生芽中有抑制作用，随着培养时间延长，丛生芽的NR相对表达量都降低。【结论】获得了桑树NR全长cDNA序列。硝态氮是桑胚轴诱导的必需营养因子，NR的表达受到桑树细胞脱分化和分化影响。

关键词: 桑树 , 硝酸还原酶 , MaNR , 细胞分化 , 实时荧光定量PCR

Abstract: 【Objective】 The objective of this study is to investigate the relationship between nitrate reductase (NR) and growth and regeneration of mulberry. A full length cDNA named MaNR was cloned from mulberry cultivar Guiyou No.62 and the sequence characteristics of MaNR were analyzed. Furthermore, the expression mode and influence factors during regeneration of mulberry hypocotyl regeneration in vitro was studied.【Method】 Specific primer sequences were designed according to the annotated gene scaffold 570 released in Morus notabilis genome database http://morus.swu.edu.cn/morusdb. Total RNA and DNA were extracted from mulberry leaf treated by KNO3, using RNAiso Plus (TaKaRa) and a modified CTAB method. cDNA and DNA were used as templates, RT-PCR was used to amplify MaNRcDNA sequence from RNA pools generated from KNO3-treated mulberry leaves. Bioinformatical tools were employed to analyze its deduced aimino acid sequence. Mulberry hypocotyls used as explants were inoculated in different nitrogen sources and growth regulating substances of the medium under aseptic conditions. Real-time PCR was used to investigate its relative expression level during hypocotyls regeneration in vitro and effects of different nitrogen sources and growth regulating substances. 【Result】 The obtained cDNA sequence of NR was 2 730 bp in length, encoding a polypeptide of 909 amino acid residues consisting of five classic conserved domain, protein molecular weight was 102.84 kD and the isoelectric point was 6.76. It showed that the sequence of NR has 95% identity compared with Morus notabilis and 78% indentity with other Rosaceae trees by NCBI BLAST. The obtained DNA sequence of NR was 5 142 bp including five exons and four introns. Cluster analysis showed that monocotyledons were clustered to one group, Mulberry and Rosaceae were clustered to one group, and the GenBank number was KF992020.1 and KF992021.1 respectively. Quantitative RT-PCR showed that this gene was highly expressed in roots, low expressed in stems. Glutamic acid significantly raised the expression of MaNR in leaves and GA3 significantly raised the expression of MaNR in stems. Hypocotyls could not be induced callus and multiple shoots in the single NH4+-N media but could be in the single NO3--N media. These nitrogen sources played a less part in subculture of shoot clumps. The shoots could grow in the single NH4+-N media and in the single NO3--N media. Real-time PCR exhibited that MaNR was highly abundant in cytoledons and hypocotyls than radicals. The relative expression level increased during hypocotyls regeneration in vitro and reduced to stable after shoot clumps formed. As time went on, the relative expression level of MaNR was reduced in shoot clumps with single NO3--N media or single NH4+-N. Glutamic acid showed a little inhibition of NR in the shoots subculture. 【Conclusion】 The full length of MaNR was cloned from mulberry trees. NO3--N was necessary for hypocotyls regeneration in vitro and the expression of NR affected by mulberry cell dedifferentiation and differentiation.

Key words: mulberry , nitrate reductase , MaNR gene , regeneration , real-time PCR

王茜龄, 余亚圣, 杨艳, 李军, 刘长英, 吕蕊花, 余茂德. 桑树硝酸还原酶基因MaNR的克隆及其表达分析[J]. 中国农业科学, 2014, 47(12): 2465-2475.

WANG Qian-Ling, YU Ya-Sheng, YANG Yan, LI Jun, LIU Chang-Ying, Lv Rui-Hua , YU Mao-De. Cloning and Expression Analysis of Nitrate Reductase Gene (MaNR) of Morus L.[J]. Scientia Agricultura Sinica, 2014, 47(12): 2465-2475.

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