桑树谷氨酸脱氢酶基因MaGDHs的克隆及表达分析

Molecular Cloning and Expression Analysis of Genes MaGDHs Encoding Glutamate Dehydrogenase in Mulberry

【目的】克隆桑树的谷氨酸脱氢酶基因GDH,了解其结构、组织特异性表达以及调控特点,并获得桑树谷氨酸脱氢酶蛋白,为进一步研究谷氨酸脱氢酶(glutamate dehydrogenase,GDH)在桑树氮代谢过程中的功能奠定基础,也为多年生木本植物的GDH研究提供参考。【方法】根据川桑基因组数据库搜索GDH同源序列设计引物,以杂交桑品种桂桑优62号(M.atropurpurea Roxb.)c DNA为模板,通过RT-PCR克隆获得Ma GDHs。利用NCBI上BLAST和CDD进行氨基酸序列比对和保守结构域分析;利用Expasy在线软件对Ma GDH氨基酸组成、分子量、等电点进行分析;采用MEGA 5.0软件构建系统进化树;通过q RT-PCR检测试管苗在不同浓度的蔗糖、铵盐、激素(6-BA)状态下Ma GDHs的表达量,用Step One Software V2.1软件根据2-△△Ct法进行基因相对表达量分析。以p ET-28a(+)、p ET-32a(+)、p Cold-TF为载体构建Ma GDH的融合蛋白重组质粒并转入到大肠杆菌中进行表达。【结果】成功获得2个Ma GDHs,序列全长均为1 236 bp,编码411个氨基酸,含一个开放阅读框,分别命名为Ma GDH1和Ma GDH2。Ma GDH1蛋白质的分子量为44.1 k D,理论等电点为5.84,Ma GDH2蛋白质的分子量为44.2 k D,理论等电点为6.68。Ma GDHs氨基酸序列含有9个外显子,8个内含子,具有线粒体转移肽、Glu/a-KG结合域和NAD(P)结合域,与川桑同源性均为99%,与双子叶植物同源性为90%左右,与单子叶植物的同源性为85%左右。重组蛋白Ma GDHs以包涵体的形式在大肠杆菌BL21(DE3)中表达,经过纯化和复性后获得了具有活性的酶蛋白,酶活性以p ET-28a(+)-Ma GDH1重组蛋白最高,为10.07 nmol·min^(-1)·m L-1。Ma GDHs的表达具有组织特异性,在桑花中表达量最高,其次是嫩叶,在果实中几乎不表达。Ma GDHs的表达受到蔗糖、NH4+和6-BA的调控。随着蔗糖浓度的增加,Ma GDHs表达量增加;过量的NH4+促进Ma GDH1的表达,而没有NH4+的情况下,Ma GDHs也有表达;细胞分裂素对Ma GDHs的表达是先抑制后促进,Ma GDH1在24 h后表达增强,Ma GDH2在48 h后表达增强。【结论】从桂桑优62号中获得了两个Ma GDHs,分别编码β和α亚基。不同载体的重组蛋白酶活性存在差异。Ma GDHs的表达具有组织特异性,在桑树生长旺盛的组织中表达量较高。过量NH4+促进Ma GDH1表达;Ma GDH1响应激素促进表达比Ma GDH2早。

【Objective】In this paper,Genes of GDH were cloned from mulberry and characteristics of their structures,tissue expressions,regulation factors were studied and GDH proteins were obtained in order to reveal the role of glutamate dehydrogenase(GDH)in nitrogen metabolism.The results could provide data for studying other perennial woody plants on GDH.【Method】Primers were designed according to the homologous sequences of GDH downloaded from the Morus genome database.Using Guisangyou 62(M.atropurpurea Roxb.)cDNA as template,Ma GDHs were cloned by RT-PCR.The similarity of GDH sequences was analyzed by BLASTN and converse structure domains were analyzed by CDD on line of NCBI.The composition of the amino acid,theoretical molecular mass and p I were deduced by Expasy software online.A phylogenetic tree was constructed by MEGA 5.0 software performed with the neighbor-joining(NJ)method.Expressions of Ma GDHs under the condition of different concentrations of sucrose,NH4+and 6-BA solutions were measured by q RT-PCR.The data were analyzed by Step One Software V2.1 using method of 2^-△△Ct.Ma GDHs recombinant plasmids were constructed with vectors of p ET-28 a(+),p ET-32 a(+),pcold-TF and transformed to E.coli to produce Ma GDHs recombinant protein.【Result】Both two obtained genes Ma GDHs were 1236 bp in length with an open reading frame encoding a polypeptide of 411 amino acids,named Ma GDH1 and Ma GDH2.The predicted molecular mass of Ma GDH1 was 44.1 k D and a predicted isoelectric point was 5.84.The predicted molecular mass of Ma GDH2 was 44.2 k D and a predicted isoelectric point was 6.68.Both two sequences have 9 exons and 8 introns including a mitochondrial transfer peptide and two features domains:a Glu/a KG binding domain and an NAD(P)binding domain.The Ma GDHs protein showed identity of 99%with Morus notabilis,about 90%with dicotyledons plants and 85%with monocotyledons plants.The proteins of Ma GDHs were successfully expressed in E.coli BL21(DE3)in the form of inclusion bodies.After purification and renaturation,active enzymes were obtained and the activity of recombinant protein p ET-28 a(+)-Ma GDH1 was the highest with 10.07 nmol·min^-1·m L^-1.Ma GDH1 and Ma GDH2 showed organ specificity but distinct transcript intensity.The expression level of flower was the highest and the expression level of young leaf was taken the second place.They hardly expressed in fruit.Transcript abundance of Ma GDHs were up-regulated by sucrose in this study,Ma GDHs were also expressed without NH4^+in media and excess NH4^+promoted the transcript of Ma GDHs.6-benayl aminopurine first inhibited and then promoted the expressions of Ma GDHs and Ma GDH1 was promoted after 24 h and Ma GDH2 was promoted after 48 h.【Conclusion】Two genes of GDH were obtained from mulberry named Ma GDH1 and Ma GDH2 respectively encodingβandαsubunit.The proteins of Ma GDHs were successfully expressed in E.coli BL21(DE3)in the form of inclusion bodies.The activities of recombinant proteins were different with different vectors.The expression of Ma GDHs showed organ specificity and expressed higher in vigorous organs than others.They were up-regulated by excess NH4+.Ma GDH1 were promoted by 6-BA earlier than Ma GDH2.