大豆Lea5基因的克隆与表达分析

何道一; 周秀杰; 张坤亮; 夏仁杰

doi:doi:10.3969/j.issn.1007-7146.2017.06.013

激光生物学报, 2017, 26 (6): 550, 网络出版: 2018-01-10

大豆Lea5基因的克隆与表达分析

Cloning and Expression Analysis of Lea5 Gene in Soybean

论文大纲

何道一 ^*周秀杰张坤亮夏仁杰

作者单位

淮北师范大学, 生命科学学院, 资源植物生物学安徽省重点实验室, 安徽淮北 235000

大豆幼胚高温处理差异显示技术 Lea5基因逆转录PCR soybean young embryos treatment by high temperature DDRT-PCR Lea5 gene RT-PCR

摘要

为了解高温处理促进大豆幼胚萌发成苗的分子生物学机制, 采用经典的基因表达差异显示技术, 分析大豆品种日本晴高温处理的幼胚与对照组材料的差异表达基因。对其中一个差异表达基因K6进行了测序和比对分析, 结果表明该基因序列与大豆Williams 82 基因组中的Lea5基因相似度高达99%, 可以确定为Lea5基因。Blastp比对分析结果表明大豆Lea5蛋白为LEA-3亚家族成员。利用蛋白质分析软件分析了Lea5基因推测的蛋白质结构的特点, 结果表明该蛋白质由113个氨基酸组成, 相对分子量为12.283 kD, 等电点高达10.12。该蛋白质不形成典型的二级结构。RT-PCR分析表明该基因表达具有组成型特点, 在大豆幼胚发育过程中稳定表达, 在大豆的根、胚轴和叶片等器官中均有表达。

Abstract

In order to investigate the molecular biological mechanism involved in germination of soybean young embryos promoted by high temperature(HT), both young embryos of soybean cultivar “Nipponbare” treated with HT and without HT were employed to screen the differential expression genes with mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). In this study, we reported K6, one of the differential expression genes. K6 was regarded as homologue of Lea5 gene (soybean cultivar Williams 82), because they shared 99% identical nucleotides. Blastp analysis showes that the putative protein of K6 contains LEA type-3 motifs and is assigned to LEA-3 superfamily. Analysis by CLC Protein workbench 5 software also showes that the putative protein has 113 amino acid residues with relative MW 12.283 kD and pI 10.12. The putative protein of K6 has no typical secondary structure element. Results of RT-PCR suggested that expression of K6 gene is constitutive. Expression levels of the gene in soybean young embryos developmental process was relatively constant. Moreover, expressions of the gene were also detected in roots, hypocotyls and leaves.

参考文献

[1] 王丕武, 张君, 姚丹, 等. 高产中熟春大豆新品种“吉农17号”选育报告［J］. 吉林农业大学学报, 2007, 29(1): 15-17.

WANG Piwu, ZHANG Jun, YAO Dan, et al. A breeding report on the new soybean variety of Jinong 17［J］. Journal of Jilin Agricultural University, 2007, 29(1): 15-17.

[2] 程琳静, 闫军辉, 钟云鹏, 等. 大豆高效体细胞胚诱导和增殖方法的研究［J］. 大豆科学, 2014, 33(3): 305-310.

CHENG Linjing,YAN Junhui, ZHONG Yunpeng, et al. Resarch to the effective methods of soybean somatic embryo induction and proliferation［J］. Soybean Science, 2014, 33(3): 305-310.

[3] LIANG P, PARDEE A B. Recent advances in differential display［J］. Current Opinion Immunology, 1995, 7(2): 274-280.

[4] TUGORES L B, LEDESMA J. Differential display of eukaryotic mRNA［J］. Methods in Molecular Biology, 1999, 97(461): 575-590.

[5] LIEVENS S, GOORMACHTIG S, HOLSTERS M A. Critical evaluation of differential display as a tool to identify genes involved in legume nodulation: Looking back and looking forward［J］. Nucleic Acids Research, 2001, 29(17): 3459-3468.

[6] 刘凯, 曾继吾, 夏瑞, 等. 柑橘mRNA 差异显示技术体系建立［J］. 基因组学与应用生物学, 2009, 28(2): 339-344.

LIU Kai, ZENG Jiwu, XIA Rui, et al. Establishing the system of Citrus mRNA differential display technology［J］. Genomics and Applied Biology, 2009, 28(2): 339-344.

[7] 衡伟, 程秀云, 贾兵, 等. mRNA差异显示法筛选‘砀山酥梨’褐皮芽变相关差异表达基因［J］. 中国农业科学, 2013, 46(15): 3172-3179.

HENG Wei, CHENG Xiuyun, JIA Bing, et al. Differential expression genes of ‘Dangshansuli’ pear russet mutant screened with DDRT-PCR［J］. Scientia Agricultura Sinica, 2013, 46(15): 3172-3179.

[8] LANG P, ZHANG C K, EBEL R C, et al. Identification of cold acclimated genes in leaves of citrus unshiu by mRNA differential display［J］. Gene, 2005, 359: 111-118.

[9] 吴海波, 郑国华, 牛先前, 等. DDRT-PCR技术分离枇杷幼果低温诱导相关基因［J］. 热带作物学报,2010, 31(3): 416-421.

WU Haibo, ZHENG Guohua, NIU Xianqian, et al. Isolation of cold stress genes in loquat fruit by DDRT-PCR technique［J］. Chinese Journal of Tropical Crops, 2010, 31(3): 416-421.

[10] 王楠, 尹俊奇, 周莹, 等. 大豆花芽mRNA差异显示技术体系反应条件的优化［J］. 吉林农业大学学报 2013, 35(4): 389-392, 397.

WANG Nan, YIN Junqi, ZHOU Ying, et al. Optimization of the system of soybean flower bud differential display technology［J］. Journal of Jilin Agricultural University, 2013, 35(4): 389-392, 397.

[11] 杨转英, 胡桂兵, 王惠聪, 等. 不同着色期荔枝果皮总RNA的提取及mRNA差显分析［J］. 果树学报, 2008, 25(2): 281-285.

YANG Zhuanyin, HU Guibing, WANG Huicong, et al. Total RNA extraction and mRNA differential display analysis of litchi pericarps at different coloring stages［J］. Journal of Fruit Science, 2008, 25(2): 281-285.

[12] 叶楠, 李永刚, 文景芝. 利用mRNA差异显示技术(DDRT-PCR)研究大豆抗疫霉根腐病的分子机制［J］.东北农业大学学报, 2008, 39(9): 6-9.

YE Nan, LI Yonggang, WEN Jingzhi. Research on molecular machanisms of soybean resistance to Phytophthora sojae by mRNA differential display PCR［J］. Journal of Northeast Agricultural University, 2008, 39(9): 6-9.

[13] PARDINAS J R, CONMBATES N J, PROUTY S M, et al. Differential subtraction display: a unified approach for isolation of cDNAs from differentially expressed genes［J］. Analytical Biochemistry, 1998, 257(2): 161-168.

[14] VOGELI-LANGE R. Rapid selection and classification of positive clones generated by mRNA differential display［J］. Nucleic Acids Research, 1996, 24(7): 1385-1386.

[15] 何道一, 张艳梅, 张坤亮, 大豆幼胚培养直接成苗影响因素研究［J］. 大豆科学, 2012, 31(1): 34-37.

HE Daoyi, ZHANG Yanmei, ZHANG Kunliang. Influence factors of plantlet regeneration directly from cultured young embryos in soybean［J］. Soybean Science, 2012, 31(1): 34-37.

[16] GALAU G A, HUGHES D W, DURE L III. Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs［J］. Plant Molecular Biology, 1986,7(3): 155-170.

[17] DURE L III, GREENWAY S C, GALAU G A. Developmental biochemistry of cottonseed embryogenesis and germination-changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis［J］. Biochemistry, 1981, 20(14): 4162-4168.

[18] GRZELCZAK Z F, SATTOLO M H, HANLEY-BOWDOIN L K, et al. Synthesis and turnover of proteins and messenger RNA in germinating wheat embryos［J］. Canadian Journal of Biochemistry, 1982, 60(3): 389-397.

[19] ROBERTS J K, DESIMONE N A, LINGLE W L, et al. Cellular concentrations and uniformity of cell-type accumulation of two LEA proteins in cotton embryos［J］. Plant Cell, 1993, 5(7): 769-780.

[20] BROWNE J, TUNNACLIFFE A, BURNELL A. Plant desiccation gene found in a nematode［J］. Nature, 2002, 416(6876): 38.

[21] SALES K, BRANDT W, RUMBAK E, et al. The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma mem-brane location and protects membranes against desiccation and ethanol-induced stress［J］. Biochimica et Biophysica Acta, 2000, 1463(1): 267-278.

[22] SOLOMON A, SALOMON R, PAPERNA I, et al. Desiccation stress of entomopathogenic nematodes induces the accumulation of a novel heat-stable protein［J］. Parasitology, 2000, 121(4): 409-416.

[23] N DONG C, DANYLUK J, WILSON K E, et al. Cold-regulated cereal chloroplast late embryo-genesis abundant-like proteins. molecular characterization and functional analyses［J］. Plant Physiology, 2002, 129(3): 1368-1381.

[24] SHAHEEN B, MOWLA, ANN C, et al. Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)-like protein in oxidative stress tolerance［J］. The Plant Journal, 2006, 48(5): 743-756.

[25] GARAY-ARROYO A, COLMENERO-FLORES J M, GARCIARRUBIO A, et al. Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit［J］. The Journal Biological Chemistry, 2000, 275(8): 5668-5674.

[26] WISE M J. Leaping to conclusions: a computational reanal-ysis of late embryogenesis abundant proteins and their possible roles［J］. BMC Bioinformatics, 2003, 4: 52.

[27] ALAN T, MICHAEL J W, The continuing conundrum of the LEA proteins［J］. Naturwissenschaften, 2007, 94(10): 791-812.

[28] 李乐, 许红亮, 杨兴露, 等. 大豆LEA基因家族全基因组鉴定、分类和表达［J］. 中国农业科学, 2011, 44(19): 3945-3954.

LI Le, XU Hongliang, YANG Xinglu, et al. Genome-wide identification, classification and expression analysis of LEA gene family in soybean［J］. Scientia Agricultura Sinica, 2011, 44(19): 3945-3954.

[29] OLIVER M J, DOWD S E, ZARAGOZA J, et al. The rehydration transcriptome of the desiccation-tolerant bryophyte Tortula ruralis: transcript classification and analysis［J］. BMC Genomics, 2004, 5: 89.

[30] BOUDET J, BUITINK J, HOEKSTRA F A, et al. Comparative analys is of the heat stable proteome o f radicles of Medicago trunculata seeds during germination identifies late embryogenesis abundant proteins associated with desiccation tolerance［J］. Plant Physiology, 2006, 140(4): 1418-1436.

何道一, 周秀杰, 张坤亮, 夏仁杰. 大豆Lea5基因的克隆与表达分析[J]. 激光生物学报, 2017, 26(6): 550. HE Daoyi, ZHOU Xiujie, ZHANG Kunliang, XIA Renjie. Cloning and Expression Analysis of Lea5 Gene in Soybean[J]. Acta Laser Biology Sinica, 2017, 26(6): 550.

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