沙柳SpsLAS2和SpsLAS3基因克隆与生物信息学及组织特异性表达分析
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摘要
为促进沙柳(Salix psammophila)的分子定向育种,研究其分枝调控遗传机制,以沙柳为试验材料,采用同源克隆技术克隆获得沙柳SpsLAS2、SpsLAS3基因并做生物信息学及组织特异性表达分析。结果表明:SpsLAS2和SpsLAS3基因序列相似性达96.7%,氨基酸序列相似性达97.6%。SpsLAS2和SpsLAS3基因ORF长分别为1 248、1 254 bp,分别编码415 aa和417 aa。其氨基酸序列具有GRAS(Gibberellin-Insensitive,Repressor of ga1-3,Scarecrow)基因家族典型的5个结构域~([1]),属LS亚家族。qRT-PCR组织特异性分析发现2个基因均在叶腋处表达量最高,表明2个基因在叶腋中是特异表达的,可能调节叶腋分生组织的发育形成。SpsLAS2、SpsLAS3相似度较高,但也存在一定差异,推测2个基因可能是同一染色体的串联基因倍增关系。
To promote the molecular breeding of Salix psammophila,branching and regulation genetic mechanism was studied. Salix psammophila was used as the experimental material,the SpsLAS2,SpsLAS3 gene of Salix psammophila was cloned by homologous cloning technique and analyzed by bioinformatics and tissue-specific expression.The similarity of SpsLAS2 and SpsLAS3 gene sequences got to 96.69%,and the amino acid sequences similarity got to 97.6%.The sequence lengths of SpsLAS2 and SpsLAS3 were 1 248 bp and 1 254 bp,coding 415 aa and 417 aa,respectively.The amino acid sequences had five typical domains of GRAS gene family and belonged to the LS subfamily.The qRT-PCR in different tissues of Salix psammophila showed that the two genes expressed the highest in the leaf axil,suggesting that the two genes specifically expressed in the leaf axil,and two genes might be involved in the regulation of axillary meristem formation by participating in leaf axillary meristem.It was suggested that the two genes may be tandem gene doubling relationship on the same chromosome.
To promote the molecular breeding of Salix psammophila,branching and regulation genetic mechanism was studied. Salix psammophila was used as the experimental material,the SpsLAS2,SpsLAS3 gene of Salix psammophila was cloned by homologous cloning technique and analyzed by bioinformatics and tissue-specific expression.The similarity of SpsLAS2 and SpsLAS3 gene sequences got to 96.69%,and the amino acid sequences similarity got to 97.6%.The sequence lengths of SpsLAS2 and SpsLAS3 were 1 248 bp and 1 254 bp,coding 415 aa and 417 aa,respectively.The amino acid sequences had five typical domains of GRAS gene family and belonged to the LS subfamily.The qRT-PCR in different tissues of Salix psammophila showed that the two genes expressed the highest in the leaf axil,suggesting that the two genes specifically expressed in the leaf axil,and two genes might be involved in the regulation of axillary meristem formation by participating in leaf axillary meristem.It was suggested that the two genes may be tandem gene doubling relationship on the same chromosome.
引文
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[22] HEERY D M.A signature motif in transcriptional coactivators mediates binding to nuclear receptor[J].Nature,1997,387:733-736.
[23] TUSKAN GA,DIFAZIN S,JANSSON S,et al.The genome fo black cottonwood,Populus trichocarpa(Torr & Gray)[J].Science,2006,313:1596-1604.
[24] 刘志刚.杨树miRNA基因家族进化与靶基因研究[D].长沙:中南林业科技大学,2013.
[25] 侯静.杨柳分化及其性别决定的基因组学基础[D].南京:南京林业大学,2016.
[26] 戴晓港.簸箕柳基因组测序及功能基因研究[D].南京:南京林业大学,2017.
[27] Salix purpurea v1.0,DOE-JGI,http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_ Spurpurea.
[2] 石瑞,曹诣斌,陈文荣,等.佛手GRAS基因的克隆及表达分析[J].浙江师范大学学报(自然科学版),2011,34(4):446-451.
[3] STUURMAN J,JAGGI F,KUHLEMEIER C.Shoot meristem maintenance is controlled by a GRAS gene mediated signal from differentiating cells[J].Genes Dev,2002,16(17):2213-2218.
[4] SCHUMACHER K,SCHMITT T,ROSSBERGM,et al.The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family[J].Proc Natl Acadsci USA,1999,96(1):290-295.
[5] THOMAS G,OLIVER C,ELISABETH S,et al.Molecular analysis of the Lateral suppressor gene in Arabidopsis reveals a conserved control mechanism for auxillary meristem formation[J].Genes & Development,2003(17):1175-1187.
[6] GREB T,CLARENZ O,SCHAFER E,et al.Molecular analysis of the Lateral suppressor gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation[J].Genes & Development,2003,17(9):1175-1187.
[7] LI X.Control of tillering in rice[J].Nature,2003,422:618-621.
[8] 路东晔,贺玉娇,金娜,等.沙柳SpsLAS基因克隆及生物信息学分析[J].分子植物育种,2017,15(2):483-491.
[9] 陈雅琼.烟草腋芽发育相关基因的克隆与功能分析[D].北京:中国农业科学院,2015.
[10] YUAN L H,PAN J S,WANG G,et al.The cucumber Lateral suppressor gene(CLS) is functionally associated with axillary meristem initiation[J].Plant Mol Biol Rep,2010,28(3):421-429.
[11] YANG D H,SUN H J,GOH C H,et al.Cloning of a Zoysia ZjLsL and its overexpression to induce axillary meristem initiation and tiller formation in Arabidopsis and bentgrass[J].Plant Biol,2012,14(3):411-419.
[12] DAE H Y,PIL-YONG Y,SO Y P,et al.Cloning,characterization,and expression of a Lateral suppressor-like gene from chrysanthemum(Dendranthema grandiflorum Kitamura)[J].Plant Physiology and Biochemistry,2005,43:1044-1051.
[13] 畅文军,刘习文,张治礼.植物GRAS蛋白结构和功能研究进展[J].生命科学,2013,25(11):1045-1052.
[14] FAMBRINI M,MARIOTTI L,PARLANTI S,et al.A GRAS-like gene of sunflower (Helianthus annuus L.) alters the gibberellin content and axillary meristem outgrowth in transgenic Arabidopsis plants[J].Plant Biology,2015,17(6):1123-1134.
[15] SILVERSTONE A L,CIAMPAGLIO C N,SUN T P.The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway[J].The Plant Cell,1998,10(2):155-169.
[16] HIRANO K,KOUKETO E,KATOH H,et al.The suppressive function of therice DELLA protein SLR1 is dependent on its transcriptional activation activity[J].The Plant Journal,2012,71(3):443-453.
[17] 何明珠.荒漠植物枝系构件及其持水力研究[D].兰州:甘肃农业大学,2004.
[18] PYSH L D,WYSOCKA-DILLER J W,CAMILLERI C,et al.The GRAS gene family in Arabidopsis:Sequence characterization and basic expression analysis of the Scarecrow-like genes[J].Plant J,1999,18(1):111-119.
[19] TIAN C G,WAN P,SUN S H.Genome-wide analysis of the GRAS gene family in rice and Arabidopsis[J].Plant Molecular Biology,2004,54(4):519-532.
[20] BOLLE C.The role of GRAS proteins in plant signal transduction and development[J].Planta,2004,218:683-692.
[21] 路东晔.沙柳SpsLAS基因克隆、组织特异性表达及表达载体构建[D].呼和浩特:内蒙古农业大学,2017.
[22] HEERY D M.A signature motif in transcriptional coactivators mediates binding to nuclear receptor[J].Nature,1997,387:733-736.
[23] TUSKAN GA,DIFAZIN S,JANSSON S,et al.The genome fo black cottonwood,Populus trichocarpa(Torr & Gray)[J].Science,2006,313:1596-1604.
[24] 刘志刚.杨树miRNA基因家族进化与靶基因研究[D].长沙:中南林业科技大学,2013.
[25] 侯静.杨柳分化及其性别决定的基因组学基础[D].南京:南京林业大学,2016.
[26] 戴晓港.簸箕柳基因组测序及功能基因研究[D].南京:南京林业大学,2017.
[27] Salix purpurea v1.0,DOE-JGI,http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_ Spurpurea.