西葫芦NCED基因家族鉴定及其响应干旱胁迫分析

Genome-wide Identification and Response to Drought Stress of NCED Genes Family in Zucchini(Cucurbita pepo L.)

NCED基因家族成员在调节植物响应干旱胁迫中发挥着关键作用,该研究通过生物信息学技术分析NCED在西葫芦基因组中的分布、结构及进化,研究家族成员在不同组织中的表达特异性及其对10%PEG 6000模拟干旱、0.1 mmol·L-1ABA激素和自然干旱胁迫的响应,以解析NCED基因家族的生物学功能。结果表明:(1)从西葫芦全基因组中鉴定出6个NCED家族基因(CpNCED1~6),且6个基因均不含内含子、分别分布于西葫芦的1、10、12、14、19和20号共6条染色体上。(2)理化性质分析发现,CpNCED1~6蛋白长度为569~590 aa,理论分子量在62.64~65.54 kD之间。(3)蛋白保守元件分析显示,除CpNCED3蛋白在遗传进化过程中出现3个基序(motif 12、motif 13和motif 15)的缺失外,其余5个蛋白都有完整的16个motif保守基序,且分布在600个氨基酸以内,同时大部分NCED蛋白序列保守性较高。(4)顺式作用元件分析显示,西葫芦CpNCED1~6基因均含ABRE、W box、MBS、P-box、TCA-element、CGTCA-motif、TGA-element和TGA-box等潜在的干旱胁迫响应元件。(5)qRT-PCR分析表明,CpNCED1~6基因在西葫芦不同组织中的表达具有组织特异性,其中,CpNCED4和CpNCED1在茎中的表达量显著高于其他4个基因,CpNCED2、CpNCED4、CpNCED6在花中的表达显著高于其余3个基因且CpNCED2表达量最高,CpNCED1~6在果实和叶中的表达量均相对较低;与对照组相比,CpNCED1~6受模拟干旱、ABA激素和自然干旱胁迫均上调表达;伴随干旱胁迫的产生,叶片中脱落酸(ABA)含量逐渐升高,暗示CpNCEDs在西葫芦干旱胁迫响应与ABA的生物合成过程中发挥着正向调控作用。研究发现,6个CpNCED1~6基因与西葫芦干旱胁迫响应密切相关,且对西葫芦干旱胁迫的响应以及ABA生物合成具有重要作用,尤其以CpNCED2和CpNCED4基因的作用更为明显。

In view of the key role of NCED gene family members in regulating plant response to drought stress,this study analyzed the distribution,structure and evolution of NCED gene family from zucchini(Cucurbita pepo L.)genome by bioinformatics technology,analyzed the expression specificity of NCED gene members in different tissues and their response to simulated drought(10%PEG 6000),hormone(0.1 mmol·L-1 ABA)and natural drought(stop watering for 0,1,2,3 and 4 d)stresses to understand the biological functions of NCED gene family.Experimental results show that:(1)a total of six NCED gene(i.e.,CpNCED1-6)members were identified from the whole genome of zucchini;The six CpNCEDs were distributed on chromosome 1,10,12,14,19 and 20 of zucchini,and none of the NCED genes contained intron.(2)Physicochemical properties analysis showed that CpNCED1-6 proteins were 569-590 amino acids,and the theoretical molecular weight ranged from 62.64-65.54 kD.(3)Protein conserved motifs analysis showed that,the CpNCEDs containing 16 complete conserved protein motifs except for the CpNCED3 had three motifs(i.e.,motif 12,motif 13 and motif 15)deletion,and CpNCED1-6 proteins conserved elements distributed within 600 amino acids,indicating most of NCED protein sequences were highly conserved.(4)The cis-acting elements analysis of NCED gene family upstream promoters(2500 bp sequence before ATG)showed that the zucchini CpNCED1-6 genes contained potential drought stress response elements such as ABRE,W box,MBS,P-box,TCA-element,CGTCA-motif,TGA-element and TGA-box.(5)Real-time fluorescent quantitative PCR(qRT-PCR)revealed that the expression of CpNCEDs gene in different zucchini tissues was tissue specific,among which,the expression levels of CpNCED4 and CpNCED1 in stems were significantly higher than those of the other three genes,the expression levels of CpNCED2,CpNCED4 and CpNCED6 in flowers were significantly higher than those of the other three genes,and the expression level of CpNCED2 was the highest,while the expression levels of CpNCED1-6 in fruits and leaves were relatively low;The expression levels of six selected family members were induced by simulated drought,ABA hormone and natural drought stresses and increased in different degrees compared with the control group;With the emergence of drought stress,the abscisic acid(i.e.,ABA)content gradually increased in leaves;This study found that six CpNCED1-6 genes played important roles in drought stress response and ABA biosynthesis of C.pepo,especially CpNCED2 and CpNCED4 genes.