荫蔽处理对不同耐荫性大豆GmPIF4s编码基因表达的影响

Effect of Shading Treatment on the Expression of GmPIF4s Encoding Genes in Different Shade Tolerance Soybean

光敏色素互作因子(PIFs)是光信号响应的关键负调控因子,其中PIF4作为该家族核心成员在响应外界光温变化,整合多种激素合成和信号转导中起着关键性作用。为了明确大豆GmPIF4s[phytochrome interaction factor 4s of Glycine max(L.)Merr.]的功能和编码基因对荫蔽诱导的表达响应,该研究以耐荫性大豆‘南豆25’(ND25)和不耐荫性大豆‘荣县冬豆’(RD)为材料,通过生物信息学和分子生物学等方法,比较分析了豆科植物PIF4s的生物信息学特点,并采用qRT-PCR方法分析大豆不同组织(根、茎、叶、茎尖分生组织)、全光照和12 h/12 h光周期下不同荫蔽时间诱导叶片GmPIF4s及其不同耐荫性大豆材料间GmPIF4s的表达特征,为培育高产、耐荫的大豆品种提供理论依据。结果表明:(1)在豆科植物中鉴定到53个同源PIF4s,其中大豆包含7个同源GmPIF4s,长度介于296~562氨基酸之间,分子量范围为41491.9~62228.39 Da,所有GmPIF4s均包含有bHLH结构域,无跨膜结构,为亲水性蛋白且定位于细胞核中;蛋白二级及三级结构预测显示,GmPIF4a、GmPIF4b与拟南芥AtPIF4结构最为相似。(2)qRT-PCR分析表明,GmPIF4s基因表达具有组织特异性;在耐荫性大豆品种ND25中,GmPIF4a、GmPIF4b、GmPIF4d相对表达量在根、叶、茎和茎尖分生组织中表达最高,除GmPIF4a、GmPIF4e、GmPIF4f外,其余GmPIF4s在地上部各组织中表达量均相对较高;不耐荫性大豆品种RD中,GmPIF4a、GmPIF4b、GmPIF4c、GmPIF4d在叶中相对表量较高,除GmPIF4f和GmPIF4g外,其余GmPIF4s在地上部各组织中表达量均相对较高。(3)全光照下荫蔽处理时间延长,大豆GmPIF4a、GmPIF4b、GmPIF4c、GmPIF4d基因均受荫蔽的强烈诱导,表达量显著上调;12 h/12 h光周期条件下无论是正常光还是荫蔽条件下,随着处理时间的延长,白天GmPIF4s表达量呈现下调趋势,夜晚却呈现上调表达趋势,推测GmPIF4s受昼夜节律调控。(4)荫蔽条件下GmPIF4s在不同耐荫性大豆的表达显示,随着荫蔽时间的延长,耐荫性大豆品种ND25与不耐荫性大豆品种RD的GmPIF4a、GmPIF4b、GmPIF4c、GmPIF4d均上调表达,但在不耐荫性大豆品种RD中表达量相对更高,并于荫蔽处理8 h后两材料的GmPIF4s表达差异达到极显著水平。

PIFs(phytochrome interaction factors)are the key negative regulators of light signal response.As the core member of the family,PIF4 plays a critical role in response to the changes of light and temperature,integrating multiple hormone synthesis and signal transduction.Shade-tolerant soybean‘Nandou 25’(ND25)and shade-intolerant soybean‘Rongxian Dongdou’(RD)were applied to clarify the function and shade-induced expression response of GmPIF4s[phytochrome interaction factor 4s of Glycine max(L.)Merr.]in soybean.In this study,the bioinformatic characteristics of leguminous plant PIF4s were compared and analyzed by bioinformatics and molecular biology methods.We analyzed the expression characteristics of GmPIF4s by qRT-PCR in different tissues of soybean(root,stem,leaf and shoot meristem)in different shading time points under full light and 12 h/12 h photoperiod,and in different shade tolerant soybean cultivars,in order to provide a theoretical basis for high-yield and shade-tolerant soybean breeding.The results showed that:(1)53 homologous PIF4s were associated with legumes by bioinformatic analysis,among them,soybean contains 7 homologous GmPIF4s.The length of amino acid is between 296-562 aa,and the molecular weight range is 41491.9-62228.39 Da.All GmPIF4s were hydrophilic proteins with bHLH domain,no transmembrane structure and located in nucleus.The secondary and tertiary structure prediction of GmPIF4s showed that the structure of GmPIF4a and GmPIF4b were most similar to the AtPIF4.(2)qRT-PCR analysis showed that the expression of GmPIF4s were tissue-specific,in the shade-tolerant soybean ND25,the relative expression levels of GmPIF4a,GmPIF4b and GmPIF4d were the highest in root,leaf,stem and shoot meristems.Except for GmPIF4a,GmPIF4e and GmPIF4f,other GmPIF4s were upregulated in all tissues above ground.In the shade-intolerant soybean RD,GmPIF4a,GmPIF4b,GmPIF4c,and GmPIF4d have relatively high levels in leaves.Except for GmPIF4f and GmPIF4g,the expression of other GmPIF4s in the above ground tissues was relatively high.(3)With the increase in shading time,GmPIF4a,GmPIF4b,GmPIF4c and GmPIF4d genes were strongly induced by shade treatment,and their expression levels are significantly up-regulated;under 12 h/12 h photoperiod condition,no matter under normal light or shade,the expression of GmPIF4s showed a downward trend during the day,but an upward-regulated expression trend at night.It speculated that GmPIF4s expression is regulated by the circadian rhythm.(4)The expression of GmPIF4s in different shade-tolerant soybean cultivars under shading condition showed that the expression of GmPIF4a,GmPIF4b,GmPIF4c and GmPIF4d in shade-tolerant and shade-intolerant soybean cultivars ND25 and RD were all up-regulated with the extension of shading time.However,the expression level of GmPIF4s was higher in the shade-intolerant soybean cultivar RD than that in shade-tolerant soybean cultivar ND25,and the difference of GmPIF4s expression between the two soybean cultivars reached a very significant level after 8 hours of shading treatment.