[目的]对木薯花青素还原酶基因(MeANR)进行克隆及表达分析，为解析木薯叶片花青素生物合成的分子机制、叶色改良及新品种培育提供理论依据。[方法]以3种叶片颜色的木薯华南9号(SC9，绿叶)、花叶木薯(黄绿叶)和紫叶木薯(紫叶)为材料，测定其花青素含量和原花青素含量，并对MeANR基因进行克隆及生物信息学分析，采用实时荧光定量PCR检测MeANR基因在不同组织及激素处理下的表达模式，通过转基因酵母胁迫试验验证MeANR基因在不同激素处理下的表达情况。[结果]SC9、花叶木薯和紫叶木薯叶片中的花青素含量和原花青素含量均存在显著差异(P<0。05)，其中紫叶木薯叶片的花青素含量最高，达444。59 ng/g，分别为SC9和花叶木薯的24。48和28。32倍。从3种叶片颜色的木薯叶片中分别克隆到1个MeANR基因，其编码区(CDS)序列为1041 bp，序列完全一致，但与Phy-tozome数据库中公布的ANR基因序列(登录号:Manes。16G016400)存在7个碱基差异。MeANR蛋白由346个氨基酸组成，属于稳定的非分泌型亲水性蛋白，二级结构主要由无规则卷曲(42。77%)和α-螺旋(37。28%)构成，延伸链和β-转角占比较小，亚细胞定位于叶绿体。MeANR基因在芽中的相对表达量最高，其次是叶片和花，在茎中的相对表达量最低。MeANR基因在不同激素处理下呈不同的表达模式，水杨酸(SA)处理后随着处理时间的延长，MeANR基因的相对表达量呈先下降后上升再下降的变化趋势，处理后24 h MeANR基因的相对表达量比处理0 h显著下降68%，而在脱落酸(ABA)和茉莉酸甲酯(MeJA)处理后MeANR基因的相对表达量总体上呈上升趋势，但ABA处理后24 h达峰值，MeJA处理后9 h达峰值。在SA处理下转MeANR基因酵母菌的存活率低于对照(转pDR196空载体的酵母菌)，而在ABA和MeJA处理下转MeANR基因酵母菌的生长情况优于对照。[结论]木薯叶片颜色深度与花青素含量存在正相关性。MeANR是木薯叶片花青素积累的负调控基因，具有明显的组织表达特异性，其表达受SA抑制，受ABA和MeJA诱导。
Cloning and expression analysis of MeANR gene in cassava(Manihot esculenta Crantz)leaves with different colors
[Objective]The purpose of the study was to clone and analyze the expression of anthocyanidin reductase gene(MeANR)in cassava,in order to provide theoretical basis for understanding the molecular mechanism of anthocya-nin biosythesis in cassava leaves,improving leave color,and cultivating new varieties.[Method]Cassava with 3 leaf co-lors:Huanan No.9(SC9,green leaves),mosaic leaf cassava(yellow and green leaves)and purple leaf cassava(purple leaves)were used as materials,the anthocyanin content and proanthocyanidin content were measured.MeANR gene was cloned and analyzed using bioinformatics.Real-time fluorescence quantitative PCR was used to detect the expression pat-tern of MeANR gene in different tissues and hormone treatments.Transgenic yeast stress experiments were conducted to verify the expression of MeANR under different hormone treatments.[Result]There were significant difference(P<0.05)between anthocyanin content and proanthocyanid content in leaves of SC9,mosaic leaf cassava and purple leaf cassava.Among them,the anthocyanin content in leaves of purple leaf cassava was the highest,reaching 444.59 ng/g,which was as 24.48 and 28.32 times as that of SC9 and mosaic leaf cassava respectively.A MeANR gene was cloned from cassava leaves with 3 different leaf colors respectively,with a coding region(CDS)of 1041 bp,the sequences were consistent,with 7 bp inconsistent with the published sequences in Phytozome database(accession number:Manes.16G016400).The MeANR protein consisted of 346 amino acids and was a stable non-secreted hydrophilic protein.Its secondary structure was mainly composed of random coils(42.77％)and α-helices(37.28％),with a small proportion of extended chains and β-turns.Subcellular localization was in the chloroplast.The expression level of MeANR gene was the highest in buds,followed by leaves and flowers,and lowest in stems.MeANR gene exhibited different expression patterns under different hormone treatments.After salicylic acid(SA)treatment,the relative expression level of MeANR showed a trend of first decreasing,then increasing,and then decreasing with the prolongation of treatment time.The relative expression level of MeANR gene at 24 h significantly decreased by 68％compared to 0 h.However,after abscisic acid(ABA)and methyl jasmonate(MeJA)treatments,the relative expression level of MeANR gene showed an upward trend,but reached its peak at 24 h of ABA treatment and 9 h of MeJA treatment.The survival rate of MeANR transgenic yeast strain under SA treatment was lower than that of control(yeast strain transformed with pDR196 empty vector),the growth of MeANR transgenic yeast strain treated with ABA and MeJA was better than the control.[Conclusion]There is a positive correlation between the cassava leaf color and anthocyanin content.MeANR is a negative regulatory gene for anthocyanin accumulation in cassava leaves,with obvious tissue expression specificity,and is inhibited by SA,induced by ABA and MeJA.
万方数据
维普
