玉米ZmGRAS13基因的克隆及功能研究

Cloning and functional analysis of ZmGRAS13 gene in maize

GRAS家族是植物特有的一类转录因子,在调控植物生长发育和响应逆境胁迫等方面发挥着重要作用。探究玉米(Zea mays L.)GRAS家族基因功能将为玉米新种质创制提供重要的基因资源。本研究克隆获得了ZmGRAS13基因(Zm00001eb401210),利用生物信息学分析、实时荧光定量PCR(qPCR)等技术对该基因的基本特性、组织表达特性及逆境胁迫下表达模式等进行分析。生物信息学分析结果显示,该基因编码序列全长为1638 bp,编码545个氨基酸;ZmGRAS13蛋白不具有跨膜结构,分子量为60.79 kD,理论等电点为5.86,具有GRAS家族所特有的保守结构域。对基因启动子上游2 kb序列进行分析,发现该序列含有与逆境胁迫、激素响应及光响应等相关的顺式作用元件。qPCR分析表明,ZmGRAS13基因在玉米不同组织中均有表达,且茎中的表达量最高;同时该基因在不同非生物胁迫处理条件下均有不同程度的诱导表达。玉米原生质体瞬时表达实验表明,ZmGRAS13蛋白定位于细胞核。在分别含有不同浓度梯度的NaCl、甘露醇(mannitol)、脱落酸(abscisic acid,ABA)、茉莉酸(jasmonic acid,JA)和水杨酸(salicylic acid,SA)的1/2 MS固体培养基上,转ZmGRAS13基因拟南芥株系的根长均显著长于野生型拟南芥;在土壤中,高盐和干旱处理下转基因拟南芥株系较野生型拟南芥生长状态更好,且绿叶率高于野生型。转ZmGRAS13基因拟南芥与野生型相比,抗逆生理指标MDA含量降低、叶绿素含量增加、POD和CAT活性增强,且差异均显著。由此推测,ZmGRAS13基因可能参与玉米生长发育调控和对逆境胁迫应答及激素信号转导途径。本研究为进一步解析ZmGRAS13在玉米中的生物学功能提供了重要的参考依据。

GRAS family is a plant-specific transcription factor,which plays an important role in regulating plant growth and de-velopment and responding to stresses.Exploring the function of GRAS family genes in maize(Zea mays L.)provides the impor-tant genetic resources for the creation of new maize germplasm.In this study,ZmGRAS13 gene(Zm00001eb401210)was cloned,and its basic characteristics,tissue expression characteristics,and the relative expression patterns under stresses were analyzed by bioinformatics and qRT-PCR.Bioinformatics showed that the full-length coding sequence of this gene was 1638 bp,encoding 545 amino acids.ZmGRAS13 protein had no transmembrane structure,and the molecular weight of 60.79 kD,the theoretical isoelec-tric point of 5.86,and had a conserved domain unique to the GRAS family.The analysis of 2 kb sequence upstream of the gene promoter indicated that the sequence contained cis-acting elements related to stresses,hormone response,and light response.The qRT-PCR analysis showed that ZmGRAS13 gene was expressed in different tissues of maize,and the relative expression level in stem was the highest.At the same time,the gene has different degrees of induced expression under different abiotic stress treat-ment conditions.The transient expression experiment of maize protoplasts demonstrated that ZmGRAS13 protein was localized in the nucleus.On 1/2 MS solid medium containing different concentrations of NaCl,mannitol,abscisic acid(ABA),jasmonic acid(JA),and salicylic acid(SA),respectively,the root length of ZmGRAS13 transgenic Arabidopsis lines was significantly longer than the control.In the soil,transgenic Arabidopsis lines grew better than the control under high salt and drought treatments,and the green leaf rate was higher than the control.Compared with the wild type,the content of stress resistance physiological index MDA decreased,the chlorophyll content increased,and the activities of POD and CAT increased in the transgenic ZmGRAS13 Arabidopsis thaliana,and the difference was significant difference.In conclusion,ZmGRAS13 gene may be involved in the regu-lation of maize growth and development,response to stresses and hormone signal transduction pathway.This study provides an important reference for the further analysis of the biological function of ZmGRAS13 in maize.