杂交鹅掌楸LhARF2基因的克隆及功能分析

生长素响应因子ARF在植物生长发育过程中发挥着重要的作用。本研究采用同源克隆和RACE相结合的方法,从杂交鹅掌楸叶片中克隆ARF2的同源基因,命名为LhARF2,对基因蛋白序列进行生物信息学分析,基因全长3193 bp,包含2589 bp的开放阅读框,编码862个氨基酸的蛋白质。蛋白质分子量为96.19 kD,理论等电点为6.21,为不稳定蛋白质;蛋白质二级结构包括α-螺旋、β-折叠、不规则卷曲和延伸链,以不规则卷曲为主;蛋白质三级结构分析显示该蛋白与拟南芥生长素响应因子(SMTL编号:4ldy.1)的序列相似性最高(67.89%)。功能结构域分析显示该蛋白质包括B3、Auxin_resp和AUX_IAA结构域。系统进化树分析表明LhARF2所编码的蛋白质与莲(Nelumbo nucifera)XP_010250544.1亲缘关系最近。激素诱导生根试验表明,拟南芥T2代幼苗相比较野生型,在没有激素处理下,根系长度差异不大;在外源激素IBA处理下,根系长度明显伸长。实时定量分析显示在IBA激素诱导下,根系和叶片表达量均上调,根系中LhARF2上调幅度分别高于WT和叶片。结果表明LhARF2基因在拟南芥中对根的生长具有促进作用,LhARF2基因受IBA激素正向调控。该研究结果为进一步分析杂交鹅掌楸LhARF2基因的功能及利用奠定基础。

Cloning and Functional Analysis of LhARF2 Gene in Liriodendron Hybrids

Auxin response factor(ARF)plays an important role in plant growth and development.In this study,we combined homologous cloning with RACE to clone the homologous gene of ARF2 from the leaves of Lliriodendron hybrids,named LhARF2,to process bioinformatics analysis of the gene protein sequence.The total length of the cloned LhARF2 gene is 3193 bp,including 2589 bp open reading frame(ORF),coding 862 amino acid.The molecular weight of this protein is 96.19 kD,the theoretical isoelectric point is 6.21,and it is an unstable protein.The secondary structure of protein consists ofα-helix,β-folding,irregular crimp and extended chain,main element was irregular crimp.The sequence similarity between the protein and Arabidopsis thaliana auxin response factor(SMTL no.4ldy.1)was up to 67.89%.Functional domain analysis of this protein includes B3,Auxin_resp and AUX_IAA domain.Phylogenetic tree analysis indicated that the genetic relationship of LhARF2 gene protein was closely related to Nelumbo nucifera(XP_010250544.1).Hormone induced rooting experiment showed that the root length of T2-generation seedlings of Arabidopsis thaliana were little different compared to wild-type Arabidopsis thaliana.Under the treatment of exogenous hormones IBA, the roots length was obvious elongation. In addition, real-timequantitative analysis was conducted on roots and leaves. Under the induction of IBA hormone, the expressions ofboth roots and leaves were up-regulated, and up-regulated range of LhARF2 in roots was higher than WT and leaves.The results indicate LhARF2 gene promoted root growth in Arabidopsis thaliana, that LhARF2 gene was positivelyregulated by IBA hormones. These results lay a foundation for further analysis of the function and utilizationof LhARF2 gene in Liriodendron hybrids.