Cloning And Functional Analysis Of Auxin Responsive And Transport-related Genes In Peanut

The phytohormone auxin plays an important role in plant development. Members of Aux/IAA family can modulate auxin-regulated gene expression negatively in the auxin signal transduction pathway. Members of AUX1/LAX gene family play important roles in plant growth and development by affecting the distribution of auxin.In this study, we isolated four Aux/IAA-like genes from the cDNA library of immature seeds of cultivated peanut Luhua-14, and denoted by PNIAA1, PNIAA2, PNIAA3and PNIAA4. Structural analysis showed that the sequence of PNIAA3is conserved and the protein product shared four conserved domains. The results by RT-PCR showed that the four genes were constitutively expressed in peanut tissues, and the PNIAA3expressed highest amount in seeds. Expression pattern analysis showed that whether it is under light or dark, the four genes were all induced by IAA long-term or short-term treatment, in which PNIAA1, PNIAA2, and PNIAA3could be induced in10minutes, while PNIAA4would be induced slightly after1-2hours. The results indicated that four Aux/IAA genes with different auxin response patterns may have different functions. In view of PNIAA3which had conserved domains with the characteristics that could be induced quickly by auxin of Aux/IAA genes, the localization and function of PNIAA3were investigated in order to explain the role of PNIAA3and auxin in signal transduction and reproductive growth of peanut.To investigate the localization of PNIAA3, the recombinant plasmid of pCAMBIA1302-PNIAA3-GFP was constructed. The results showed that PNIAA3located in the nucleus and PNIAA3belongs to the nucleoprotein family. The results shed some clues for the identification and function prediction of PNIAA3.To investigate gene function of PNIAA3, the sense and antisense expression vectors were constructed and were then transformed into Arabidopsis. By analyzing phenotypes of transgenic lines, we found that the length of main roots and hypocotyls of sense transgenic lines were shorter than those of wild-type plants, while in antisense transgenic lines, those were longer than wild-type plants. It showed that overexpression of PNIAA3inhibits the growth of main roots and hypocotyls, while inhibiting the expression of PNIAA3can promote the growth of main roots and hypocotyls, suggesting that PNIAA3may be negative regulator in auxin transduction, and the increased expression of PNIAA3could hinder the auxin transduction and affect plant growth. At the same time, we analyzed the phenotypes of two IAA3mutants and found that the phenotypes of them were similar with the antisense transgenic lines, the results also proved that PNIAA3was negative regulator in auxin transduction. The further results showed that there were almost no changes in sensitivity of transgenic Arabidopsis in response to exogenous IAA And there were almost no changes of auxin in the distribution in roots of transgenic Arabidopsis and mutants by observing the gravitropism of plants. The content of IAA in wild-type and transgenic plants were also measured, and the results showed that PNIAA3could result in phenotypic transition by influencing the content of IAA.At the same time, we isolated a AUXI/LAX-like gene from the cDNA library of immature seeds of cultivated peanut Luhua-14, and denoted by PNAUX1. The expression pattern analysis showed that the gene was constitutively expressed in peanut tissues, and expressed highly in roots and stems, and results indicated that PNAUX1which could control the different distribution of auxin may play vary roles in different tissues and organs through different channels and mechanisms. In order to investigate function of PNAUX1, the sense and antisense expression vectors were constructed and the vectors were then transformed into Arabidopsis plants. The transgenic plants will be used for further gene function analysis.