| The GH3 family is an important class of early auxin-response genes involved in the maintenance of appropriate free IAA levels in plants.Several studies have suggested that the GH3 protein play importanr roles in the development of the hypocotyl and roots,plant defense response and light signaling pathways in Arabidopsis thaliana,but the role of this gene family in woody plants is poorly understood.In this study,we cloned the BpGH3.5 gene from Betula platyphylla x B.pendula and produced transgenic birch lines that overexpressed either a sense or antisense version of the BpGH3.5 gene.We took note of the phenotype of the transgenic plants and detected the concentration of auxin of the lines while we carried out transcriptome analysis on NT and BpGH3.5 transgenic lines.Our results may provide useful insights for further study of the role of the BpGH3.5 gene in the regulation of birch tree development.(1)We cloned a GH3-like gene from Betula platyphylla x B.pendula and named it the BpGH3.5 gene which sequence is most similar to GH3.5 of Ricinus communis by multiple alignment analysis.The phylogenetic tree of BpGH3 protein showed that BpGH3.5 belongs to the Group I class of the GH3 family,evolutionarily close to AtGH3.10 and AtGH3.11.(2)qRT-PCR results showed that the expression of BpGH3.5 was found obviously higher in leaves than in birch roots and stems.In addition,the expression of the gene can rapidly respond to TIBA,MeJA,ABA and SA treatment.(3)The upstream sequence 1275bp of BpGH3.5 promotor was cloned.The analysis of cis-element showed that the promotor contained many binding sites relative with plant hormones.The promoter of BpGH3.5 was cloned into the vector pGH3.5-GUS and was used to infect the birch leaves using Agrobacterium-mediated transformation.Transient infection in birch showed that the promoter of BpGH3.5 has GUS obviously activities in the cotyledon and leaf,and exhibited activity in hypocotyl after seed germination while there was no activity in the roots and stems of birch.(4)Sense and antisense sequences of BpGH3.5 were cloned into the pGWB2 vector with the 35S promoter.The construct was delivered into the Agrobacterium which was used to infect the birch leaves,and transgenic birch lines that overexpressed either a sense or antisense version of the BpGH3.5 gene were detected by PCR,northern blot and western blot analysis.(5)We observed consistently short primary roots(PR)and lateral roots(LR)in seedlings expressing sense and anti-sense BpGH3.5 transcripts compared to NT seedlings.In contrast,plant height was not significantly different between the NT and transgenic lines grown on WPM for 25 days and in the greenhouse for 2 years.In addition,all BpGH3.5 transgenic lines had more short root hairs than NT.(6)Further explore of the dissected root tips of NT and BpGH3.5 transgenic lines showed that reduced root apical meristem length and a reduction in cortex-cell number in the meristematic zone.(7)RNA-Seq results indicated that compared with the NT line,3,320 and 5,212 unigenes were found to be differentially expressed in the BpGH3.5 overexpressed and suppressed lines,respectively,and many differentially expressed unigenes related to cell proliferation and growth were downregulated in BpGH3.5 transgenic lines.(8)We detected the concentration of IAA by ultra-performance liquid chromatography-tandem mass spectrometry.Quantitation of IAA results showed that IAA level decreased in both the BpGH3.5 overexpressed and suppressed plants. |
PDF Full Text Request