Validation And Functional Analysis Of MiR166 And Its Target Gene HDZⅢ In Betula Luminifera

Betula luminifera is a precious timber species with excellent properties which is distributed in south of China.It is an ideal species for forest genetic research.miR166 genes play crucial roles in apical meristems and vascular tissues development in plants.It regulate vascular tissues growth according to the combination of target genes of HD-ZIP Ⅲ.Validation and function analysis of miR166 and HD-ZIP Ⅲgene family laida significant foundation for wood formation mechanismand genetic improvement.In our study,gene validation and function of miR166 and its target gene HDZ Ⅲ were analyzed in Betula luminifera.The major conclusion are listed as following.1.Eight miR166 genes named BlmiR166a~BlmiR166g were cloned from Betula luminifera based on transcriptome and genomic data.The sequences of these genes ranged from 0.3Kb to 2.4Kb.Primary bioinformatic analysis of miR166 and HDZ Ⅲ is involved in gene structure and cis-regulatory elements.BlmiR166 b,BlmiR166c and BlmiR166 e were consist of one intron and others were no intron.All BlHD-ZIP Ⅲ were consist of eighteen exons except BlHDZ1(nineteen exons).The statistics of cis-regulatory elements in all genes show that the number of light responsive element were the most.The phylogenetic analysis of miR166 mature and pre-miR166 sequence showed all eight mature sequence were in one clade while pre-miR166 belonged to three different clades.2.The qpcr results elucidated that the expression of BlmiR166 a and BlmiR166 h were affluent in phloem and may be relevant to vascular development element;BlmiR166d and BlmiR166 e transcripts were highly expressed in bud and expressed low in other tissues.BlmiR166b、BlmiR166c and BlmiR166 f were highly expressed in male flowers.BlmiR166 g transcripts were expressed in bud,male and female inflorescence with low quantity.We found that all BlHD-ZIP Ⅲ genes were expressed in different tissues andexhibited low levels of expression in phloem,seed and root.Expression pattern of BlHDZ2 and BlHDZ5 are very similar to a high expression abundance in xylem and may involved in xylem development.Expression of BlHDZ3 and BlHDZ4 in meristem is much higher than other organs and may involved in meristem development.Some of HD-ZIP Ⅲ were negatively regulated by miR166.The expression of BlmiR166 a and BlmiR166 h in xylem were extremely low and in phloem were high.And target gene BlHDZ2 and BlHDZ5exhibited opposite expression mode which indicated that these genes have balanced activity in xylem and phloem development.3.The expression of HD-ZIP Ⅲ genes in different hormone treated stems showed five BlHD-ZIP Ⅲ were all up-regulated obviously treated by 0.1mM IAA.It suggests that interaction between BlHDZ Ⅲ and IAA may play a significant role in xylem growth.The expression of BlHDZ1,BlHDZ3 and BlHDZ4 are consistent and they were all up-regulated by IAA,ET and GA.It suggests that the phenomena may be relevant to the hormone element.BlHDZ2 and BlHDZ5 were induced only treated by 0.1mM IAA.4.Five BlHD-ZIP Ⅲ overexpression vectors were constructed and transformed by Agrobacterium-mediated methodinto Arabidopsis.And PCR was used to validate the target gene and reporter gene at the molecular level.At present,we have BlHDZ1,BlHDZ2,BlHDZ4 and BlHDZ5 positive transgenic plants.And T2 generation of BlHDZ1 and BlHDZ5 were 8 lines,BlHDZ2 and BlHDZ4 were 2 lines.We also found BlHDZ4 and BlHDZ5 exhibit differences inPhenotypic traits compared to wild type phenotype.The specific function of these genes has yet to be further in-depth study.