| White birch(Betula platyphylla)is one of the important broad-leaved native tree species in Northeast China.So far,people have obtained a number of excellent single white birch plants through provenance selection,family selection and excellent tree selection.Due to the difficulty in rooting from cuttings,it has not yet been established.A set of easy-to-promote vegetative propagation technology system limits the popularization and application of fine birch plants.WUSCHEL-related homeobox11(WOX)transcription factor is a widespread transcription factor in plants.Studies have shown that WOX11 is mainly involved in the regulation of adventitious root formation.It can promote the formation of root primordia to generate more adventitious roots.If the BpWOX11 gene is introduced into Overexpression in the birch genome will hopefully solve the problem of difficult rooting of birch cuttings.Therefore,the experiment used wild-type birch as material,cloned the BpWOX11 gene,constructed an overexpression vector,and carried out the transformation of mature birch embryos by Agrobacterium-mediated method.Analysis to explore the function of BpWOX11 gene.The results of the study are as follows:(1)Cloning of birch BpWOX11 gene and analysis of bioinformatics.Using the c DNA of birch tissue culture rooting seedlings as a template,primers were designed to clone the BpWOX11 gene.The NCBI online website was used to analyze the results.The results showed that the BpWOX11 gene consists of 744 bp and encodes 247 amino acids.The phylogenetic analysis shows that the amino acid sequence of birch BpWOX11 is similar to that of Juglans regia Jr WOX11.The amino acid sequence has the highest homology.(2)Study on overexpression of BpWOX11 gene in white birch.Using the mature embryos of birch as the receptor,the genetic transformation of birch was carried out by Agrobacteriummediated method,and 10 resistant strains were successfully obtained.PCR and fluorescence quantitative PCR technology were used to detect the obtained resistant strains.The genes have been integrated into the birch genomes of 10 resistant strains and can be expressed at the m RNA level.(3)The number and growth of adventitious roots of transgenic birch.Using BpWOX11 transgenic white birch as material,the rooting analysis of tissue culture seedlings showed that the adventitious root length,adventitious root fresh weight and adventitious root dry weight of transgenic strains were significantly higher than those of WT strain(P<0.05).The analysis showed that the rooting rate of transgenic strains was significantly higher than that of WT strain,and the average length of adventitious roots was significantly lower than that of WT strains(P<0.05).The investigation on the relative growth of plant height at 30 d after transplanting showed that the relative growth of OE2,OE6 and OE7 strains was significantly higher than that of WT strain,and the other 6 overexpression strains had no significant difference with WT strains;Only the OE6 strain was significantly higher than the WT strain in the relative growth of the plant height at the last 90 days,and the other 7 overexpression strains were significantly lower than the WT strain.In terms of leaf length/leaf width,the transgenic strains were significantly higher than the WT strain.(4)Analysis of 2 downstream target genes regulated by BpWOX11.Using the transgenic birch c DNA as the template,the expression characteristics of key downstream target genes regulated by BpWOX11 were analyzed by real-time quantitative PCR.The expression was upregulated in the expression strains.In conclusion,the adventitious root length and fresh weight of the adventitious root of the white birch BpWOX11 overexpressing line were significantly higher than those of the WT strain at the rooting stage of tissue culture,and the field cutting rooting rate of the transgenic strains was also significantly higher than that of the WT strain,indicating that BpWOX11 could promote the formation of birch adventitious roots and improve the rooting rate of cuttings. |
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