Cloning And Functional Studies Of KNOX Genes In Betula Luminifera

Betula luminifera is one of the excellent fast-growing timber species,which was classified as a national first grade material.At the same time,it's also a kind of fine material for forest genetic studying.In this study,bioinformatics tools and biological techniques,such as gene cloning and quantitative PCR,were used for excavating the KNOX genes related to wood properties from B.luminifera and explore the mechanism,which will provide an important basis for molecular breeding of B.luminifera.The main results are as follows:In this study,10 BlKNOX genes were isolated from B.luminifera by using RACE technique.The length of the sequence was between 4332 and 9208 bp.The length of ORF was between 408 and 1220 bp.The predicted coding of 10 protein sequences consisted of 136-376 amino acid residues,and the similarity of amino acid sequence was from 12.7 to 64.8%.Bioinformatics analysis revealed that they had typical KNOX domain,with the exception of BlKNOX6,the other genes had a complete KNOX domain.Systemic evolution analysis found that KONX protein were divided into two main categories:KNOX ? and KNOX ? class.KNOX Class I includes: BlSTM,BlKNOX1,BlKNOX2,BlKNOX3,BlKNOX4 and BlKNOX9.KNOX Class II includes: BlKNOX5,BlKNOX7 and BlKNOX8,where BlKNOX6 with incomplete KNOX domains belonged to the miniKNOX class.From the genetic structure,the gene structure of the 10 BlKNOX was obviously different,which indicated that their function might be different in the process of growth and development of B.luminifera.The results of the up-regulation elements of BlKNOX genes showed that the number and types of cis-acting elements contained in the promoter region of 10 BlKNOX genes were different,which indicated that they could play different roles in regulating different metabolic pathways.Quantitative PCR results showed that 10 BlKNOX genes were specifically expressed in different tissues and organs in B.luminifera.BlKNOX1 had the highest expression level in mature male flowers,followed by stems and female flowers,and BlKNOX1 expression levels were higher expressed in different stem degree lignification.BlKNOX2 had a higher expression level in all stems and flowers,and raised with the increase of the degree of lignification of the stem,and had the lowest expression in leaves.The expression level of BlKNOX3 in stem was significantly higher than that of other organs,and increased with the increase of lignification degree.BlKNOX4,BlSTM have a similar expression pattern,which both have a high expression level in stem and young male flowers and is hardly expressed in leaves.BlKNOX5,BlKNOX7 also have a similar expression pattern,respectively,in mature leaves and young leaves with the highest expression.Both BlKNOX6 and BlKNOX9 were highly expressed in female flowers and leaves.BlKNOX8 was only expressed in female flowers,especially in young female flowers.The results of steroid stress demonstrated that the induction effect of IAA on BlKNOX2 and BlSTM was obvious,and the induction effect was more obvious with the increase of lignification degree.6-BA only inhibited the expression of BlKNOX4 gene.Treated by GA and ACC,the expression of 5BlKNOX genes was similar,except that the increased expression of BlSTM gene and the expression of other genes was inhibited to different degree.NAA treatment of five BlKNOX gene induction wascontrary to GA and ACC.The results of hormone treatment demonstrated that the induction of BlKNOX gene was different by different hormone treatments,and the regulatory pathway of BlKNOX gene may not be identical.The expressions of BlKNOX1 and BlSTM were decreased in both TW and OW,and the expression of BlKNOX2,BlKNOX7 and BlKNOX4 were increased in TW,but the expression between OW and the control were same nearly.The BlKNOX1?BlKNOX3 overexpression vector were constructed,and the positive plants were obtained by converting tobacco and B.luminifera.From tobacco transgenic positive plants,we found that transgenic lines stem more erect,leaf edge wrinkling and deeper veins,compared with the wild type.GUS staining also found that tobacco-positive plant stem segments can be dyed blue.Maule staining showed that the degree of lignification of transgenic tobacco was higher than that of control.Transgenic B.luminifera plants were only detected in the positive plants,phenotypic observation and staining analysis need further study.The above experimental results not only explored the function of BlKNOX gene,but also laid a foundation for the analysis of the molecular basis of the woody formation of B.luminifera,and provided the molecular assisted breeding and seed promotion of B.Luminifera.