Functional Analysis Of LBD1-30 Gene In Stem Secondary Growth Of Populus

Secondary growth is the biological basis of perennial wood growth.During this process,vascular cambium stem cells undergo continuous cell division and differentiation,cell expansion,secondary cell wall synthesis,and programmed cell death,and then produce secondary phloem and xylem.At present,most researches about secondary growth focused on genes with higher expression in xylem than in phloem,and few studies focused on genes with higher expression in phloem than in xylem.This study uses the latter as a starting point.We study the regulation of genes on secondary growth through genetic engineering.By analyzing the previous RNA-seq data,we characteriazed the whole gene expression level of xylem and phloem in the stem of the poplar,and focused on the analysis of genes in the phloem higher than xylem.We found a LBD gene that was expressed significantly higher in the secondary phloem than secondary xylem.In this study,The stem segments of84 K poplar was used as material,and the LBD1-30 gene(Potri.008G043900)was cloned.The overexpression vector of 35S: LBD1-30 was constructed.Using Agrobacterium-mediated gene transformation system,LBD1-30 gene was transferred into84 K poplar,and the effect of LBD1-30 on the development of secondary vascular tissue of poplar was preliminary verified by phenotypic analysis and section analysis.It is proved that it has a significant regulatory effect on the secondary growth of poplars.This research provides important practical significance and theoretical value for cultivating new varieties of fast-growing poplar trees in the future.The main findings are as follows:(1)Cloning and sequence analysis of 84 K poplar LBD1-30 gene.The CDNA sequence of the LBD1-30 gene was searched from the LBD1-30 gene bank of Populus trichocarpa.The CDNA sequence of the LBD1-30 gene is 666 bp in length and encodes 221 amino acids.Primers were designed and total RNA was extracted from 84 K poplar stems.The LBD1-30gene(Potri.008G043900)was cloned by RT-PCR.The results of alignment analysis showedthat there were more than one single nucleotide polymorphism(SNP)in the coding sequence of LBD1-30 in the 84 K poplar compared to the Populus trichocarpa reference sequence,but the similarity of the protein sequence was as high as 96.4 %.(2)We constructed an overexpression vector: 35S: LBD1-30,and successfully transferred to 84 K poplar by Agrobacterium-mediated transformation.The rooted transgenic seedlings generated from the callus were transferred to a new rooting medium containing 30mg/L Kana and then screened.The rooted transgenic plants were selected for DNA extraction for PCR identification,and the transgenic positive plants were finally obtained.(3)Quantitative and phenotypic analysis of LBD1-30 gene expression level in transgenic seedlings.The expression level of LBD1-30 gene in transgenic seedlings was much higher than that of wild type,and there was a significant variations in the expression level between different lines.Transgenic plants can be categorized into high-thick and low-thick phenotypes.With the increase in gene expression,the low-thick phenotypes became more and more obviously.But this transgenic lines rooting slow,which is difficult to prepare materials.The gene expression level of the high-thick lines was slightly lower than that of the low-thick lines,but the rooting,growth were the same as wild type,and the later growth was appreciable.(4)Regulation of secondary growth by LBD1-30 transgene.Compared with the wild type,LBD1-30 overexpression transgenic seedlings showed increased lignification of secondary phloem fibers,while secondary xylem lignification was inhibited and even irregular lignification was evident.It was suggested that LBD1-30 gene may play an important role in the regulation of mitotic proliferation and secondary xylem/secondary phloem partial process of stratum stem cells in the secondary growth process.