Effects On Growth And Development Of Populus By Overexpression Of BAS1 Gene From Arabidopsis Thaliana

The BAS1(phy B activation-tagged suppressor1)gene encodes a monooxygenase CYP72B1 containing cytochrome P450,which has the activity of hydroxylase and can catalyze the hydroxylation of brassinosteroid 26 C to hydroxylate the brassinosteroid.Lactone bioactivity is reduced,so the BAS1 gene acts as a C-26-hydroxylase,an important brassinosteroid inactivation gene.Brassinosteroids(brassican steroids,BR)affect plant growth and development and play an important regulatory role in cell elongation and division,vascular bundle differentiation,leaf morphogenesis,plant fertility,senescence and resistance.Poplar is an important afforestation and industrial tree species in China.It is considered as a model plant for genetic engineering of forest trees.Wood-based products originate from forest trees,improve the quality of the wood,and achieve directional cultivation,which can increase the utilization rate of wood and realize the efficient use of resources on limited forest land.Plant vascular development is the basis of wood formation.Phytohormones affect the development of plant vascular tissue.Therefore,it is of great significance to study the developmental regulatory mechanism of plant vascular tissue.In this study,a plant expression vector of BAS1 gene driven by p LXM5 and35 S promoters was constructed using Populus tomentosa as a material.At BAS1 gene was genetically transformed into poplar by Agrobacterium tumefaciens-mediated leaf disc transformation method to study the growth of Populus tomentosa.The effect of development obtained the following findings:1 Construction of Plant Expression Vectors and Acquisition of Transgenic PlantsTwo plant expression vectors were constructed.The gus::npt II fusion gene was used as a screening marker gene and a reporter gene.Kan+ was a screening marker gene and was named p SH-35S-BAS1 and p SH-p LXM5-BAS1,respectively.The expression vector was transformed into Agrobacterium tumefaciens strain LBA4404,and transformed into Populus tomentosa by leaf disc transformation.Fifty-four strains of poplar transgenic to p LXM5::BAS1 gene and 34 strains of transgenic35S::BAS1 poplar were obtained through screening.2 Agronomic Traits of Transgenic P.tomentosaBy measuring the height and diameter of the independent strains,it was found that the height and diameter of the transgenic plants grew faster than the wild type,and the ratio of diameter to plant height was found to be consistent with the increase in the diameter and height of the transgenic poplars,indicating that the transgenic plants were increased.At the same time,it is also increasing.The diameter of the plant at a height of 0 cm from the soil and the diameter at a diameter at the plant's DBH(130 cm)were measured.The results showed that the stalks of the transgenic p LXM5::BAS1 were increased by 50% and 89%,respectively,and the 35S::BAS1gene was transferred to Yang.Tree stems were increased by 46% and 70%,respectively.The length of the section of breast diameter(130 cm)and the length and width of the upper and lower leaves of the upper and lower leaves at breast height(130 cm)were measured.However,the stem length of the transgenic plants was not significantly different from that of the control group.The transgenic white poplar leaves were in contrast to the control group.In comparison,the increase in leaf length was not significant and the leaf width increased by 42% and 40% respectively.3 Determination of fresh weight and dry weight of transgenic white poplarThrough the determination of the fresh and dry weights of Populus tomentosa,it was found that the fresh weight of poplars transformed with p LXM5::BAS1 gene was 1.8 times that of the control group,and the dry weight was 1.7 times that of the control group;the transgenic 35S::BAS1 poplar was The fresh weight was 2 times that of the control group,and the dry weight was 2.2 times that of the control group.4 Effects of overexpression of BAS1 gene on the Tissue Structure of Stem and Vein of Populus tomentosaBy sectioning and microscopic observation of stems and veins of transgenic plants of Populus tremuloides,the number of xylem cell layers in p LXM5::BAS1and dwarfing poplars was reduced by 38% and 23% compared with the wild type;35S::BAS1 gene Yang The number of myeloid cells in the stem increased by 37%compared to the control group.This indicates that BAS1 gene mainly inhibits the differentiation of xylem and promotes the differentiation of phloem and pith.The length of the cortical cells of the p LXM5::BAS1 gene was increased by 20%,the length of the myeloid cells was increased by 22%,the length of the cortical cells of the 35S::BAS1 gene was increased by 32%,and the length of the myeloid cells was increased by 66% in the dwarfed plants.The cell length was reduced by 19% and the length of myeloid cells was reduced by 38%,indicating that the BAS1 gene expression can regulate cell length.The number of xylem vessels in the p LXM5::BAS1 gene was decreased by 54% compared with the control group,and the number of xylem vessels in the dwarf hairy white stem of the 35S::BAS1 gene was reduced by 39% compared to the control group.The number of myeloid cells increased but not significant,indicating that the expression of the BAS1 gene affects cell division.In leaves,there was no significant difference between p LXM5::BAS1gene and the control group;the 35S::BAS1 gene poplar veins were significantly thicker than the controls,and the xylem was well developed.The number of cortical cells increased,but the leaf thickness was not.obvious change.The dwarf poplar veins were coarser than the control,the xylem of the veins was small,and the ductal cells were few.The number of palisade cells and sponge cells increased.5 Expression of Brassinolide Related Genes in the Expression of BAS1 GeneThrough the determination of brassinosteroid content,it was found that the BRs content in transgenic p PLX transgenic plants of transgenic p LXM5::BAS1 and transgenic 35S::BAS1 were 1.1-fold and 1.13-fold higher than those of wild-type plants,respectively.The content is only 1/10 of the wild type,and it is significantly reduced.The expression analysis of brassinosteroid-related genes revealed that the expression of BAS1 gene reduced the BR content,and BR synthesis pathway initiated the expression of BR synthetic genes CYP90A1/CPD,CYP90C1/ROT3 and CYP90D1,and the expression of BAS1 gene.The degree will affect the expression of BR metabolic gene CYP734A1.It was found that proper expression of the BAS1 gene suppressed the expression of the BR metabolic gene CYP734A1,resulting in an increase in the BR content.Overexpression of the BAS1 gene promoted the expression of the BR metabolic gene CYP734A1,resulting in a lower BR content than the wild type Yang.tree.The results showed that the expression of BAS1 gene not only affected the synthesis of BR synthesis and metabolic pathway related genes,but also affected the negative feedback regulatory elements of BR synthesis.The up-regulation or down-regulation of these genes may promote cell differentiation and plant growth and development.