Analysis On Biological Function Of PtrUNE12 In Secondary Growth And Salt Stress Response Of Populus Alba × P.glandulosa ’84k’

As one of the most abundant renewable resources and the main fixer of atmospheric CO2,wood plays an important role in responding to the global energy crisis and climate change.At the same time,cultivating new tree varieties with strong stress resistance,excellent material properties and large growth is a powerful measure to alleviate the ecological safety problems caused by soil salinization.Plant bHLH transcription factor family genes play an important role in multiple biological processes of plants.However,so far no bHLH family genes have been found that have regulatory functions on the secondary growth of trees and salt stress.This study investigated the biological functions of the bHLH family PtrUNE12 of Populus trichocarpa in the secondary growth of poplar trees and the response to salt stress,and the conclusions are as follows:(1)Analysis of the expression characteristics of PtrUNE12 showed that PtrUNE12 is expressed to varying degrees in the roots,stems and leaves of poplars at different developmental stages,but the expression level is in the root tips,secondary stems,and mature leaves where the secondary growth is vigorous relatively high.PtrUNE12 is tissue-specific in response to salt stress,and the expression of PtrUNE12 is the highest in leaf and root tissues.PtrUNE12 cDNA is 903 bp in length,and its promoter region contains the high-frequency elements SMRE,SNBE,AC-1 and AC-2 in the biological pathway of SCW formation,and stress ABRE,MYC,MYB,MeJA response,GA response,SA response and ABA response element.The above results preliminarily indicate that PtrUNE12 participates in the secondary growth of poplar trees and the response to salt stress.(2)Transient transformation studies of tobacco epidermal cells show that PtrUNE12 is located in the nucleus.Yeast hybridization analysis found that PtrUNE12 has transcriptional autoactivation,and the activation region is located at the N-terminus of its encoded protein.(3)The transgenic plants of P.alba× P.glandulosa ’84K’ with the overexpressed PtrUNE12 and the significantly inhibited pROK Ⅱ-PtrUNE12-SRDX were obtained by the Agrobacterium infection method.The PtrUNE12 overexpression transgenic line has a normal phenotype and vigorous growth.while the dominant inhibitory transgenic line showed abnormal growth status and weak growth potential.Root character analysis showed that the PtrUNE12 overexpression transgenic line had vigorous root development,the root length,root fresh weight and dry weight,and adventitious root number increased,and the number and diameter of xylem vessels of the main root and the thickness of cellulose SCW increased significantly compared with the control.However,the significantly inhibited transgenic lines decreased significantly.Observation of leaf traits showed that the leaf length and leaf width of the overexpression transgenic lines increased significantly.Significant inhibition of the above traits is just the opposite,the leaf development is slow and there is obvious curling phenomenon.Stem trait analysis showed that the number of stem nodes,plant height and ground diameter of the overexpression transgenic line were significantly higher than those of the control,and the suppressed expression transgenic line was significantly lower than the control;the cellulose SCW thickness of the overexpression transgenic line and the number and diameter of the vessel The size is significantly higher than that of the control,and the traits of the fiber and vessel of the significantly inhibited strain are significantly lower than that of the control(Figure 5-4);Phloroglucinol staining showed that the secondary growth and lignin content of the stem node of the overexpression transgenic line IN4-11 increased,while the inhibition of the expression transgenic line was the opposite.Quantitative PCR analysis of secondary xylem showed that the expression of SCW components(lignin,cellulose and hemicellulose)of the overexpression transgenic lines increased significantly,while the expression of the SCW components of the significantly inhibited transgenic lines did not change significantly or significantly decrease(Figure 5-11).Further studies using yeast one-hybrid technology showed that PtrUNE12 has a direct binding function to the promoters of lignin synthesis genes PAL1 and CCR2.In summary,PtrUNE12 can not only promote the primary growth of plants(such as better root,stem and leaf development),but also increase the secondary growth of poplars and the formation of SCW.(4)The results of histochemical staining of leaves showed that:compared with the wild-type control,under normal growth conditions,the PtrUNE12 overexpression transgenic line and the significantly inhibited transgenic line had no significant changes in the active oxygen content in the body and the cell structure was intact.When the salt stress is 24h,the staining intensity of NBT and DAB in the leaves of the overexpression transgenic lines becomes weaker.while the dominant suppression expression vector increases.The determination of the activity of the main active oxygen scavenging enzymes showed that under normal growth conditions,the PtrUNE12 overexpression transgenic lines and the transgenic lines significantly inhibited the content of the main active oxygen scavenging enzymes SOD and POD,superoxide anion(OFR)and malondialdehyde(MDA).Compared with the control,there was no significant change;after salt stress for 24 hours,the activity of SOD、POD scavenging enzymes of the PtrUNE12 overexpression transgenic line increased significantly,while the content of OFR and MDA decreased significantly;the above-mentioned traits of the transgenic line significantly inhibited the opposite(Figure 6-6.Figure 6-7).Quantitative PCR studies on genes related to osmotic stress show that:Under normal growth conditions,PtrUNE12 overexpression transgenic lines showed no significant difference in expression of osmotic stress-related genes(RD22,RD29A,AREB1A.AREB1B,and DREB2A)compared with significantly suppressed transgenic lines and wild-type controls;under salt stress,PtrUNE12 overexpression The expression of the above-mentioned genes in the transgenic lines was significantly increased,while the above-mentioned genes in the expression-inhibited lines were significantly decreased.The above results indicate that under normal conditions,PtrUNE12 does not participate in the regulation of active oxygen metabolism pathways and stress response pathways in poplars,under salt stress,PtrUNE12 can respond to salt stress by regulating the biological pathways of active oxygen metabolism and stress-responsive gene expression in poplar trees.