Molecular Mechanism Of Wood Formation Effected By Growth-related Gene PdTYDC2 In Populus Deltoides

Forest is an important non-crop energy plant,an important part of national forest resources,and a basic resource for sustainable use.Studies have shown that PdTYDC2,a gene related to poplar growth,participates in an important metabolic synthesis pathway of poplar and is related to the formation of xylem.This paper mainly focused on the molecular mechanism of growth-related gene PdTYDC2 participating in wood formation in poplar.We used 84K(Populus deltoides)as experimental material,obtained the overexpression and knockout transgenic plants of PdTYDC2 to study the gene function,and screened the possible upstream genes of PdTYDC2 by yeast-one-hybrid to study the regulation network.The main research results obtained in this paper are as follows:1.Obtained the PdTYDC2 sequence information from phytozome3.1 database and designed primers,the PdTYDC2 gene was isolated and cloned by reverse-transcription.The full length of PdTYDC2 gene was 1485 bp,coding 494 amino acid and containing a complete open reading frame of 1485 bp with no 5 ’non-coding region(5’ UTR)and no 3’non-coding region(3’ UTR).And analysis revealed that it contains a conserved domain of pyridoxal-dependent tyrosine decarboxylase.2.Constructed ProC4H: : PdTYDC2 vector and 35 s: : PdTYDC2 vector,carriered out genetic transformation of poplar,the count of received ProC4H: : PdTYDC2 transgenic line is 13,the growth parameters of the 120-day-old plants were found the plant height of overexpressed lines decreased by 15%,and the stem diameter of overexpressed lines increased by 21%.It showed that PdTYDC2 could positively regulate stem thickening and suppresses stem increase.3.The plant expression of PdTYDC2 was knocked out by CRISPR/Cas9,and two knockout sites were designed.Currently,the vector has been obtained.The poplar genetic transformation is under way and no transgenic plants have been obtained.4.266 MYB family transcription factors were obtained from the poplar transcription factor library.By comparing their expression levels on the website,48 MYB family transcription factors with high expression in xylem were finally selected.Forty-seven transcription factors were amplified,and the transcription factors were constructed in p GADT7 vector by infusion,and the plasmid was transformed into yeast Y187.Finally,a MYB family transcription factor yeast library with specific xylem expression capacity of47 was obtained.5.A cis-element sequence of PdTYDC2 is SMER: ACCAAAT constructed three in series in the construction of the pHIS2.1 vector as a bait and carried out the yeast-one-hybrid by mating.Finally obtained 20 candidate clones that possible regulation of PdTYDC2,including PdMYB199 reported earlier,suggesting that PdMYB199 can be combined with the promoter sequence of PdTYDC2.This study systematically analyzed the gene function and related genes upstream and downstream of the regulatory network through the genetic transformation system,and found that PdTYDC2 may be regulated by multiple upstream regulatory factors to affect the thickening of poplar stalks,including the focus of PdMYB199.The research will help to deepen the understanding of the molecular mechanism and regulatory network of poplar wood formation,and will provide theoretical reference for the future design and cultivation of new fast-growing,high-yielding,high-quality forest trees using genetic engineering.