Functional Characterization Of Transcription Factor BpMYB60 From Broussonetia Papyrifera In Response To Cd Stress

The rehabilitation of soil heavy metals,especially cadmium（Cd）pollution,has become an urgent problem.The phytoremediation of soil Cd pollution is not only green and low-carbon,but also cost-effective and beneficial to local ecological restoration,and the use of genetic engineered high quality and efficient Cd-tolerant plants is one of the important ways.Broussonetia papyrifera is a well-adapted plant species that is regarded as an excellent candidate for the management of heavy metal contamination in soil,but little is known about its molecular mechanisms of resistance.MYB is a very important transcription factor family in plants involving in various physiological and biochemical processes,such as secondary metabolism and stress.However,there is still a lack of research on the functional mechanisms of the MYB family members of B papyrifera.Therefore,in this study,a R2R3-like MYB gene,BpMYB60,with elevated expression in response to Cd stress was selected from the B.papyrifera transcriptome under Cd treatment,and BpMYB60 was inserted into B.papyrifera by Agrobacterium-mediated method to obtain overexpression transgenic plant lines.The non-transformed wild-type（NT）was used as a control.Then the growth and physiological related indexes of the overexpression and NT lines before and after Cd Cl₂ treatment were determined to explore the function of BpMYB60 in response to Cd stress.Meanwhile,the downstream target genes and metabolic pathways involved in the regulation of BpMYB60 were further identified by transcriptome sequencing technology,and combined with yeast one-hybrid（Y1H）method,GUS expression system as well as q RT-PCR method,the regulatory role of BpMYB60under Cd stress was systematically annotated,the molecular mechanism of BpMYB60regulating the response to Cd stress was revealled.The main findings are as follows:1.The open reading frame（ORF）of BpMYB60 is 1080 bp in length and encodes a protein containing 359 amino acids with a molecular weight of 39.57 KDa,the theoretical isoelectric point of 6.27.Analysis of the conserved structural domains of the protein revealed that BpMYB60 has a conserved SANT structural domain at the N-terminal 13～63 and 66～114amino acids,respectively（MYB structural domain）,which belongs to R2R3-MYB.Phylogenetic analysis showed that BpMYB60 protein shared the highest homology with Gs MYB60 protein.Under Cd Cl₂ stress,BpMYB60 was significantly induced in different tissues,such as the peak level in the root was 10.09-fold of the control,and the peak expression appeared sequentially as root-stem-leaf.2.BpMYB60 was inserted into the plant expression vector pROKII to form the reconstituted expression vector 35S::BpMYB60,and 35S::BpMYB60 was transformed into B.papyrifera by Agrobacterium-mediated method to acquire overexpression transgenic plants,and 11 transgenic lines were identified by DNA detection,the expression level was determined by q RT-PCR analysis and two lines（B60#1 and B60#2）with BpMYB60 expression to wild-type B.papyrifera over 100 were selected for further analysis.It was found that under Cd stress,B60#1 and B60#2 grew better than NT,maintained high levels of chlorophyll content,lower accumulation of reactive oxygen species（ROS）（H₂O₂ content）,lighter degree of cell damage（electrolyte leakage rate and MDA content）and higher antioxidant enzyme（CAT,GST,SOD,POD）activities.Meanwhile,the Cd transporter protein activity（NPT）,the Cd enrichment coefficient and transfer coefficient of B60#1 and B60#2 were higher than those of NT under Cd stress;the expression activities of antioxidant defense response-related genes（SOD,NHX,GST,GPX）were remarkably greater in B60#1 and B60#2 than those in NT,such as the relative expression of GST and GPX was 89.93 and 26.44-fold higher than those of NT,respectively.These results suggested that BpMYB60 can regulate the accumulation and transport of Cd through ROS accumulation,antioxidant protection and Cd transport system then to enhance the plant resistance to Cd stress.4.Y1H and GUS expression system were used to effectively verify that BpMYB60 could regulate the downstream target genes through recogning the elements of MYBCORE and TATCCAOSAMY,and the representative gene BpFC2 among the downstream candidate genes related to Cd transport was identified.QRT-PCR analysis confirmed that the expression of BpFC2 in the B60#2 was 10.56-fold of that in NT.Under Cd stress,the peak expression of BpFC2 in the root was 21.32-fold of the control at 96 h,indicating that BpFC2 is also a Cd-responsive gene.As a representative gene downstream of BpMYB60,the expression profile of BpFC2 under Cd Cl₂ stress tended to be consistent with that of BpMYB60,indicating that BpMYB60 and BpFC2 may have similar functional realization pathways in Cd response regulation.In conclusion,the BpMYB60 gene btained in this study can effectively regulate the transfer of Cd enrichment through the regulation of downstream BpFC2 and other genes based on multiple pathways such as ROS metabolism and MAPK signaling,which can provide theoretical basis and genetic resources for revealing the molecular mechanism of Cd response and constructing Cd-accumulated trees for Cd pollution remediation.