| Cadmium(Cd)is a highly toxic environmental pollutant.In recent years,Cd has been released into soil and water due to various anthropogenic activities such as mining and industrialization,resulted in many Cd-contaminated lands unable to be used normally.Cd can not only enter the plant system through soil and affects the growth and development of plants,but also threatens human health through the food chain.Phytoremediation is an important remediation method for soil Cd pollution.The construction of high-quality and efficient plants through genetic engineering is an important way to break through the limitations of wild plants.Therefore,it will become crucial to clarify the mechanism of plant regulation of Cd pollution and to cultivate new Cd-resistant plant varieties.Broussonetia papyrifera is considered to be an important candidate plant for heavy metal-contaminated soil remediation.Although many physiological and biochemical studies have been carried out to prove that B.papyrifera has a strong tolerance to Cd,the molecular mechanism of the tree resistance to Cd is still unknown.Transcriptional regulation is an important molecular mechanism of plant stress response.In response to the challenge of environmental stress,plant cells activate several signaling pathways and trigger the expression of transcription factors(TFs).MYB protein is one of the largest families of plant TFs,which is specific to plants and participate in the regulation of plant cell development,secondary metabolism,and responses to environmental stresses.In this study,the R2R3-MYB family gene BpTT2 in response to Cd stress,was overexpressed,and then transformed into wild-type B.papyrifera(WT)by agrobacterium-mediated method using genetic engineering to obtain BpTT2-overexpressed B.papyrifera.Through pot experiments under natural growth conditions(CK)and 500 μmol/L CdCl2 stress,the possible physiological and biochemical processes and heavy metal Cd enrichment of BpTT2-overexpressed B.papyrifera were analyzed,and the anti-Cd function of BpTT2 gene was verified.The downstream genes and metabolic pathways regulated by BpTT2 under Cd stress were further screened by transcriptome sequencing technology(RNA-Seq),and the expression regulation mechanism of related genes under Cd stress was analyzed by qRT-PCR.The anti-Cd molecular function of BpTT2 gene was expounded.The main results are as follows:(1)The seeds of B.papyrifera from different sources were collected,and the excellent germplasm resources with the maximum length,width,1000-seed weight and moisture content were selected for seed germination.A large number of B.papyrifera tissue culture seedlings were obtained by plant tissue culture.The plant expression vector containing overexpressed BpTT2 and the empty plasmid vector pROKII were introduced into the leaves and stem segments of B.papyrifera by agrobacterium-mediated method,and the BpTT2-overexpressed B.papyrifera was obtained by co-cultivation,sterilization,screening of resistant buds.The qRT-PCR verification showed that the expression of BpTT2 gene in BpTT2-overexpressed B.papyrifera was significantly higher than that of WT and empty vector pROKII B.papyrifera.Among them,the transgenic line with the highest relative expression was 7.22 times that of WT.(2)The pot experiment confirmed the anti-Cd function of BpTT2-overexpressed B.papyrifera,that is,under the stress of 500 μmol/L CdCl2 for three months,the plant height,biomass,soluble protein content,chlorophyll a and total chlorophyll content of BpTT2-overexpressed B.papyrifera seedlings were significantly higher than WT and pROKII B.papyrifera seedlings(P<0.001).Meanwhile,more substances related to ROS accumulation,such as MDA and H2O2,were detected in non-transformed B.papyrifera lines.Not only that,Cd reduced the stomatal conductance(Gs)of non-transformed B.papyrifera leaves,affected the absorption of CO2,thereby prevented photosynthesis.The three antioxidant enzymes activities of SOD,POD and CAT were significantly increased in the non-transformed lines(P<0.01),reflected the active state for scavenging more ROS.All in all,from the physiological and biochemical characteristics,BpTT2-overexpressed B.papyrifera can protect against oxidative stress caused by Cd stress by regulating osmotic pressure and reducing ROS accumulation.(3)The enrichment of heavy metal Cd in three lines of B.papyrifera under CK and Cd stress was analyzed,the results showed that:there were no significant differences in Cd content,translocation factor(TF)and biocon centration factor(BCF)in soil,roots,stems and leaves under CK condition(P>0.05).Under Cd stress,the WT had the highest enrichment of Cd,with a BCF of 3.41±0.72;the BpTT2-overexpressed B.papyrifera had the least Cd enrichment,with a BCF of 1.52±0.05,which was significantly lower than that of the non-transformed lines(P<0.05).Cd was enriched in the B.papyrifera tissues as follows:root>stem>leaf,and the residual Cd content in the soil was significantly increased in the BpTT2-overexpressed B.papyrifera(P>0.05).It indicated that the overexpression of BpTT2 was mainly through inhibiting Cd2+in soil and plant roots,prevented more Cd from entering cells and inhibiting growth and development.(4)Transcriptome analysis was performed on leaves of BpTT2-overexpressed B.papyrifera and WT B.papyrifera under Cd stress.In the WT-Cd vs BpTT2-Cd comparison group,a total of 2626 DEGs were screened with significantly different expression(q-value<0.05),and more than 3/4 of the genes were up-regulated.GO functional annotation revealed that these DEGs were mainly involved in "cell wall macromolecule catabolic process" and "integral component of membrane".KEGG enrichment analysis showed that a large number of genes related to Cd stress response were significantly enriched in the metabolic pathways of "Plant-pathogen interaction","MAPK signaling pathway-plant","Plant hormone signal transduction" and "Flavonoid biosynthesis".Among them,the ABA signal transduction genes SNRK2 and PP2C were also involved in the regulation of "MAPK signaling pathway-plant" and"Plant-pathogen interaction".Many TFs genes were also induced to be up-regulated,including WRKY TFs genes WRKY29,WRKY33,AP2/ERF TFs gene ERF1 and bHLH TFs gene MYC2,which also regulated multiple signaling cascades simultaneously and enhanced the defense ability of BpTT2-overexpressed B.papyrifera against Cd.(5)Some downstream representative genes induced by BpTT2 overexpression were verified by qRT-PCR,and the results confirmed the reliability of transcriptome sequencing results.Among them,the plant hormone AUX/IAA signal transduction gene ARF5 and ABA signal transduction genes PP2C and SNRK2,protein kinase-related genes(PHOT1,WAKL4,PK1 and MPK3),ROS accumulation-related genes(RbohD and CAT1),and a large number of TFs family genes highly sensitive to Cd stress(WRKY33,WRKY29,MYB2,MYB4,ERF1,ABR1,CRF4 and MYC2)were significantly up-regulated in BpTT2-overexpressed B.papyrifera leaves under Cd stress(P<0.01);while the glutathione S-transferase(GST)genes GSTU22 and GSTL3 function mainly by inhibiting their expression.These genes were consistent in the regulation of Cd stress in the same protein family,which are not only involved in the regulation of plant signal transduction pathways,but also involved in scavenging ROS and feedback regulation of H2O2 signal.The candidate genes related to Cd enrichment and tolerance screened and identified in this study are the prerequisites for phytoremediation using genetic engineering.The results elucidated the basic biological functions and molecular regulatory mechanisms of BpTT2-overexpressed B.papyrifera under Cd stress,especially provided important genomic resources for subsequent functional studies of MYB protein,and field trials of transgenic B.papyrifera will contribute to future phytoremediation applications. |
